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tubulin beta antibody

RRID:AB_528499

Antibody ID

AB_528499

Target Antigen

Proper Citation

(DSHB Cat# E7, RRID:AB_528499)

Clonality

unknown

Comments

consolidated with AB_2315513 and AB_2315515 on 02/2018 by curator.

Vendor

DSHB

Cat Num

E7

Publications that use this research resource

Collision of Expanding Actin Caps with Actomyosin Borders for Cortical Bending and Mitotic Rounding in a Syncytium.

  • Zhang Y
  • Dev. Cell
  • 2018 Jun 4

Literature context:


Abstract:

The early Drosophila embryo is a large syncytial cell that compartmentalizes mitotic spindles with furrows. Before furrow ingression, an Arp2/3 actin cap forms above each nucleus and is encircled by actomyosin. We investigated how these networks transform a flat cortex into a honeycomb-like compartmental array. The growing caps circularize and ingress upon meeting their actomyosin borders, which become the furrow base. Genetic perturbations indicate that the caps physically displace their borders and, reciprocally, that the borders resist and circularize their caps. These interactions create an actomyosin cortex arrayed with circular caps. The Rac-GEF Sponge, Rac-GTP, Arp3, and actin coat the caps as a growing material that can drive cortical bending for initial furrow ingression. Additionally, laser ablations indicate that actomyosin contraction squeezes the cytoplasm, producing counterforces that swell the caps. Thus, Arp2/3 caps form clearances of the actomyosin cortex and control buckling and swelling of these clearances for metaphase compartmentalization.

Funding information:
  • NIGMS NIH HHS - R01 GM099773(United States)

A Drosophila Tumor Suppressor Gene Prevents Tonic TNF Signaling through Receptor N-Glycosylation.

  • de Vreede G
  • Dev. Cell
  • 2018 Jun 4

Literature context:


Abstract:

Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. Here we report that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. We show that ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, which results in increased binding to the continually circulating TNF. Our results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues.

Funding information:
  • NICHD NIH HHS - U54 HD07495-26/30(United States)
  • NIGMS NIH HHS - R01 GM090150()

FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

  • Saridaki T
  • J. Neurochem.
  • 2018 May 10

Literature context:


Abstract:

Parkinson disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that overexpression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson disease we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 overexpression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy, and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7 induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7 induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by the dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of α-synuclein and could be beneficial in patients with Parkinson disease. This article is protected by copyright. All rights reserved.

Funding information:
  • NIAAA NIH HHS - P20 AA017837(United States)

Dynamic Fluctuations in Subcellular Localization of the Hippo Pathway Effector Yorkie In Vivo.

  • Manning SA
  • Curr. Biol.
  • 2018 May 21

Literature context:


Abstract:

The Hippo pathway is an evolutionarily conserved signaling network that integrates diverse cues to control organ size and cell fate. The central downstream pathway protein in Drosophila is the transcriptional co-activator Yorkie (YAP and TAZ in humans), which regulates gene expression with the Scalloped/TEA domain family member (TEAD) transcription factors [1-8]. A central regulatory step in the Hippo pathway is phosphorylation of Yorkie by the NDR family kinase Warts, which promotes Yorkie cytoplasmic localization by stimulating association with 14-3-3 proteins [9-12]. Numerous reports have purported a static model of Hippo signaling whereby, upon Hippo activation, Yorkie/YAP/TAZ become cytoplasmic and therefore inactive, and upon Hippo repression, Yorkie/YAP/TAZ transit to the nucleus and are active. However, we have little appreciation for the dynamics of Yorkie/YAP/TAZ subcellular localization because most studies have been performed in fixed cells and tissues. To address this, we used live multiphoton microscopy to investigate the dynamics of an endogenously tagged Yorkie-Venus protein in growing epithelial organs. We found that the majority of Yorkie rapidly traffics between the cytoplasm and nucleus, rather than being statically localized in either compartment. In addition, discrete cell populations within the same organ display different rates of Yorkie nucleo-cytoplasmic shuttling. By assessing Yorkie dynamics in warts mutant tissue, we found that the Hippo pathway regulates Yorkie subcellular distribution by regulating its rate of nuclear import. Furthermore, Yorkie's localization fluctuates dramatically throughout the cell cycle, being predominantly cytoplasmic during interphase and, unexpectedly, chromatin enriched during mitosis. Yorkie's association with mitotic chromatin is Scalloped dependent, suggesting a potential role in mitotic bookmarking.

Funding information:
  • Wellcome Trust - 090532(United Kingdom)

Reduced TDP-43 Expression Improves Neuronal Activities in a Drosophila Model of Perry Syndrome.

  • Hosaka Y
  • EBioMedicine
  • 2018 Apr 11

Literature context:


Abstract:

Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150Glued and TDP-43 is largely unknown. Here, we report that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150G50R in Drosophila. Dopaminergic expression of p150G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43 TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities.

Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals.

  • Jee D
  • Mol. Cell
  • 2018 Jan 18

Literature context:


Abstract:

While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.

Funding information:
  • NIAID NIH HHS - AI075257(United States)

E2F/DP Prevents Cell-Cycle Progression in Endocycling Fat Body Cells by Suppressing dATM Expression.

  • Guarner A
  • Dev. Cell
  • 2017 Dec 18

Literature context:


Abstract:

To understand the consequences of the complete elimination of E2F regulation, we profiled the proteome of Drosophila dDP mutants that lack functional E2F/DP complexes. The results uncovered changes in the larval fat body, a differentiated tissue that grows via endocycles. We report an unexpected mechanism of E2F/DP action that promotes quiescence in this tissue. In the fat body, dE2F/dDP limits cell-cycle progression by suppressing DNA damage responses. Loss of dDP upregulates dATM, allowing cells to sense and repair DNA damage and increasing replication of loci that are normally under-replicated in wild-type tissues. Genetic experiments show that ectopic dATM is sufficient to promote DNA synthesis in wild-type fat body cells. Strikingly, reducing dATM levels in dDP-deficient fat bodies restores cell-cycle control, improves tissue morphology, and extends animal development. These results show that, in some cellular contexts, dE2F/dDP-dependent suppression of DNA damage signaling is key for cell-cycle control and needed for normal development.

Funding information:
  • British Heart Foundation - WT091310(United Kingdom)

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

  • Sigg MA
  • Dev. Cell
  • 2017 Dec 18

Literature context:


Abstract:

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

Funding information:
  • NIA NIH HHS - R01 AG027849(United States)
  • NIAMS NIH HHS - R01 AR054396()
  • NIGMS NIH HHS - R01 GM095941()

Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity.

  • Clark JP
  • Curr. Biol.
  • 2017 Sep 11

Literature context:


Abstract:

To test the directness of factors in initiating PIWI-directed gene silencing, we employed a Piwi-interacting RNA (piRNA)-targeted reporter assay in Drosophila ovary somatic sheet (OSS) cells [1]. This assay confirmed direct silencing roles for piRNA biogenesis factors and PIWI-associated factors [2-12] but suggested that chromatin-modifying proteins may act downstream of the initial silencing event. Our data also revealed that RNA-polymerase-II-associated proteins like PAF1 and RTF1 antagonize PIWI-directed silencing. PAF1 knockdown enhances PIWI silencing of reporters when piRNAs target the transcript region proximal to the promoter. Loss of PAF1 suppresses endogenous transposable element (TE) transcript maturation, whereas a subset of gene transcripts and long-non-coding RNAs adjacent to TE insertions are affected by PAF1 knockdown in a similar fashion to piRNA-targeted reporters. Additionally, transcription activation at specific TEs and TE-adjacent loci during PIWI knockdown is suppressed when PIWI and PAF1 levels are both reduced. Our study suggests a mechanistic conservation between fission yeast PAF1 repressing AGO1/small interfering RNA (siRNA)-directed silencing [13, 14] and Drosophila PAF1 opposing PIWI/piRNA-directed silencing.

Mechano-dependent signaling by Latrophilin/CIRL quenches cAMP in proprioceptive neurons.

  • Scholz N
  • Elife
  • 2017 Aug 8

Literature context:


Abstract:

Adhesion-type G protein-coupled receptors (aGPCRs), a large molecule family with over 30 members in humans, operate in organ development, brain function and govern immunological responses. Correspondingly, this receptor family is linked to a multitude of diverse human diseases. aGPCRs have been suggested to possess mechanosensory properties, though their mechanism of action is fully unknown. Here we show that the Drosophila aGPCR Latrophilin/dCIRL acts in mechanosensory neurons by modulating ionotropic receptor currents, the initiating step of cellular mechanosensation. This process depends on the length of the extended ectodomain and the tethered agonist of the receptor, but not on its autoproteolysis, a characteristic biochemical feature of the aGPCR family. Intracellularly, dCIRL quenches cAMP levels upon mechanical activation thereby specifically increasing the mechanosensitivity of neurons. These results provide direct evidence that the aGPCR dCIRL acts as a molecular sensor and signal transducer that detects and converts mechanical stimuli into a metabotropic response.

Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye.

  • Kim W
  • Dev. Cell
  • 2017 Aug 21

Literature context:


Abstract:

Target of rapamycin complex 1 (TORC1) regulates cell growth in response to nutrients and growth factors. Although TORC1 signaling has been thoroughly studied at the cellular level, the regulation of TORC1 in multicellular tissues and organs has remained elusive. Here we found that TORC1 is selectively activated in the second mitotic wave (SMW), the terminal synchronous cell division, of the developing Drosophila eye. We demonstrated that Hedgehog (Hh) signaling regulates TORC1 through E2F1 and the cyclin D/Cdk4 complex in the SMW, and this regulation is independent from insulin and amino acid signaling pathways. TORC1 is necessary for the proper G1/S transition of the cells, and the activation of TORC1 rescues the cell-cycle defect of Hh signaling-deficient cells in the SMW. Based on this evolutionarily conserved regulation of TORC1 by Hh signaling, we propose that Hh-dependent developmental signaling pathways spatially regulate TORC1 activity in multicellular organisms.

Funding information:
  • NIMHD NIH HHS - G12 MD007592(United States)

MUC1 and HIF-1alpha Signaling Crosstalk Induces Anabolic Glucose Metabolism to Impart Gemcitabine Resistance to Pancreatic Cancer.

  • Shukla SK
  • Cancer Cell
  • 2017 Jul 10

Literature context:


Abstract:

Poor response to cancer therapy due to resistance remains a clinical challenge. The present study establishes a widely prevalent mechanism of resistance to gemcitabine in pancreatic cancer, whereby increased glycolytic flux leads to glucose addiction in cancer cells and a corresponding increase in pyrimidine biosynthesis to enhance the intrinsic levels of deoxycytidine triphosphate (dCTP). Increased levels of dCTP diminish the effective levels of gemcitabine through molecular competition. We also demonstrate that MUC1-regulated stabilization of hypoxia inducible factor-1α (HIF-1α) mediates such metabolic reprogramming. Targeting HIF-1α or de novo pyrimidine biosynthesis, in combination with gemcitabine, strongly diminishes tumor burden. Finally, reduced expression of TKT and CTPS, which regulate flux into pyrimidine biosynthesis, correlates with better prognosis in pancreatic cancer patients on fluoropyrimidine analogs.

Funding information:
  • NCI NIH HHS - P01 CA117969()
  • NCI NIH HHS - P30 CA036727()
  • NCI NIH HHS - P30 CA046934()
  • NCI NIH HHS - P50 CA127297()
  • NCI NIH HHS - R01 CA163649()
  • NCI NIH HHS - R01 CA210439()
  • NCI NIH HHS - R01 CA216853()
  • NCI NIH HHS - U54 CA163120()
  • NIGMS NIH HHS - P30 GM106397()
  • None - 13-20-25-SING()

Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division.

  • Banerjee JJ
  • Elife
  • 2017 Jun 30

Literature context:


Abstract:

Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. We show that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery.

Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration.

  • Saini JS
  • Cell Stem Cell
  • 2017 May 4

Literature context:


Abstract:

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins, and those from the AMD donors show significantly increased complement and inflammatory factors, which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening, combined with transcriptome analysis, we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome, ribosome, and chromatin-modifying genes. Thus, targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD.

Funding information:
  • NEI NIH HHS - F32 EY025931()
  • NEI NIH HHS - R01 EY022079()
  • NIA NIH HHS - RF1 AG042932()

Regulatory Role of RNA Chaperone TDP-43 for RNA Misfolding and Repeat-Associated Translation in SCA31.

  • Ishiguro T
  • Neuron
  • 2017 Apr 5

Literature context:


Abstract:

Microsatellite expansion disorders are pathologically characterized by RNA foci formation and repeat-associated non-AUG (RAN) translation. However, their underlying pathomechanisms and regulation of RAN translation remain unknown. We report that expression of expanded UGGAA (UGGAAexp) repeats, responsible for spinocerebellar ataxia type 31 (SCA31) in Drosophila, causes neurodegeneration accompanied by accumulation of UGGAAexp RNA foci and translation of repeat-associated pentapeptide repeat (PPR) proteins, consistent with observations in SCA31 patient brains. We revealed that motor-neuron disease (MND)-linked RNA-binding proteins (RBPs), TDP-43, FUS, and hnRNPA2B1, bind to and induce structural alteration of UGGAAexp. These RBPs suppress UGGAAexp-mediated toxicity in Drosophila by functioning as RNA chaperones for proper UGGAAexp folding and regulation of PPR translation. Furthermore, nontoxic short UGGAA repeat RNA suppressed mutated RBP aggregation and toxicity in MND Drosophila models. Thus, functional crosstalk of the RNA/RBP network regulates their own quality and balance, suggesting convergence of pathomechanisms in microsatellite expansion disorders and RBP proteinopathies.

LSM12 and ME31B/DDX6 Define Distinct Modes of Posttranscriptional Regulation by ATAXIN-2 Protein Complex in Drosophila Circadian Pacemaker Neurons.

  • Lee J
  • Mol. Cell
  • 2017 Apr 6

Literature context:


Abstract:

ATAXIN-2 (ATX2) has been implicated in human neurodegenerative diseases, yet it remains elusive how ATX2 assembles specific protein complexes to execute its physiological roles. Here we employ the posttranscriptional co-activator function of Drosophila ATX2 to demonstrate that LSM12 and ME31B/DDX6 are two ATX2-associating factors crucial for sustaining circadian rhythms. LSM12 acts as a molecular adaptor for the recruitment of TWENTY-FOUR (TYF) to ATX2. The ATX2-LSM12-TYF complex thereby stimulates TYF-dependent translation of the rate-limiting clock gene period (per) to maintain 24 hr periodicity in circadian behaviors. In contrast, ATX2 contributes to NOT1-mediated gene silencing and associates with NOT1 in a ME31B/DDX6-dependent manner. The ME31B/DDX6-NOT1 complex does not affect PER translation but supports high-amplitude behavioral rhythms along with ATX2, indicating a PER-independent clock function of ATX2. Taken together, these data suggest that the ATX2 complex may switch distinct modes of posttranscriptional regulation through its associating factors to control circadian clocks and ATX2-related physiology.

Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering.

  • Liu L
  • Dev. Cell
  • 2017 Apr 10

Literature context:


Abstract:

Columnar restriction of neurites is critical for forming nonoverlapping receptive fields and preserving spatial sensory information from the periphery in both vertebrate and invertebrate nervous systems, but the underlying molecular mechanisms remain largely unknown. Here, we demonstrate that Drosophila homolog of α-neurexin (DNrx) plays an essential role in columnar restriction during L4 axon branching. Depletion of DNrx from L4 neurons resulted in misprojection of L4 axonal branches into neighboring columns due to impaired ephrin clustering. The proper ephrin clustering requires its interaction with the intracellular region of DNrx. Furthermore, we find that Drosophila neuroligin 4 (DNlg4) in Tm2 neurons binds to DNrx and initiates DNrx clustering in L4 neurons, which subsequently induces ephrin clustering. Our study demonstrates that DNrx promotes ephrin clustering and reveals that ephrin/Eph signaling from adjacent L4 neurons restricts axonal branches of L4 neurons in columns.

Metazoan Nuclear Pores Provide a Scaffold for Poised Genes and Mediate Induced Enhancer-Promoter Contacts.

  • Pascual-Garcia P
  • Mol. Cell
  • 2017 Apr 6

Literature context:


Abstract:

Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. We identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, we uncovered a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Although changing levels of Nup98 disrupted enhancer-promoter contacts, it did not affect ongoing transcription but instead compromised subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, our data identify Nups as a class of architectural proteins for enhancers and supports a model in which animal genomes use the nuclear pore as an organizing scaffold for inducible poised genes.

Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification.

  • Hu D
  • Mol. Cell
  • 2017 Feb 2

Literature context:


Abstract:

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.

Funding information:
  • NCI NIH HHS - P30 CA060553()
  • NCI NIH HHS - R01 CA101774()
  • NCI NIH HHS - R01 CA214035()
  • NCI NIH HHS - R35 CA197569()
  • NCI NIH HHS - R50 CA211428()
  • NCI NIH HHS - T32 CA080621()

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

  • Liang K
  • Cell
  • 2017 Jan 12

Literature context:


Abstract:

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

Funding information:
  • NCATS NIH HHS - UL1 TR001082()
  • NCI NIH HHS - P30 CA060553()
  • NCI NIH HHS - R01 CA101774()
  • NCI NIH HHS - R01 CA117907()
  • NCI NIH HHS - R01 CA214035()
  • NCI NIH HHS - R35 CA197569()
  • NCI NIH HHS - R50 CA211428()
  • NCI NIH HHS - T32 CA080621()
  • NIGMS NIH HHS - R01 GM120109()

Netrin-1 induces local translation of down syndrome cell adhesion molecule in axonal growth cones.

  • Jain S
  • Dev Neurobiol
  • 2016 Jun 9

Literature context:


Abstract:

Down syndrome cell adhesion molecule (DSCAM) plays an important role in many neurodevelopmental processes such as axon guidance, dendrite arborization, and synapse formation. DSCAM is located in the Down syndrome trisomic region of human chromosome 21 and may contribute to the Down syndrome brain phenotype, which includes a reduction in the formation of long-distance connectivity. The local translation of a select group of mRNA transcripts within growth cones is necessary for the formation of appropriate neuronal connectivity. Interestingly, we have found that Dscam mRNA is localized to growth cones of mouse hippocampal neurons, and is dynamically regulated in response to the axon guidance molecule, netrin-1. Furthermore, netrin-1 stimulation results in an increase in locally translated DSCAM protein in growth cones. Deleted in colorectal cancer (DCC), a netrin-1 receptor, is required for the netrin-1-induced increase in Dscam mRNA local translation. We also find that two RNA-binding proteins-fragile X mental retardation protein (FMRP) and cytoplasmic polyadenylation element binding protein (CPEB)-colocalize with Dscam mRNA in growth cones, suggesting their regulation of Dscam mRNA localization and translation. Finally, overexpression of DSCAM in mouse cortical neurons results in a severe stunting of axon outgrowth and branching, suggesting that an increase in DSCAM protein results in a structural change having functional consequences. Taken together, these results suggest that netrin-1-induced local translation of Dscam mRNA during embryonic development may be an important mechanism to regulate axon growth and guidance in the developing nervous system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 799-816, 2016.

Funding information:
  • NICHD NIH HHS - N01-HD-6-2915(United States)
  • NINDS NIH HHS - R01NS023022-23(United States)

Immunoblotting validation of research antibodies generated against HS1-associated protein X-1 in the human neutrophil model cell line PLB-985.

  • Cavnar P
  • F1000Res
  • 2015 Jun 29

Literature context:


Abstract:

HS1-associated protein X-1 (Hax1) is a 35 kDa protein that is ubiquitously expressed. Hax1 is an anti-apoptotic protein with additional roles in cell motility, and autosomal recessive loss of Hax1 results in Kostmann syndrome, a form of severe congenital neutropenia. Because of the important role of Hax1 in neutrophils we demonstrate here validation of two commercially available research antibodies directed against human Hax1 in the human myeloid leukemia cell line PLB-985 cells. We show that both the mouse anti-Hax1 monoclonal IgG directed against amino acids 10-148 of Hax1 and a rabbit anti-Hax1 polyclonal IgG antibody directed against full-length Hax1 reliably and consistently detect Hax1 during immunoblotting of three different PLB-985 cell densities. Using shRNA mediated Hax1 knockdown, we demonstrate the specificity of both Hax1 antibodies. In addition, our results suggest that the rabbit anti-Hax1 polyclonal antibody provides a stronger intensity in detecting Hax1 protein, with detection in as few as 0.1 x 10 (6) cells in 6 total replicates we have performed.

Trehalase regulates neuroepithelial stem cell maintenance and differentiation in the Drosophila optic lobe.

  • Chen X
  • PLoS ONE
  • 2015 Mar 30

Literature context:


Abstract:

As one of the major hydrolases in Drosophila, trehalase (Treh) catalyzes the hydrolysis of trehalose into glucose providing energy for flight muscle activity. Treh is highly conserved from bacteria to humans, but little is known about its function during animal development. Here, we analyze the function of Treh in Drosophila optic lobe development. In the optic lobe, neuroepithelial cells (NEs) first divide symmetrically to expand the stem cell pool and then differentiate into neuroblasts, which divide asymmetrically to generate medulla neurons. We find that the knockdown of Treh leads to a loss of the lamina and a smaller medulla. Analyses of Treh RNAi-expressing clones and loss-of-function mutants indicate that the lamina and medulla phenotypes result from neuroepithelial disintegration and premature differentiation into medulla neuroblasts. Although the principal role of Treh is to generate glucose, the Treh loss-of-function phenotype cannot be rescued by exogenous glucose. Thus, our results indicate that in addition to being a hydrolase, Treh plays a role in neuroepithelial stem cell maintenance and differentiation during Drosophila optic lobe development.

Funding information:
  • NEI NIH HHS - EY017319(United States)
  • NINDS NIH HHS - R01-NS051874-16(United States)

The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43.

  • Liachko NF
  • PLoS Genet.
  • 2014 Dec 5

Literature context:


Abstract:

Pathological aggregates of phosphorylated TDP-43 characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), two devastating groups of neurodegenerative disease. Kinase hyperactivity may be a consistent feature of ALS and FTLD-TDP, as phosphorylated TDP-43 is not observed in the absence of neurodegeneration. By examining changes in TDP-43 phosphorylation state, we have identified kinases controlling TDP-43 phosphorylation in a C. elegans model of ALS. In this kinome-wide survey, we identified homologs of the tau tubulin kinases 1 and 2 (TTBK1 and TTBK2), which were also identified in a prior screen for kinase modifiers of TDP-43 behavioral phenotypes. Using refined methodology, we demonstrate TTBK1 and TTBK2 directly phosphorylate TDP-43 in vitro and promote TDP-43 phosphorylation in mammalian cultured cells. TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states. Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord. These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.

Funding information:
  • NIDCD NIH HHS - R01 DC012085(United States)

Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development.

  • Wang WL
  • Epigenetics Chromatin
  • 2014 Oct 10

Literature context:


Abstract:

BACKGROUND: Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). RESULTS: We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are enriched on nucleosomes containing both active and repressive histone PTMs in human A549 cells and Xenopus embryos. CONCLUSIONS: Significantly, we demonstrated that H2A phosphorylation and H4 arginine methylation form a new class of bona fide pre-deposition modifications in the vertebrate embryo. We show that S1ph and R3me containing chromatin domains are not correlated with H3 regulatory PTMs, suggesting a unique role for phosphorylation and arginine methylation.

Funding information:
  • NIGMS NIH HHS - R35 GM118110(United States)

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.

  • Werner ME
  • J. Cell Biol.
  • 2014 Aug 4

Literature context:


Abstract:

The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. We use the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. We identify a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, we show that Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.

Funding information:
  • NIMH NIH HHS - T32 MH067631(United States)

Proteomic analysis of laser capture microscopy purified myotendinous junction regions from muscle sections.

  • Can T
  • Proteome Sci
  • 2014 Jul 29

Literature context:


Abstract:

The myotendinous junction is a specialized structure of the muscle fibre enriched in mechanosensing complexes, including costameric proteins and core elements of the z-disc. Here, laser capture microdissection was applied to purify membrane regions from the myotendinous junctions of mouse skeletal muscles, which were then processed for proteomic analysis. Sarcolemma sections from the longitudinal axis of the muscle fibre were used as control for the specificity of the junctional preparation. Gene ontology term analysis of the combined lists indicated a statistically significant enrichment in membrane-associated proteins. The myotendinous junction preparation contained previously uncharacterized proteins, a number of z-disc costameric ligands (e.g., actinins, capZ, αB cristallin, filamin C, cypher, calsarcin, desmin, FHL1, telethonin, nebulin, titin and an enigma-like protein) and other proposed players of sarcomeric stretch sensing and signalling, such as myotilin and the three myomesin homologs. A subset were confirmed by immunofluorescence analysis as enriched at the myotendinous junction, suggesting that laser capture microdissection from muscle sections is a valid approach to identify novel myotendinous junction players potentially involved in mechanotransduction pathways.

Funding information:
  • Medical Research Council - MC_U142684168(United Kingdom)
  • NCI NIH HHS - T32 CA117846(United States)

Ear2 deletion causes early memory and learning deficits in APP/PS1 mice.

  • Kummer MP
  • J. Neurosci.
  • 2014 Jun 25

Literature context:


Abstract:

To assess the consequences of locus ceruleus (LC) degeneration and subsequent noradrenaline (NA) deficiency in early Alzheimer's disease (AD), mice overexpressing mutant amyloid precursor protein and presenilin-1 (APP/PS1) were crossed with Ear2(-/-) mice that have a severe loss of LC neurons projecting to the hippocampus and neocortex. Testing spatial memory and hippocampal long-term potentiation revealed an impairment in APP/PS1 Ear2(-/-) mice, whereas APP/PS1 or Ear2(-/-) mice showed only minor changes. These deficits were associated with distinct synaptic changes including reduced expression of the NMDA 2A subunit and increased levels of NMDA receptor 2B in APP/PS1 Ear2(-/-) mice. Acute pharmacological replacement of NA by L-threo-DOPS partially restored phosphorylation of β-CaMKII and spatial memory performance in APP/PS1 Ear2(-/-) mice. These changes were not accompanied by altered APP processing or amyloid β peptide (Aβ) deposition. Thus, early LC degeneration and subsequent NA reduction may contribute to cognitive deficits via CaMKII and NMDA receptor dysfunction independent of Aβ and suggests that NA supplementation could be beneficial in treating AD.

The role of spartin and its novel ubiquitin binding region in DALIS occurrence.

  • Karlsson AB
  • Mol. Biol. Cell
  • 2014 Apr 11

Literature context:


Abstract:

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains--a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively--have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin's UBR and defined the UBR's amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63-linked ubiquitin chains but does not bind Lys-48-linked ubiquitin chains. Finally, we demonstrate that spartin's role in DALIS formation depends on key residues within its UBR.

Funding information:
  • Cancer Research UK - P30 CA021765(United Kingdom)

Genetic and functional studies implicate synaptic overgrowth and ring gland cAMP/PKA signaling defects in the Drosophila melanogaster neurofibromatosis-1 growth deficiency.

  • Walker JA
  • PLoS Genet.
  • 2013 Nov 26

Literature context:


Abstract:

Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila melanogaster Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3'-5' cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1(st) and 2(nd) chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that human ALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man.

Funding information:
  • NCI NIH HHS - 5U24 CA143883-04(United States)

A genetic RNAi screen for IP₃/Ca²⁺ coupled GPCRs in Drosophila identifies the PdfR as a regulator of insect flight.

  • Agrawal T
  • PLoS Genet.
  • 2013 Oct 7

Literature context:


Abstract:

Insect flight is regulated by various sensory inputs and neuromodulatory circuits which function in synchrony to control and fine-tune the final behavioral outcome. The cellular and molecular bases of flight neuromodulatory circuits are not well defined. In Drosophila melanogaster, it is known that neuronal IP3 receptor mediated Ca²⁺ signaling and store-operated Ca²⁺ entry (SOCE) are required for air-puff stimulated adult flight. However, G-protein coupled receptors (GPCRs) that activate intracellular Ca²⁺ signaling in the context of flight are unknown in Drosophila. We performed a genetic RNAi screen to identify GPCRs that regulate flight by activating the IPIP₃ receptor. Among the 108 GPCRs screened, we discovered 5 IPIP₃/Ca²⁺ linked GPCRs that are necessary for maintenance of air-puff stimulated flight. Analysis of their temporal requirement established that while some GPCRs are required only during flight circuit development, others are required both in pupal development as well as during adult flight. Interestingly, our study identified the Pigment Dispersing Factor Receptor (PdfR) as a regulator of flight circuit development and as a modulator of acute flight. From the analysis of PdfR expressing neurons relevant for flight and its well-defined roles in other behavioral paradigms, we propose that PdfR signaling functions systemically to integrate multiple sensory inputs and modulate downstream motor behavior.

Funding information:
  • NIGMS NIH HHS - GM60585(United States)

Recycling of kinesin-1 motors by diffusion after transport.

  • Blasius TL
  • PLoS ONE
  • 2013 Oct 7

Literature context:


Abstract:

Kinesin motors drive the long-distance anterograde transport of cellular components along microtubule tracks. Kinesin-dependent transport plays a critical role in neurogenesis and neuronal function due to the large distance separating the soma and nerve terminal. The fate of kinesin motors after delivery of their cargoes is unknown but has been postulated to involve degradation at the nerve terminal, recycling via retrograde motors, and/or recycling via diffusion. We set out to test these models concerning the fate of kinesin-1 motors after completion of transport in neuronal cells. We find that kinesin-1 motors are neither degraded nor returned by retrograde motors. By combining mathematical modeling and experimental analysis, we propose a model in which the distribution and recycling of kinesin-1 motors fits a "loose bucket brigade" where individual motors alter between periods of active transport and free diffusion within neuronal processes. These results suggest that individual kinesin-1 motors are utilized for multiple rounds of transport.

Funding information:
  • Wellcome Trust - WT077044/Z/05/Z(United Kingdom)

Stratifin (14-3-3 σ) limits plakophilin-3 exchange with the desmosomal plaque.

  • Roberts BJ
  • PLoS ONE
  • 2013 Oct 14

Literature context:


Abstract:

Desmosomes are prominent cell-cell adhesive junctions in stratified squamous epithelia and disruption of desmosomal adhesion has been shown to have dramatic effects on the function and integrity of these tissues. During normal physiologic processes, such as tissue development and wound healing, intercellular adhesion must be modified locally to allow coordinated cell movements. The mechanisms that control junction integrity and adhesive strength under these conditions are poorly understood. We utilized a proteomics approach to identify plakophilin-3 associated proteins and identified the 14-3-3 family member stratifin. Stratifin interacts specifically with plakophilin-3 and not with other plakophilin isoforms and mutation analysis demonstrated the binding site includes serine 285 in the amino terminal head domain of plakophilin-3. Stratifin interacts with a cytoplasmic pool of plakophilin-3 and is not associated with the desmosome in cultured cells. FRAP analysis revealed that decreased stratifin expression leads to an increase in the exchange rate of cytoplasmic plakophilin-3/GFP with the pool of plakophilin-3/GFP in the desmosome resulting in decreased desmosomal adhesion and increased cell migration. We propose a model by which stratifin plays a role in regulating plakophilin-3 incorporation into the desmosomal plaque by forming a plakophilin-3 stratifin complex in the cytosol and thereby affecting desmosome dynamics in squamous epithelial cells.

Funding information:
  • NIGMS NIH HHS - 1-R01-GM65466-01(United States)

The NADPH metabolic network regulates human αB-crystallin cardiomyopathy and reductive stress in Drosophila melanogaster.

  • Xie HB
  • PLoS Genet.
  • 2013 Jun 2

Literature context:


Abstract:

Dominant mutations in the alpha-B crystallin (CryAB) gene are responsible for a number of inherited human disorders, including cardiomyopathy, skeletal muscle myopathy, and cataracts. The cellular mechanisms of disease pathology for these disorders are not well understood. Among recent advances is that the disease state can be linked to a disturbance in the oxidation/reduction environment of the cell. In a mouse model, cardiomyopathy caused by the dominant CryAB(R120G) missense mutation was suppressed by mutation of the gene that encodes glucose 6-phosphate dehydrogenase (G6PD), one of the cell's primary sources of reducing equivalents in the form of NADPH. Here, we report the development of a Drosophila model for cellular dysfunction caused by this CryAB mutation. With this model, we confirmed the link between G6PD and mutant CryAB pathology by finding that reduction of G6PD expression suppressed the phenotype while overexpression enhanced it. Moreover, we find that expression of mutant CryAB in the Drosophila heart impaired cardiac function and increased heart tube dimensions, similar to the effects produced in mice and humans, and that reduction of G6PD ameliorated these effects. Finally, to determine whether CryAB pathology responds generally to NADPH levels we tested mutants or RNAi-mediated knockdowns of phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), and malic enzyme (MEN), the other major enzymatic sources of NADPH, and we found that all are capable of suppressing CryAB(R120G) pathology, confirming the link between NADP/H metabolism and CryAB.

Funding information:
  • NCRR NIH HHS - S10 RR031599(United States)

The evolutionarily conserved protein CG9186 is associated with lipid droplets, required for their positioning and for fat storage.

  • Thiel K
  • J. Cell. Sci.
  • 2013 May 15

Literature context:


Abstract:

Lipid droplets (LDs) are specialized cell organelles for the storage of energy-rich lipids. Although lipid storage is a conserved feature of all cells and organisms, little is known about fundamental aspects of the cell biology of LDs, including their biogenesis, structural assembly and subcellular positioning, and the regulation of organismic energy homeostasis. We identified a novel LD-associated protein family, represented by the Drosophila protein CG9186 and its murine homolog MGI:1916082. In the absence of LDs, both proteins localize at the endoplasmic reticulum (ER). Upon lipid storage induction, they translocate to LDs using an evolutionarily conserved targeting mechanism that acts through a 60-amino-acid targeting motif in the center of the CG9186 protein. Overexpression of CG9186, and MGI:1916082, causes clustering of LDs in both tissue culture and salivary gland cells, whereas RNAi knockdown of CG9186 results in a reduction of LDs. Organismal RNAi knockdown of CG9186 results in a reduction in lipid storage levels of the fly. The results indicate that we identified the first members of a novel and evolutionarily conserved family of lipid storage regulators, which are also required to properly position LDs within cells.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

The C8ORF38 homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit.

  • Zhang K
  • J. Cell Biol.
  • 2013 Mar 18

Literature context:


Abstract:

Mitochondrial complex I (CI) is an essential component in energy production through oxidative phosphorylation. Most CI subunits are encoded by nuclear genes, translated in the cytoplasm, and imported into mitochondria. Upon entry, they are embedded into the mitochondrial inner membrane. How these membrane-associated proteins cope with the hydrophilic cytoplasmic environment before import is unknown. In a forward genetic screen to identify genes that cause neurodegeneration, we identified sicily, the Drosophila melanogaster homologue of human C8ORF38, the loss of which causes Leigh syndrome. We show that in the cytoplasm, Sicily preprotein interacts with cytosolic Hsp90 to chaperone the CI subunit, ND42, before mitochondrial import. Loss of Sicily leads to loss of CI proteins and preproteins in both mitochondria and cytoplasm, respectively, and causes a CI deficiency and neurodegeneration. Our data indicate that cytosolic chaperones are required for the subcellular transport of ND42.

Funding information:
  • Intramural NIH HHS - Z99 HG999999(United States)

Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice.

  • Iliff AJ
  • Hum. Mol. Genet.
  • 2013 Mar 15

Literature context:


Abstract:

Fragile X premutation-associated disorders, including Fragile X-associated Tremor Ataxia Syndrome, result from unmethylated CGG repeat expansions in the 5' untranslated region (UTR) of the FMR1 gene. Premutation-sized repeats increase FMR1 transcription but impair rapid translation of the Fragile X mental retardation protein (FMRP), which is absent in Fragile X Syndrome (FXS). Normally, FMRP binds to RNA and regulates metabotropic glutamate receptor (mGluR)-mediated synaptic translation, allowing for dendritic synthesis of several proteins. FMRP itself is also synthesized at synapses in response to mGluR activation. However, the role of activity-dependent translation of FMRP in synaptic plasticity and Fragile X-premutation-associated disorders is unknown. To investigate this question, we utilized a CGG knock-in mouse model of the Fragile X premutation with 120-150 CGG repeats in the mouse Fmr1 5' UTR. These mice exhibit increased Fmr1 mRNA production but impaired FMRP translational efficiency, leading to a modest reduction in basal FMRP expression. Cultured hippocampal neurons and synaptoneurosomes derived from CGG KI mice demonstrate impaired FMRP translation in response to the group I mGluR agonist 3,5-dihydroxyphenylglycine. Electrophysiological analysis reveals enhanced mGluR-mediated long-term depression (mGluR-LTD) at CA3-CA1 synapses in acute hippocampal slices prepared from CGG KI mice relative to wild-type littermates, similar to Fmr1 knockout mice. However, unlike mGluR-LTD in mice completely lacking FMRP, mGluR-LTD in CGG knock-in mice remains dependent on new protein synthesis. These studies demonstrate partially overlapping synaptic plasticity phenotypes in mouse models of FXS and Fragile X premutation disorders and support a role for activity-dependent synthesis of FMRP in enduring forms of synaptic plasticity.

Funding information:
  • NIDDK NIH HHS - U01 DK070200-01(United States)

Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage.

  • Sen A
  • PLoS ONE
  • 2013 Jan 23

Literature context:


Abstract:

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.

Funding information:
  • NLM NIH HHS - R01 LM010129-01(United States)

The CPEB protein Orb2 has multiple functions during spermatogenesis in Drosophila melanogaster.

  • Xu S
  • PLoS Genet.
  • 2012 Dec 4

Literature context:


Abstract:

Cytoplasmic Polyadenylation Element Binding (CPEB) proteins are translational regulators that can either activate or repress translation depending on the target mRNA and the specific biological context. There are two CPEB subfamilies and most animals have one or more genes from each. Drosophila has a single CPEB gene, orb and orb2, from each subfamily. orb expression is only detected at high levels in the germline and has critical functions in oogenesis but not spermatogenesis. By contrast, orb2 is broadly expressed in the soma; and previous studies have revealed important functions in asymmetric cell division, viability, motor function, learning, and memory. Here we show that orb2 is also expressed in the adult male germline and that it has essential functions in programming the progression of spermatogenesis from meiosis through differentiation. Like the translational regulators boule (bol) and off-schedule (ofs), orb2 is required for meiosis and orb2 mutant spermatocytes undergo a prolonged arrest during the meiotic G2-M transition. However, orb2 differs from boule and off-schedule in that this arrest occurs at a later step in meiotic progression after the synthesis of the meiotic regulator twine. orb2 is also required for the orderly differentiation of the spermatids after meiosis is complete. The differentiation defects in orb2 mutants include abnormal elongation of the spermatid flagellar axonemes, a failure in individualization and improper post-meiotic gene expression. Amongst the orb2 differentiation targets are orb and two other mRNAs, which are transcribed post-meiotically and localized to the tip of the flagellar axonemes. Additionally, analysis of a partial loss of function orb2 mutant suggests that the orb2 differentiation phenotypes are independent of the earlier arrest in meiosis.

β-secretase cleavage of the fly amyloid precursor protein is required for glial survival.

  • Bolkan BJ
  • J. Neurosci.
  • 2012 Nov 14

Literature context:


Abstract:

β-secretase (or BACE1) is the key enzyme in the production of β-amyloid (Aβ), which accumulates in the senile plaques characteristic for Alzheimer's disease. Consequently, the lack of BACE1 prevents β-processing of the amyloid precursor protein and Aβ production, which made it a promising target for drug development. However, the loss of BACE1 is also detrimental, leading to myelination defects and altered neuronal activity, functions that have been associated with the cleavage of Neuregulin and a voltage-gated sodium channel subunit. Here we show that the Drosophila ortholog of BACE, dBACE, is required for glial survival. Cell-specific knockdown experiments reveal that this is a non-cell autonomous function, as a knockdown of dBACE in photoreceptor neurons leads to progressive degeneration of glia in their target zone, the lamina. Interestingly, this phenotype is suppressed by the loss of the fly amyloid precursor protein (APPL), whereas a secretion-deficient form of APPL enhances the degeneration. This shows that full-length APPL in neurons promotes the death of neighboring glial cells and that β-processing of APPL is needed to prevent glial death. These results therefore not only demonstrate a novel function for an APP protein in glia, but they also show this function specifically requires regulation by β-cleavage.

Funding information:
  • NINDS NIH HHS - R01 NS39600(United States)

The C-terminal unique region of desmoglein 2 inhibits its internalization via tail-tail interactions.

  • Chen J
  • J. Cell Biol.
  • 2012 Nov 12

Literature context:


Abstract:

Desmosomal cadherins, desmogleins (Dsgs) and desmocollins, make up the adhesive core of intercellular junctions called desmosomes. A critical determinant of epithelial adhesive strength is the level and organization of desmosomal cadherins on the cell surface. The Dsg subclass of desmosomal cadherins contains a C-terminal unique region (Dsg unique region [DUR]) with unknown function. In this paper, we show that the DUR of Dsg2 stabilized Dsg2 at the cell surface by inhibiting its internalization and promoted strong intercellular adhesion. DUR also facilitated Dsg tail-tail interactions. Forced dimerization of a Dsg2 tail lacking the DUR led to decreased internalization, supporting the conclusion that these two functions of the DUR are mechanistically linked. We also show that a Dsg2 mutant, V977fsX1006, identified in arrhythmogenic right ventricular cardiomyopathy patients, led to a loss of Dsg2 tail self-association and underwent rapid endocytosis in cardiac muscle cells. Our observations illustrate a new mechanism desmosomal cadherins use to control their surface levels, a key factor in determining their adhesion and signaling roles.

Funding information:
  • NIAID NIH HHS - R01 AI077599(United States)

Luminal localization of α-tubulin K40 acetylation by cryo-EM analysis of fab-labeled microtubules.

  • Soppina V
  • PLoS ONE
  • 2012 Oct 30

Literature context:


Abstract:

The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

Funding information:
  • NHGRI NIH HHS - HG00750(United States)

Drosophila Tel2 is expressed as a translational fusion with EpsinR and is a regulator of wingless signaling.

  • Lee JH
  • PLoS ONE
  • 2012 Oct 2

Literature context:


Abstract:

Tel2, a protein conserved from yeast to vertebrates, is an essential regulator of diverse cellular processes including telomere maintenance, DNA damage checkpoints, DNA repair, biological clocks, and cell signaling. The Drosophila Tel2 protein is produced as a translational fusion with EpsinR, a Clathrin adapter that facilitates vesicle trafficking between the Golgi and endosomes. EpsinR and Tel2 are encoded by a Drosophila gene called lqfR. lqfR is required for viability, and its specific roles include cell growth, proliferation, and planar cell polarity. We find that all of these functions of lqfR are attributed entirely to Tel2, not EpsinR. In addition, we find that Drosophila LqfR/Tel2 is a component of one or more protein complexes that contain E-cadherin and Armadillo. Moreover, Tel2 modulates E-cadherin and Armadillo cellular dynamics. We propose that at least one of the functions of Drosophila Tel2 is regulation of Wingless signaling.

Funding information:
  • NIDDK NIH HHS - U01 DK058231(United States)

Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis.

  • Vourekas A
  • Nat. Struct. Mol. Biol.
  • 2012 Aug 8

Literature context:


Abstract:

Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind Piwi-interacting RNAs (piRNAs), small germline RNAs whose biogenesis and functions are still largely elusive. We used high-throughput sequencing after cross-linking and immunoprecipitation (HITS-CLIP) coupled with RNA-sequencing (RNA-seq) to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrated that Miwi binds spermiogenic mRNAs directly, without using piRNAs as guides, and independent biochemical analyses of testis mRNA ribonucleoproteins (mRNPs) established that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.

Funding information:
  • NIBIB NIH HHS - R37 EB002784(United States)

The protein kinase KIS impacts gene expression during development and fear conditioning in adult mice.

  • Manceau V
  • PLoS ONE
  • 2012 Aug 31

Literature context:


Abstract:

The brain-enriched protein kinase KIS (product of the gene UHMK1) has been shown to phosphorylate the human splicing factor SF1 in vitro. This phosphorylation in turn favors the formation of a U2AF(65)-SF1-RNA complex which occurs at the 3' end of introns at an early stage of spliceosome assembly. Here, we analyzed the effects of KIS knockout on mouse SF1 phosphorylation, physiology, adult behavior, and gene expression in the neonate brain. We found SF1 isoforms are differently expressed in KIS-ko mouse brains and fibroblasts. Re-expression of KIS in fibroblasts restores a wild type distribution of SF1 isoforms, confirming the link between KIS and SF1. Microarray analysis of transcripts in the neonate brain revealed a subtle down-regulation of brain specific genes including cys-loop ligand-gated ion channels and metabolic enzymes. Q-PCR analyses confirmed these defects and point to an increase of pre-mRNA over mRNA ratios, likely due to changes in splicing efficiency. While performing similarly in prepulse inhibition and most other behavioral tests, KIS-ko mice differ in spontaneous activity and contextual fear conditioning. This difference suggests that disregulation of gene expression due to KIS inactivation affects specific brain functions.

Funding information:
  • NHGRI NIH HHS - P41HG004059(United States)

A modular toolset for recombination transgenesis and neurogenetic analysis of Drosophila.

  • Wang JW
  • PLoS ONE
  • 2012 Jul 31

Literature context:


Abstract:

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.

Funding information:
  • NHGRI NIH HHS - R01 HG005692(United States)

Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover.

  • Lange S
  • Mol. Biol. Cell
  • 2012 Jul 29

Literature context:


Abstract:

Protein turnover through cullin-3 is tightly regulated by posttranslational modifications, the COP9 signalosome, and BTB/POZ-domain proteins that link cullin-3 to specific substrates for ubiquitylation. In this paper, we report how potassium channel tetramerization domain containing 6 (KCTD6) represents a novel substrate adaptor for cullin-3, effectively regulating protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5). Binding of sAnk1.5 to KCTD6, and its subsequent turnover is regulated through posttranslational modification by nedd8, ubiquitin, and acetylation of C-terminal lysine residues. The presence of the sAnk1.5 binding partner obscurin, and mutation of lysine residues increased sAnk1.5 protein levels, as did knockdown of KCTD6 in cardiomyocytes. Obscurin knockout muscle displayed reduced sAnk1.5 levels and mislocalization of the sAnk1.5/KCTD6 complex. Scaffolding functions of obscurin may therefore prevent activation of the cullin-mediated protein degradation machinery and ubiquitylation of sAnk1.5 through sequestration of sAnk1.5/KCTD6 at the sarcomeric M-band, away from the Z-disk-associated cullin-3. The interaction of KCTD6 with ankyrin-1 may have implications beyond muscle for hereditary spherocytosis, as KCTD6 is also present in erythrocytes, and erythrocyte ankyrin isoforms contain its mapped minimal binding site.

Funding information:
  • NINDS NIH HHS - R01 NS057811(United States)

Drosophila heat shock response requires the JNK pathway and phosphorylation of mixed lineage kinase at a conserved serine-proline motif.

  • Gonda RL
  • PLoS ONE
  • 2012 Jul 31

Literature context:


Abstract:

Defining context specific requirements for proteins and pathways is a major challenge in the study of signal transduction. For example, the stress-activated protein kinase (SAPK) pathways are comprised of families of closely related transducers that are activated in a variety of tissues and contexts during development and organismal homeostasis. Consequently, redundant and pleiotropic effects have hampered a complete understanding of the individual contributions of transducers in distinct contexts. Here, we report on the function of a context-specific regulatory phosphorylation site, PXSP, in the Drosophila mixed lineage kinase protein, Slpr, a mitogen-activated protein kinase kinase kinase (MAP3K) in the Jun Kinase (JNK) pathway. Genetic analysis of the function of non-phosphorylatable (PXAP) and phosphomimetic mutant (PXEP) Slpr transgenes in several distinct contexts revealed minimal effects in JNK-dependent tissue closure processes but differential requirements in heat stress response. In particular, PXAP expression resulted in sensitivity of adults to sustained heat shock, like p38 and JNK pathway mutants. In contrast, PXEP overexpression conferred some resistance. Indeed, phosphorylation of the PXSP motif is enriched under heat shock conditions and requires in part, the p38 kinases for the enrichment. These data suggest that coordination of signaling between p38 and Slpr serves to maintain JNK signaling during heat stress. In sum, we demonstrate a novel role for JNK signaling in the heat shock response in flies and identify a posttranslational modification on Slpr, at a conserved site among MAP3K mixed lineage kinase family members, which bolsters stress resistance with negligible effects on JNK-dependent developmental processes.

Funding information:
  • NIGMS NIH HHS - R01GM10974(United States)

Internalized gap junctions are degraded by autophagy.

  • Fong JT
  • Autophagy
  • 2012 May 1

Literature context:


Abstract:

Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously-and effectively after treatment with natural inflammatory mediators-internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.

Funding information:
  • NCI NIH HHS - R01 CA069202(United States)

Silencing of amyloid precursor protein expression using a new engineered delta ribozyme.

  • Ben Aissa M
  • Int J Alzheimers Dis
  • 2012 Apr 6

Literature context:


Abstract:

Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40-42 residue peptide (Aβ(40-42)) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/I001735/1(United Kingdom)
  • NHLBI NIH HHS - UC2 HL102925(United States)

N-cadherin mediates neuronal cell survival through Bim down-regulation.

  • Lelièvre EC
  • PLoS ONE
  • 2012 Mar 19

Literature context:


Abstract:

N-cadherin is a major adhesion molecule involved in the development and plasticity of the nervous system. N-cadherin-mediated cell adhesion regulates neuroepithelial cell polarity, neuronal precursor migration, growth cone migration and synaptic plasticity. In vitro, it has been involved in signaling events regulating processes such as cell mobility, proliferation and differentiation. N-cadherin has also been implicated in adhesion-dependent protection against apoptosis in non-neuronal cells. In this study, we investigated if the engagement of N-cadherin participates to the control of neuronal cells survival/death balance. We observed that plating either primary mouse spinal cord neurons or primary rat hippocampal neurons on N-cadherin recombinant substrate greatly enhances their survival compared to non-specific adhesion on poly-L-lysine. We show that N-cadherin engagement, in the absence of other survival factors (cell-matrix interactions and serum), protects GT1-7 neuronal cells against apoptosis. Using this cell line, we then searched for the signaling pathways involved in the survival effect of N-cadherin engagement. The PI3-kinase/Akt survival pathway and its downstream effector Bad are not involved, as no phosphorylation of Akt or Bad proteins in response to N-cadherin engagement was observed. In contrast, N-cadherin engagement activated the Erk1/2 MAP kinase pathway. Moreover, N-cadherin ligation mediated a 2-fold decrease in the level of the pro-apoptotic protein Bim-EL whereas the level of the anti-apoptotic protein Bcl-2 was unchanged. Inhibition of Mek1/2 kinases with U0126, and the resulting inhibition of Erk1/2 phosphorylation, induced the increase of both the level of Bim-EL and apoptosis of cells seeded on the N-cadherin substrate, suggesting that Erk phosphorylation is necessary for cell survival. Finally, the overexpression of a phosphorylation defective form of Bim-EL prevented N-cadherin-engagement induced cell survival. In conclusion, our results show that N-cadherin engagement mediates neuronal cell survival by enhancing the MAP kinase pathway and down-regulating the pro-apoptotic protein Bim-EL.

Funding information:
  • NIA NIH HHS - R01 AG021133(United States)
  • NIMH NIH HHS - R01 MH086920(United States)

Small molecule inhibition of RISC loading.

  • Tan GS
  • ACS Chem. Biol.
  • 2012 Feb 17

Literature context:


Abstract:

Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 μM), suramin (IC(50) = 0.69 μM), and oxidopamine HCL (IC(50) = 1.61 μM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.

Funding information:
  • NEI NIH HHS - R01 EY020533(United States)

Protein interacting with C kinase (PICK1) is a suppressor of spinocerebellar ataxia 3-associated neurodegeneration in Drosophila.

  • McGurk L
  • Hum. Mol. Genet.
  • 2012 Jan 1

Literature context:


Abstract:

Spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant ataxia. The disease is caused by an expansion of a CAG-trinucelotide repeat region within the coding sequence of the ATXN3 gene, and this results in an expanded polyglutamine (polyQ) tract within the Ataxin-3 protein. The polyQ expansion leads to neuronal dysfunction and cell death. Here, we tested the ability of a number of proteins that interact with Ataxin-3 to modulate SCA3 pathogenicity using Drosophila. Of 10 candidates, we found four novel enhancers and one suppressor. The suppressor, PICK1 (Protein interacting with C kinase 1), is a transport protein that regulates the trafficking of ion channel subunits involved in calcium homeostasis to and from the plasma membrane. In line with calcium homeostasis being a potential pathway mis-regulated in SCA3, we also found that down-regulation of Nach, an acid sensing ion channel, mitigates SCA3 pathogenesis in flies. Modulation of PICK1 could be targeted in other neurodegenerative diseases, as the toxicity of SCA1 and tau was also suppressed when PICK1 was down-regulated. These findings indicate that interaction proteins may define a rich source of modifier pathways to target in disease situations.

Funding information:
  • Wellcome Trust - 660060(United Kingdom)

Decreased microRNA-214 levels in breast cancer cells coincides with increased cell proliferation, invasion and accumulation of the Polycomb Ezh2 methyltransferase.

  • Derfoul A
  • Carcinogenesis
  • 2011 Dec 22

Literature context:


Abstract:

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression by inhibiting translation or promoting degradation of specific target messenger RNAs (mRNAs). Alteration of the levels of a number of miRNAs is common in solid and hematological tumors. We have shown previously that miR-214 regulates Ezh2 in skeletal muscle and embryonic stem cells. The current study was aimed at examining the role of miR-214 in breast cancer where miR-214 levels are reduced but whether this phenomenon bears a functional relevance is unknown. MiR-214 expression was inversely correlated with Ezh2 mRNA and protein levels in breast cancer cell lines and at least one copy of the miR-214 alleles was found to be deleted in 24% (6/25) of primary breast tumors. Experimental increase of miR-214 in breast cancer cell lines correlated with reduction of Ezh2 protein levels, a known marker of invasion and aggressive breast cancer behavior. Supporting a direct targeting mechanism, miR-214 decreased luciferase activity from a construct containing the Ezh2 3' untranslated region. Expression of miR-214 specifically reduced cell proliferation of breast cancer cells and inhibited the invasive potential of a highly metastatic breast cancer cell line. These findings indicate that reduced miR-214 levels may contribute to breast tumorigenesis by allowing abnormally elevated Ezh2 accumulation and subsequent unchecked cell proliferation and invasion.

Funding information:
  • NIDDK NIH HHS - 1U24DK097771-01(United States)

Drosophila Dynein intermediate chain gene, Dic61B, is required for spermatogenesis.

  • Fatima R
  • PLoS ONE
  • 2011 Dec 6

Literature context:


Abstract:

This study reports the identification and characterization of a novel gene, Dic61B, required for male fertility in Drosophila. Complementation mapping of a novel male sterile mutation, ms21, isolated in our lab revealed it to be allelic to CG7051 at 61B1 cytogenetic region, since two piggyBac insertion alleles, CG7051(c05439) and CG7051(f07138) failed to complement. CG7051 putatively encodes a Dynein intermediate chain. All three mutants, ms21, CG7051(c05439) and CG7051(f07138), exhibited absolute recessive male sterility with abnormally coiled sperm axonemes causing faulty sperm individualization as revealed by Phalloidin staining in Don Juan-GFP background. Sequencing of PCR amplicons uncovered two point mutations in ms21 allele and confirmed the piggyBac insertions in CG7051(c05439) and CG7051(f07138) alleles to be in 5'UTR and 4(th) exon of CG7051 respectively, excision of which reverted the male sterility. In situ hybridization to polytene chromosomes demonstrated CG7051 to be a single copy gene. RT-PCR of testis RNA revealed defective splicing of the CG7051 transcripts in mutants. Interestingly, expression of cytoplasmic dynein intermediate chain, α, β, γ tubulins and α-spectrin was normal in mutants while ultra structural studies revealed defects in the assembly of sperm axonemes. Bioinformatics further highlighted the homology of CG7051 to axonemal dynein intermediate chain of various organisms, including DNAI1 of humans, mutations in which lead to male sterility due to immotile sperms. Based on these observations we conclude that CG7051 encodes a novel axonemal dynein intermediate chain essential for male fertility in Drosophila and rename it as Dic61B. This is the first axonemal Dic gene of Drosophila to be characterized at molecular level and shown to be required for spermatogenesis.

Funding information:
  • NHLBI NIH HHS - HL071158(United States)
  • NIDDK NIH HHS - T32 DK007319(United States)

ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B-dependent CRMP2 phosphorylation.

  • Wakatsuki S
  • Nat. Cell Biol.
  • 2011 Nov 6

Literature context:


Abstract:

Wallerian degeneration is observed in many neurological disorders, and it is therefore important to elucidate the axonal degeneration mechanism to prevent, and further develop treatment for, such diseases. The ubiquitin-proteasome system (UPS) has been implicated in Wallerian degeneration, but the underlying molecular mechanism remains unclear. Here we show that ZNRF1, an E3 ligase, promotes Wallerian degeneration by targeting AKT to degrade through the UPS. AKT phosphorylates glycogen synthase kinase-3β (GSK3B), and thereby inactivates it in axons. AKT overexpression significantly delays axonal degeneration. Overexpression of the active (non-phosphorylated) form of GSK3B induces CRMP2 phosphorylation, which is required for the microtubule reorganization observed in the degenerating axon. The inhibition of GSK3B and the overexpression of non-phosphorylated CRMP2 both protected axons from Wallerian degeneration. These findings indicate that the ZNRF1-AKT-GSK3B-CRMP2 pathway plays an important role in controlling Wallerian degeneration.

Funding information:
  • European Research Council - 281699(International)

Liquid facets-related (lqfR) is required for egg chamber morphogenesis during Drosophila oogenesis.

  • Leventis PA
  • PLoS ONE
  • 2011 Nov 1

Literature context:


Abstract:

Clathrin interactor 1 [CLINT1] (also called enthoprotin/EpsinR) is an Epsin N-terminal homology (ENTH) domain-containing adaptor protein that functions in anterograde and retrograde clathrin-mediated trafficking between the trans-Golgi network and the endosome. Removal of both Saccharomyces cerevisiae homologs, Ent3p and Ent5p, result in yeast that are viable, but that display a cold-sensitive growth phenotype and mistrafficking of various vacuolar proteins. Similarly, either knock-down or overexpression of vertebrate CLINT1 in cell culture causes mistrafficking of proteins. Here, we have characterized Drosophila CLINT1, liquid-facets Related (lqfR). LqfR is ubiquitously expressed throughout development and is localized to the Golgi and endosome. Strong hypomorphic mutants generated by imprecise P-element excision exhibit extra macrochaetae, rough eyes and are female sterile. Although essentially no eggs are laid, the ovaries do contain late-stage egg chambers that exhibit abnormal morphology. Germline clones reveal that LqfR expression in the somatic follicle cells is sufficient to rescue the oogenesis defects. Clones of mutant lqfR follicle cells have a decreased cell size consistent with a downregulation of Akt1. We find that while total Akt1 levels are increased there is also a significant decrease in activated phosphorylated Akt1. Taken together, these results show that LqfR function is required to regulate follicle cell size and signaling during Drosophila oogenesis.

Funding information:
  • NICHD NIH HHS - U54 HD083091(United States)

Central nervous system inflammation induces muscle atrophy via activation of the hypothalamic-pituitary-adrenal axis.

  • Braun TP
  • J. Exp. Med.
  • 2011 Nov 21

Literature context:


Abstract:

Skeletal muscle catabolism is a co-morbidity of many chronic diseases and is the result of systemic inflammation. Although direct inflammatory cytokine action on muscle promotes atrophy, nonmuscle sites of action for inflammatory mediators are less well described. We demonstrate that central nervous system (CNS)-delimited interleukin 1β (IL-1β) signaling alone can evoke a catabolic program in muscle, rapidly inducing atrophy. This effect is dependent on hypothalamic-pituitary-adrenal (HPA) axis activation, as CNS IL-1β-induced atrophy is abrogated by adrenalectomy. Furthermore, we identified a glucocorticoid-responsive gene expression pattern conserved in models of acute and chronic inflammatory muscle atrophy. In contrast with studies suggesting that the direct action of inflammatory cytokines on muscle is sufficient to induce catabolism, adrenalectomy also blocks the atrophy program in response to systemic inflammation, demonstrating that glucocorticoids are requisite for this process. Additionally, circulating levels of glucocorticoids equivalent to those produced under inflammatory conditions are sufficient to cause profound muscle wasting. Together, these data suggest that a significant component of inflammation-induced muscle catabolism occurs indirectly via a relay in the CNS.

Funding information:
  • NIDA NIH HHS - R13 DA034437(United States)

Signal transducer and activator of transcription-6 (STAT6) inhibition suppresses renal cyst growth in polycystic kidney disease.

  • Olsan EE
  • Proc. Natl. Acad. Sci. U.S.A.
  • 2011 Nov 1

Literature context:


Abstract:

Autosomal-dominant (AD) polycystic kidney disease (PKD) is a leading cause of renal failure in the United States, and currently lacks available treatment options to slow disease progression. Mutations in the gene coding for polycystin-1 (PC1) underlie the majority of cases but the function of PC1 has remained poorly understood. We have previously shown that PC1 regulates the transcriptional activity of signal transducer and activator of transcription-6 (STAT6). Here we show that STAT6 is aberrantly activated in cyst-lining cells in PKD mouse models. Activation of the STAT6 pathway leads to a positive feedback loop involving auto/paracrine signaling by IL13 and the IL4/13 receptor. The presence of IL13 in cyst fluid and the overexpression of IL4/13 receptor chains suggests a mechanism of sustained STAT6 activation in cysts. Genetic inactivation of STAT6 in a PKD mouse model leads to significant inhibition of proliferation and cyst growth and preservation of renal function. We show that the active metabolite of leflunomide, a drug approved for treatment of arthritis, inhibits STAT6 in renal epithelial cells. Treatment of PKD mice with this drug leads to amelioration of the renal cystic disease similar to genetic STAT6 inactivation. These results suggest STAT6 as a promising drug target for treatment of ADPKD.

Funding information:
  • NIGMS NIH HHS - T32 GM008061(United States)

Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein.

  • Bhogal B
  • Nat. Neurosci.
  • 2011 Oct 30

Literature context:


Abstract:

Loss of FMR1 gene function results in fragile X syndrome, the most common heritable form of intellectual disability. The protein encoded by this locus (FMRP) is an RNA-binding protein that is thought to primarily act as a translational regulator; however, recent studies have implicated FMRP in other mechanisms of gene regulation. We found that the Drosophila fragile X homolog (dFMR1) biochemically interacted with the adenosine-to-inosine RNA-editing enzyme dADAR. Adar and Fmr1 mutant larvae exhibited distinct morphological neuromuscular junction (NMJ) defects. Epistasis experiments based on these phenotypic differences revealed that Adar acts downstream of Fmr1 and that dFMR1 modulates dADAR activity. Furthermore, sequence analyses revealed that a loss or overexpression of dFMR1 affects editing efficiency on certain dADAR targets with defined roles in synaptic transmission. These results link dFMR1 with the RNA-editing pathway and suggest that proper NMJ synaptic architecture requires modulation of dADAR activity by dFMR1.

Funding information:
  • NIGMS NIH HHS - T32 GM007184(United States)

A non-canonical start codon in the Drosophila fragile X gene yields two functional isoforms.

  • Beerman RW
  • Neuroscience
  • 2011 May 5

Literature context:


Abstract:

Fragile X syndrome is caused by the loss of expression of the fragile X mental retardation protein (FMRP). As a RNA binding protein, FMRP functions in translational regulation, localization, and stability of its neuronal target transcripts. The Drosophila homologue, dFMR1, is well conserved in sequence and function with respect to human FMRP. Although dFMR1 is known to express two main isoforms, the mechanism behind production of the second, more slowly migrating isoform has remained elusive. Furthermore, it remains unknown whether the two isoforms may also contribute differentially to dFMR1 function. We have found that this second dFMR1 isoform is generated through an alternative translational start site in the dfmr1 5'UTR. This 5'UTR coding sequence is well conserved in the melanogaster group. Translation of the predominant, smaller form of dFMR1 (dFMR1-S(N)) begins at a canonical start codon (ATG), whereas translation of the minor, larger form (dFMR1-L(N)) begins upstream at a non-canonical start codon (CTG). To assess the contribution of the N-terminal extension toward dFMR1 activity, we generated transgenic flies that exclusively express either dFMR1-S(N) or dFMR1-L(N). Expression analyses throughout development revealed that dFMR1-S(N) is required for normal dFMR1-L(N) expression levels in adult brains. In situ expression analyses showed that either dFMR1-S(N) or dFMR1-L(N) is individually sufficient for proper dFMR1 localization in the nervous system. Functional studies demonstrated that both dFMR1-S(N) and dFMR1-L(N) can function independently to rescue dfmr1 null defects in synaptogenesis and axon guidance. Thus, dfmr1 encodes two functional isoforms with respect to expression and activity throughout neuronal development.

Funding information:
  • NIAMS NIH HHS - R01 AR049288(United States)

Transcriptional profiling of intrinsic PNS factors in the postnatal mouse.

  • Smith RP
  • Mol. Cell. Neurosci.
  • 2011 Jan 10

Literature context:


Abstract:

Neurons in the peripheral nervous system (PNS) display a higher capacity to regenerate after injury than those in the central nervous system, suggesting cell specific transcriptional modules underlying axon growth and inhibition. We report a systems biology based search for PNS specific transcription factors (TFs). Messenger RNAs enriched in dorsal root ganglion (DRG) neurons compared to cerebellar granule neurons (CGNs) were identified using subtractive hybridization and DNA microarray approaches. Network and transcription factor binding site enrichment analyses were used to further identify TFs that may be differentially active. Combining these techniques, we identified 32 TFs likely to be enriched and/or active in the PNS. Twenty-five of these TFs were then tested for an ability to promote CNS neurite outgrowth in an overexpression screen. Real-time PCR and immunohistochemical studies confirmed that one representative TF, STAT3, is intrinsic to PNS neurons, and that constitutively active STAT3 is sufficient to promote CGN neurite outgrowth.

Funding information:
  • NHLBI NIH HHS - HL66682(United States)

Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis.

  • Franke JD
  • Dev. Biol.
  • 2010 Sep 15

Literature context:


Abstract:

Metazoan development involves a myriad of dynamic cellular processes that require cytoskeletal function. Nonmuscle myosin II plays essential roles in embryonic development; however, knowledge of its role in post-embryonic development, even in model organisms such as Drosophila melanogaster, is only recently being revealed. In this study, truncation alleles were generated and enable the conditional perturbation, in a graded fashion, of nonmuscle myosin II function. During wing development they demonstrate novel roles for nonmuscle myosin II, including in adhesion between the dorsal and ventral wing epithelial sheets; in the formation of a single actin-based wing hair from the distal vertex of each cell; in forming unbranched wing hairs; and in the correct positioning of veins and crossveins. Many of these phenotypes overlap with those observed when clonal mosaic analysis was performed in the wing using loss of function alleles. Additional requirements for nonmuscle myosin II are in the correct formation of other actin-based cellular protrusions (microchaetae and macrochaetae). We confirm and extend genetic interaction studies to show that nonmuscle myosin II and an unconventional myosin, encoded by crinkled (ck/MyoVIIA), act antagonistically in multiple processes necessary for wing development. Lastly, we demonstrate that truncation alleles can perturb nonmuscle myosin II function via two distinct mechanisms--by titrating light chains away from endogenous heavy chains or by recruiting endogenous heavy chains into intracellular aggregates. By allowing myosin II function to be perturbed in a controlled manner, these novel tools enable the elucidation of post-embryonic roles for nonmuscle myosin II during targeted stages of fly development.

Funding information:
  • NINDS NIH HHS - 1 R01 NS054911-01A1(United States)

Axonal pathology precedes demyelination in a mouse model of X-linked demyelinating/type I Charcot-Marie Tooth neuropathy.

  • Vavlitou N
  • J. Neuropathol. Exp. Neurol.
  • 2010 Sep 15

Literature context:


Abstract:

The X-linked demyelinating/type I Charcot-Marie-Tooth neuropathy (CMT1X) is an inherited peripheral neuropathy caused by mutations in GJB1, the gene that encodes the gap junction protein connexin32. Connexin32 is expressed by myelinating Schwann cells and forms gap junctions in noncompact myelin areas, but axonal involvement is more prominent in X-linked compared with other forms of demyelinating Charcot-Marie-Tooth disease. To clarify the cellular and molecular mechanisms of axonal pathology in CMT1X, we studied Gjb1-null mice at early stages (i.e. 2-4 months old) of the neuropathy, when there is minimal or no demyelination. The diameters of large myelinated axons were progressively reduced in Gjb1-null mice compared with those in wild-type littermates. Furthermore, neurofilaments were relatively more dephosphorylated and more densely packed starting at 2 months of age. Increased expression of β-amyloid precursor protein, a marker of axonal damage, was also detected in Gjb1-null nerves. Finally, fast axonal transport, assayed by sciatic nerve ligation experiments, was slower in distal axons of Gjb1-null versus wild-type animals with reduced accumulation of synaptic vesicle-associated proteins. These findings demonstrate that axonal abnormalities including impaired cytoskeletal organization and defects in axonal transport precede demyelination in this mouse model of CMT1X.

Funding information:
  • NIBIB NIH HHS - R03 EB008673(United States)

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms.

  • Hammond JW
  • J. Cell Biol.
  • 2010 Jun 14

Literature context:


Abstract:

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.

Funding information:
  • NHLBI NIH HHS - N01-HV-28186(United States)

A chemical screen identifies novel compounds that overcome glial-mediated inhibition of neuronal regeneration.

  • Usher LC
  • J. Neurosci.
  • 2010 Mar 31

Literature context:


Abstract:

A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced four "hit compounds," which act with nanomolar potency on several different neuronal types and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR (epidermal growth factor receptor) activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.

Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation.

  • Wong YH
  • J. Neurosci.
  • 2010 Mar 24

Literature context:


Abstract:

Many Ig superfamily members are expressed in the developing nervous system, but the functions of these molecules during neurogenesis are not all clear. Here, we explore the expression and function of one of members of this superfamily, protogenin (PRTG), in the developing nervous system. Expression of PRTG protein is strong in the neural tube of mouse embryos between embryonic days 7.75 and 9.5 but disappears after embryonic day 10.5 when the neural progenitor marker nestin expresses prominently. Perturbation of PRTG activity in P19 embryonal carcinoma cells and in chick embryos, by either RNA interference or a dominant-negative PRTG mutant, increases neuronal differentiation. Using yeast two-hybrid screening and an in situ binding assay, we were able to identify ERdj3 (a stress-inducible endoplasmic reticulum DnaJ homolog) as a putative PRTG ligand. Addition of purified ERdj3 protein into the P19 differentiation assay reduced neurogenesis. This effect was blocked by addition of either a neutralizing antibody against PRTG or purified PRTG ectodomain protein, indicating that the effect of ERdj3 on neurogenesis is mediated through PRTG. Forced expression of ERdj3 in the chick neural tube also impairs neuronal differentiation. Together, these results suggest that expression of PRTG defines a stage between pluripotent epiblasts and committed neural progenitors, and its signaling plays a critical role in suppressing premature neuronal differentiation during early neural development.

Funding information:
  • NIH HHS - DP1 OD003958(United States)

Expression and regulation of vitamin D3 upregulated protein 1 (VDUP1) is conserved in mammalian and insect brain.

  • Levendusky MC
  • J. Comp. Neurol.
  • 2009 Dec 10

Literature context:


Abstract:

Originally characterized as a cell-cycle inhibitor induced by vitamin D(3), the tumor suppressor vitamin-D(3) upregulated protein 1 (VDUP1) has increasingly been shown to play major physiological roles in cell differentiation and glucose metabolism. Here we show evolutionarily conserved expression patterns of VDUP1 in Drosophila and rat nervous systems, including subcellular localization--cytoplasmic enrichment in neurons and nuclear expression in glia. These anatomical correlates suggested conservation of VDUP1 regulation, which was investigated both functionally and through promoter studies. Characterization of orthologous vdup1 cis-regulatory regions identified evolutionarily conserved sequence blocks (CSBs) with similarities to neural enhancers, including basic helix-loop-helix (bHLH) transcription factor Neurogenin/Math/atonal and Mash/achaete-scute family members. E-boxes (CANNTG), the binding sites for bHLH proteins, were associated with these CSBs as well, including E-boxes known to mediate glucose-dependent upregulation of VDUP1 in nonneuronal cells. Hyperglycemia-induced upregulation of VDUP1 was observed in brain tumor cells and in the Drosophila nervous system, which resulted in developmental arrest. Taken together, these data demonstrate evolutionary conservation of VDUP1 regulation and function, and suggest an expanding role for VDUP1 in nervous system development.

Funding information:
  • Canadian Institutes of Health Research - MOP-79411(Canada)

Regulation of fat cell mass by insulin in Drosophila melanogaster.

  • DiAngelo JR
  • Mol. Cell. Biol.
  • 2009 Dec 25

Literature context:


Abstract:

A phylogenetically conserved response to nutritional abundance is an increase in insulin signaling, which initiates a set of biological responses dependent on the species. Consequences of augmented insulin signaling include developmental progression, cell and organ growth, and the storage of carbohydrates and lipids. Here, we address the evolutionary origins of insulin's positive effects on anabolic lipid metabolism by selectively modulating insulin signaling in the fat body of the fruit fly, Drosophila melanogaster. Analogous to the actions of insulin in higher vertebrates, those in Drosophila include expansion of the insect fat cell mass both by increasing the adipocyte number and by promoting lipid accumulation. The ability of insulin to accomplish the former depends on its capacity to bring about phosphorylation and inhibition of the transcription factor Drosophila FOXO (dFOXO) and the serine/threonine protein kinase shaggy, the fly ortholog of glycogen synthase kinase 3 (GSK3). Increasing the amount of triglyceride per cell also depends on the phosphorylation of shaggy but is independent of dFOXO. Thus, the findings of this study provide evidence that the control of fat mass by insulin is a conserved process and place dFOXO and shaggy/GSK3 downstream of the insulin receptor in controlling adipocyte cell number and triglyceride storage, respectively.

A tail-anchored myotonic dystrophy protein kinase isoform induces perinuclear clustering of mitochondria, autophagy, and apoptosis.

  • Oude Ophuis RJ
  • PLoS ONE
  • 2009 Nov 25

Literature context:


Abstract:

BACKGROUND: Studies on the myotonic dystrophy protein kinase (DMPK) gene and gene products have thus far mainly concentrated on the fate of length mutation in the (CTG)n repeat at the DNA level and consequences of repeat expansion at the RNA level in DM1 patients and disease models. Surprisingly little is known about the function of DMPK protein products. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate here that transient expression of one major protein product of the human gene, the hDMPK A isoform with a long tail anchor, results in mitochondrial fragmentation and clustering in the perinuclear region. Clustering occurred in a variety of cell types and was enhanced by an intact tubulin cytoskeleton. In addition to morphomechanical changes, hDMPK A expression induces physiological changes like loss of mitochondrial membrane potential, increased autophagy activity, and leakage of cytochrome c from the mitochondrial intermembrane space accompanied by apoptosis. Truncation analysis using YFP-hDMPK A fusion constructs revealed that the protein's tail domain was necessary and sufficient to evoke mitochondrial clustering behavior. CONCLUSION/SIGNIFICANCE: Our data suggest that the expression level of the DMPK A isoform needs to be tightly controlled in cells where the hDMPK gene is expressed. We speculate that aberrant splice isoform expression might be a codetermining factor in manifestation of specific DM1 features in patients.

Neurogenesis in an annelid: characterization of brain neural precursors in the polychaete Capitella sp. I.

  • Meyer NP
  • Dev. Biol.
  • 2009 Nov 1

Literature context:


Abstract:

Intertaxonomic comparisons are important for understanding neurogenesis and evolution of nervous systems, but high-resolution, cellular studies of early central nervous system development and the molecular mechanisms controlling this process in lophotrochozoans are still lacking. We provide a detailed cellular and molecular description of early brain neurogenesis in a lophotrochozoan annelid, Capitella sp. I. We utilized different approaches including DiI lineage tracing, immunohistochemistry, BrdU labeling, and gene expression analyses to characterize neural precursor cells in Capitella sp. I. Brain neurogenesis proceeds by the ingression of single cells from the anterior ectoderm to generate a stratified epithelial layer. Most cell divisions are restricted to apically localized cells with mitotic spindles oriented parallel to the epithelial layer. Prior to and during this process, an achaete-scute complex homolog, CapI-ash1, is expressed in clusters of surface cells in the anterior ectoderm, consistent with a proneural function for CapI-ash1. In contrast, a homolog of the neural differentiation marker elav, CapI-elav1, is restricted to basally localized cells within the forming brain. Unlike insects, Capitella sp. I does not have morphologically obvious enlarged neuroblasts, although Capitella sp. I brain neurogenesis displays several similarities with non-insect arthropod and vertebrate neurogenesis, providing a useful lophotrochozoan model for comparison.

A second-site noncomplementation screen for modifiers of Rho1 signaling during imaginal disc morphogenesis in Drosophila.

  • Patch K
  • PLoS ONE
  • 2009 Oct 23

Literature context:


Abstract:

BACKGROUND: Rho1 is a small GTPase of the Ras superfamily that serves as the central component in a highly conserved signaling pathway that regulates tissue morphogenesis during development in all animals. Since there is tremendous diversity in the upstream signals that can activate Rho1 as well as the effector molecules that carry out its functions, it is important to define relevant Rho1-interacting genes for each morphogenetic event regulated by this signaling pathway. Previous work from our lab and others has shown that Rho signaling is necessary for the morphogenesis of leg imaginal discs during metamorphosis in Drosophila, although a comprehensive identification of Rho1-interacting genes has not been attempted for this process. METHODOLOGY/PRINCIPAL FINDINGS: We characterized an amorphic allele of Rho1 that displays a poorly penetrant dominant malformed leg phenotype and is capable of being strongly enhanced by Rho1-interacting heterozygous mutations. We then used this allele in a second-site noncomplementation screen with the Exelixis collection of molecularly defined deficiencies to identify Rho1-interacting genes necessary for leg morphogenesis. In a primary screen of 461 deficiencies collectively uncovering approximately 50% of the Drosophila genome, we identified twelve intervals harboring Rho1-interacting genes. Through secondary screening we identified six Rho1-interacting genes including three that were previously identified (RhoGEF2, broad, and stubbloid), thereby validating the screen. In addition, we identified Cdc42, Rheb and Sc2 as novel Rho1-interacting genes involved in adult leg development. CONCLUSIONS/SIGNIFICANCE: This screen identified well-known and novel Rho1-interacting genes necessary for leg morphogenesis, thereby increasing our knowledge of this important signaling pathway. We additionally found that Rheb may have a unique function in leg morphogenesis that is independent of its regulation of Tor.

Negative regulation of active zone assembly by a newly identified SR protein kinase.

  • Johnson EL
  • PLoS Biol.
  • 2009 Sep 22

Literature context:


Abstract:

Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc) that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

Desmoglein 1-dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis.

  • Getsios S
  • J. Cell Biol.
  • 2009 Jun 29

Literature context:


Abstract:

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor-Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

A multicomponent assembly pathway contributes to the formation of acentrosomal microtubule arrays in interphase Drosophila cells.

  • Rogers GC
  • Mol. Biol. Cell
  • 2009 Feb 25

Literature context:


Abstract:

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both gamma-tubulins. However, gamma-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted gamma-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.

TGF-beta coordinately activates TAK1/MEK/AKT/NFkB and SMAD pathways to promote osteoclast survival.

  • Gingery A
  • Exp. Cell Res.
  • 2008 Sep 10

Literature context:


Abstract:

To better understand the roles of TGF-beta in bone metabolism, we investigated osteoclast survival in response TGF-beta and found that TGF-beta inhibited apoptosis. We examined the receptors involved in promotion of osteoclast survival and found that the canonical TGF-beta receptor complex is involved in the survival response. The upstream MEK kinase TAK1 was rapidly activated following TGF-beta treatment. Since osteoclast survival involves MEK, AKT, and NFkappaB activation, we examined TGF-beta effects on activation of these pathways and observed rapid phosphorylation of MEK, AKT, IKK, IkappaB, and NFkappaB. The timing of activation coincided with SMAD activation and dominant negative SMAD expression did not inhibit NFkappaB activation, indicating that kinase pathway activation is independent of SMAD signaling. Inhibition of TAK1, MEK, AKT, NIK, IKK, or NFkappaB repressed TGF-beta-mediated osteoclast survival. Adenoviral-mediated TAK1 or MEK inhibition eliminated TGF-beta-mediated kinase pathway activation and constitutively active AKT expression overcame apoptosis induction following MEK inhibition. TAK1/MEK activation induces pro-survival BclX(L) expression and TAK1/MEK and SMAD pathway activation induces pro-survival Mcl-1 expression. These data show that TGF-beta-induced NFkappaB activation is through TAK1/MEK-mediated AKT activation, which is essential for TGF-beta to support of osteoclast survival.

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila.

  • Arama E
  • PLoS Biol.
  • 2007 Oct 1

Literature context:


Abstract:

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3-dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3(Testis)), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3(Testis) that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis-like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation.

Funding information:
  • NEI NIH HHS - R01 EY020535(United States)

DA-Raf1, a competent intrinsic dominant-negative antagonist of the Ras-ERK pathway, is required for myogenic differentiation.

  • Yokoyama T
  • J. Cell Biol.
  • 2007 Jun 4

Literature context:


Abstract:

Ras activates Raf, leading to the extracellular-regulated kinase (ERK)-mitogen-activated protein kinase pathway, which is involved in a variety of cellular, physiological, and pathological responses. Thus, regulators of this Ras-Raf interaction play crucial roles in these responses. In this study, we report a novel regulator of the Ras-Raf interaction named DA-Raf1. DA-Raf1 is a splicing isoform of A-Raf with a wider tissue distribution than A-Raf. It contains the Ras-binding domain but lacks the kinase domain, which is responsible for activation of the ERK pathway. As inferred from its structure, DA-Raf1 bound to activated Ras as well as M-Ras and interfered with the ERK pathway. The Ras-ERK pathway is essential for the negative regulation of myogenic differentiation induced by growth factors. DA-Raf1 served as a positive regulator of myogenic differentiation by inducing cell cycle arrest, the expression of myogenin and other muscle-specific proteins, and myotube formation. These results imply that DA-Raf1 is the first identified competent, intrinsic, dominant-negative antagonist of the Ras-ERK pathway.

Funding information:
  • NINDS NIH HHS - NS35165(United States)
  • NINDS NIH HHS - R01 NS085093(United States)

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm.

  • Marquez B
  • Dev. Biol.
  • 2007 Mar 1

Literature context:


Abstract:

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.

Funding information:
  • NCRR NIH HHS - UL1 RR025774(United States)

Substitution of critical isoleucines in the KH domains of Drosophila fragile X protein results in partial loss-of-function phenotypes.

  • Banerjee P
  • Genetics
  • 2007 Mar 26

Literature context:


Abstract:

Fragile X mental retardation proteins (FMRP) are RNA-binding proteins that interact with a subset of cellular RNAs. Several RNA-binding domains have been identified in FMRP, but the contribution of these individual domains to FMRP function in an animal model is not well understood. In this study, we have generated flies with point mutations in the KH domains of the Drosophila melanogaster fragile X gene (dfmr1) in the context of a genomic rescue fragment. The substitutions of conserved isoleucine residues within the KH domains with asparagine are thought to impair binding of RNA substrates and perhaps the ability of FMRP to assemble into mRNP complexes. The mutants were analyzed for defects in development and behavior that are associated with deletion null alleles of dfmr1. We find that these KH domain mutations result in partial loss of function or no significant loss of function for the phenotypes assayed. The phenotypes resulting from these KH domain mutants imply that the capacities of the mutant proteins to bind RNA and form functional mRNP complexes are not wholly disrupted and are consistent with biochemical models suggesting that RNA-binding domains of FMRP can function independently.

Funding information:
  • NHGRI NIH HHS - R01-HG004885(United States)

Adult neurogenesis: a common strategy across diverse species.

  • Sullivan JM
  • J. Comp. Neurol.
  • 2007 Jan 20

Literature context:


Abstract:

Adult neurogenesis, the generation of new neurons from adult precursor cells, occurs in the brains of a phylogenetically diverse array of animals. In the higher (amniotic) vertebrates, these precursor cells are glial cells that reside within specialized regions, known as neurogenic niches, the elements of which both support and regulate neurogenesis. The in vivo identity and location of the precursor cells responsible for adult neurogenesis in nonvertebrate taxa, however, remain largely unknown. Among the invertebrates, adult neurogenesis has been particularly well characterized in freshwater crayfish (Arthropoda, Crustacea), although the identity of the precursor cells sustaining continuous neuronal proliferation in these animals has yet to be established. Here we provide evidence suggesting that, as in the higher vertebrates, the precursor cells maintaining adult neurogenesis in the crayfish Procambarus clarkii are glial cells. These precursor cells reside within a specialized region, or niche, on the ventral surface of the brain, and their progeny migrate from this niche along glial fibers and then proliferate to form new neurons in the central olfactory pathway. The niche in which these precursor cells reside has many features in common with the neurogenic niches of higher vertebrates. These commonalities include: glial cells functioning as both precursor and support cells, directed migration, close association with the brain vasculature, and specialized basal laminae. The cellular machinery maintaining adult neurogenesis appears, therefore, to be shared by widely disparate taxa. These extensive structural and functional parallels suggest a common strategy for the generation of new neurons in adult brains.

Funding information:
  • PHS HHS - EF-0850100(United States)

Phosphorylation and activity of the tumor suppressor Merlin and the ERM protein Moesin are coordinately regulated by the Slik kinase.

  • Hughes SC
  • J. Cell Biol.
  • 2006 Oct 23

Literature context:


Abstract:

Merlin and Moesin are closely related members of the 4.1 Ezrin/Radixin/Moesin domain superfamily implicated in regulating proliferation and epithelial integrity, respectively. The activity of both proteins is regulated by head to tail folding that is controlled, in part, by phosphorylation. Few upstream regulators of these phosphorylation events are known. In this study, we demonstrate that in Drosophila melanogaster, Slik, a Ste20 kinase, controls subcellular localization and phosphorylation of Merlin, resulting in the coordinate but opposite regulation of Merlin and Moesin. These results suggest the existence of a novel mechanism for coordinate regulation of cell proliferation and epithelial integrity in developing tissues.

Funding information:
  • Intramural NIH HHS - P50CA/DA84718(United States)

Drosophila Rho-kinase (DRok) is required for tissue morphogenesis in diverse compartments of the egg chamber during oogenesis.

  • Verdier V
  • Dev. Biol.
  • 2006 Sep 15

Literature context:


Abstract:

The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. We have determined that the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, "dumping" of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis.

Funding information:
  • NHGRI NIH HHS - 2P50 HG002790-06(United States)

Compensation of inositol 1,4,5-trisphosphate receptor function by altering sarco-endoplasmic reticulum calcium ATPase activity in the Drosophila flight circuit.

  • Banerjee S
  • J. Neurosci.
  • 2006 Aug 9

Literature context:


Abstract:

Ionic Ca2+ functions as a second messenger to control several intracellular processes. It also influences intercellular communication. The release of Ca2+ from intracellular stores through the inositol 1,4,5-trisphosphate receptor (InsP3R) occurs in both excitable and nonexcitable cells. In Drosophila, InsP3R activity is required in aminergic interneurons during pupal development for normal flight behavior. By altering intracellular Ca2+ and InsP3 levels through genetic means, we now show that signaling through the InsP3R is required at multiple steps for generating the neural circuit required in air puff-stimulated Drosophila flight. Decreased Ca2+ release in aminergic neurons during development of the flight circuit can be compensated by reducing Ca2+ uptake from the cytosol to intracellular stores. However, this mode of increasing intracellular Ca2+ is insufficient for maintenance of flight patterns over time periods necessary for normal flight. Our study suggests that processes such as maintenance of wing posture and formation of the flight circuit require InsP3 receptor function at a slow timescale and can thus be modulated by altering levels of cytosolic Ca2+ and InsP3. In contrast, maintenance of flight patterns probably requires fast modulation of Ca2+ levels, in which the intrinsic properties of the InsP3R play a pivotal role.

Funding information:
  • Howard Hughes Medical Institute - R01 NS054814-06(United States)

Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation.

  • Chen HH
  • Mol. Cell. Biol.
  • 2006 Apr 15

Literature context:


Abstract:

CrkRS is a Cdc2-related protein kinase that contains an arginine- and serine-rich (SR) domain, a characteristic of the SR protein family of splicing factors, and is proposed to be involved in RNA processing. However, whether it acts together with a cyclin and at which steps it may function to regulate RNA processing are not clear. Here, we report that CrkRS interacts with cyclin L1 and cyclin L2, and thus rename it as the long form of cyclin-dependent kinase 12 (CDK12(L)). A shorter isoform of CDK12, CDK12(S), that differs from CDK12(L) only at the carboxyl end, was also identified. Both isoforms associate with cyclin L1 through interactions mediated by the kinase domain and the cyclin domain, suggesting a bona fide CDK/cyclin partnership. Furthermore, CDK12 isoforms alter the splicing pattern of an E1a minigene, and the effect is potentiated by the cyclin domain of cyclin L1. When expression of CDK12 isoforms is perturbed by small interfering RNAs, a reversal of the splicing choices is observed. The activity of CDK12 on splicing is counteracted by SF2/ASF and SC35, but not by SRp40, SRp55, and SRp75. Together, our findings indicate that CDK12 and cyclin L1/L2 are cyclin-dependent kinase and cyclin partners and regulate alternative splicing.

Funding information:
  • NHLBI NIH HHS - 5P01 HL-67105(United States)
  • NHLBI NIH HHS - HL66621(United States)

The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila.

  • Cygnar KD
  • Genetics
  • 2005 Jun 27

Literature context:


Abstract:

The protein phosphatase 2A (PP2A) regulatory subunit Tap42 is essential for target of rapamycin (TOR)-mediated signaling in yeast, but its role in higher eukaryotes has not been established. Here we show that Tap42 does not contribute significantly to TOR signaling in Drosophila, as disruption of the Tap42 gene does not cause defects in cell growth, metabolism, or S6-kinase activity characteristic of TOR inactivation. In addition, Tap42 is not required for increased cell growth in response to activation of TOR signaling. Instead, we find that Tap42 mutations cause disorganization of spindle microtubules in larval neuroblasts, leading to a preanaphase mitotic arrest in these cells. Loss of Tap42 ultimately results in increased JNK signaling, caspase activation, and cell death. These phenotypes are associated with increased accumulation and nuclear localization of PP2A in Tap42 mutant cells. Our results demonstrate that the role of Tap42 in TOR signaling has not been conserved in higher eukaryotes, indicating fundamental differences in the mechanisms of TOR signaling between yeast and higher eukaryotes.

Funding information:
  • Wellcome Trust - 1R24OD011883-01(United Kingdom)

The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation.

  • Caretti G
  • Genes Dev.
  • 2004 Nov 1

Literature context:


Abstract:

The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. We report that Ezh2 expression was developmentally regulated in the myotome compartment of mouse somites and that its down-regulation coincided with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle differentiation, and this property was conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their presence correlated with methylation of K27 of histone H3. YY1 was required for Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF were recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation.

Funding information:
  • Biotechnology and Biological Sciences Research Council - BB/H018123/2(United Kingdom)

Role of peroxidoxins in Leishmania chagasi survival. Evidence of an enzymatic defense against nitrosative stress.

  • Barr SD
  • J. Biol. Chem.
  • 2003 Mar 21

Literature context:


Abstract:

The mechanisms by which Leishmania parasites survive exposure to highly reactive oxygen (ROS) and nitrogen (RNS) species within phagosomes of macrophages are not well known. Recently it has been shown that RNS alone is sufficient and necessary to control Leishmania donovani infection in mice (Murray, H. W., and Nathan, C. F. (1999) J. Exp. Med. 189, 741-746). No enzymatic defense against RNS has been discovered in Leishmania to date. We have previously isolated two peroxidoxins (LcPxn1 and LcPxn2) from Leishmania chagasi and showed that recombinant LcPxn1 protein was capable of detoxifying hydrogen peroxide, hydroperoxide, and hydroxyl radicals (Barr, S. D., and Gedamu, L. (2001) J. Biol. Chem. 276, 34279-34287). In further characterizing the physiological role of peroxidoxins in Leishmania survival, we show here that recombinant LcPxn1 protein can detoxify RNS in addition to ROS, whereas recombinant LcPxn2 protein can only detoxify hydrogen peroxide. LcPxn1 and LcPxn2 are localized to the cytoplasm, and overexpression of LcPxn1 in L. chagasi parasites enhanced survival when exposed to exogenous ROS and RNS and enhanced survival within U937 macrophage cells. Site-directed mutagenesis studies revealed that the conserved Cys-52 residue is essential for detoxifying hydrogen peroxide, t-butyl hydroperoxide, and hydroxyl radicals, whereas the conserved Cys-173 residue is essential for detoxifying t-butyl hydroperoxide and peroxynitrite. This is the first report of an enzymatic defense against RNS in Leishmania.

Funding information:
  • Intramural NIH HHS - ZIA ES102805(United States)

Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression.

  • Mazroui R
  • Hum. Mol. Genet.
  • 2002 Nov 15

Literature context:


Abstract:

Absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein, is responsible for the Fragile X syndrome, the most common form of inherited mental retardation. FMRP is a cytoplasmic protein associated with mRNP complexes containing poly(A)+mRNA. As a step towards understanding FMRP function(s), we have established the immortal STEK Fmr1 KO cell line and showed by transfection assays with FMR1-expressing vectors that newly synthesized FMRP accumulates into cytoplasmic granules. These structures contain mRNAs and several other RNA-binding proteins. The formation of these cytoplasmic granules is dependent on determinants located in the RGG domain. We also provide evidence that FMRP acts as a translation repressor following co-transfection with reporter genes. The FMRP-containing mRNPs are dynamic structures that oscillate between polyribosomes and cytoplasmic granules reminiscent of the Stress Granules that contain repressed mRNAs. We speculate that, in neurons, FMRP plays a role as a mRNA repressor in incompetent mRNP granules that have to be translocated from the cell body to distal locations such as dendritic spines and synaptosomes.

Funding information:
  • NIGMS NIH HHS - 5P20GM103636(United States)
  • NINDS NIH HHS - NS-39050(United States)

Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2.

  • Stevaux O
  • EMBO J.
  • 2002 Sep 16

Literature context:


Abstract:

RBF1, a Drosophila pRB family homolog, is required for cell cycle arrest and the regulation of E2F-dependent transcription. Here, we describe the properties of RBF2, a second family member. RBF2 represses E2F transcription and is present at E2F-regulated promoters. Analysis of in vivo protein complexes reveals that RBF1 and RBF2 interact with different subsets of E2F proteins. dE2F1, a potent transcriptional activator, is regulated specifically by RBF1. In contrast, RBF2 binds exclusively to dE2F2, a form of E2F that functions as a transcriptional repressor. We find that RBF2-mediated repression requires dE2F2. More over, RBF2 and dE2F2 act synergistically to antagonize dE2F1-mediated activation, and they co-operate to block S phase progression in transgenic animals. The network of interactions between RBF1 or RBF2 and dE2F1 or dE2F2 reveals how the activities of these proteins are integrated. These results suggest that there is a remarkable degree of symmetry in the arrangement of E2F and RB family members in mammalian cells and in DROSOPHILA.

Funding information:
  • Canadian Institutes of Health Research - MOP-38854(Canada)

Identification of immunological reagents for use in the study of freshwater planarians by means of whole-mount immunofluorescence and confocal microscopy.

  • Robb SM
  • Genesis
  • 2002 Apr 11

Literature context:


Abstract:

In recent years, interest in planarians as a model system for the study of metazoan regeneration, adult stem cell biology, and the evolution of metazoan body plans has been growing steadily. The availability of RNA interference (RNAi), BrdU-labeling of planarian stem cells, and thousands of planarian cDNA sequences soon to be released into public databases has opened planarians to molecular dissection. However, the successful application of large-scale RNAi-based screens, for example, will depend in part on the availability of markers to characterize the resulting phenotypes. Given the paucity of antibodies available for the study of planarian biology, we have screened various public and commercial antibody resources to identify immunoreagents capable of cross-reacting with planarian tissues. Here we report the identification and characterization of 33 such antibodies recognizing a wide variety of tissues in freshwater planarians.

Funding information:
  • NIA NIH HHS - AG039669(United States)

Developmental expression of the fragile X-related 1 proteins in mouse testis: association with microtubule elements.

  • Huot ME
  • Hum. Mol. Genet.
  • 2001 Nov 15

Literature context:


Abstract:

Fragile X mental retardation 1 protein (FMRP) is the archetype of a class of cytoplasmic mRNA-binding proteins that includes the fragile X-related 1 and 2 proteins (FXR1P and FXR2P). Whereas absence of FMRP is the cause of fragile X syndrome, it is not known if FXR1P and FXR2P are associated with any pathology. It is also still elusive whether these homologous proteins can partially compensate for the absence of FMRP in the case of the fragile X syndrome. FXR1 is widely expressed in mammals and its expression pattern is complex since several mRNA variants and protein isoforms are detected. In mouse, we observed that the highest level of FXR1 is found in the adult testis. This tissue is an exception, since all known FXR1P isoforms, some of which have been considered as tissue specific, are detected in it. In young animals, changes in mRNA-spliced variants and their corresponding protein isoforms occur during spermatogenesis. Using biochemical, immunohistochemical and electron microscopic techniques, we show that FXR1P is associated with microtubule elements. Since the cytoskeletal framework is implicated in cellular plasticity as well as in mRNA transport, we propose new possibilities for the function(s) of the FXR proteins.

Funding information:
  • NIDA NIH HHS - DA09082(United States)
  • NINDS NIH HHS - 1 ZIA NS003119 05(United States)

T lymphocyte-triggering factor of african trypanosomes is associated with the flagellar fraction of the cytoskeleton and represents a new family of proteins that are present in several divergent eukaryotes.

  • Hill KL
  • J. Biol. Chem.
  • 2000 Dec 15

Literature context:


Abstract:

The trypanosome cytoskeleton consists almost entirely of microtubule-based structures. Although alpha- and beta-tubulin from Trypanosoma brucei have been well characterized, much less is known about other cytoskeleton-associated proteins in trypanosomes. Using biochemical fractionation, we demonstrate here that T lymphocyte-triggering factor (TLTF) from T. brucei is a component of the detergent-resistant and Ca(2+)-resistant fraction of the parasite cytoskeleton. This fraction contains the flagellar apparatus and a subset of cytoskeletal protein complexes that together function in cell motility, cytokinesis, and organelle inheritance. We also show that TLTF-related genes are present in several highly divergent eukaryotic organisms. Although the function of the corresponding proteins is not known, the mammalian TLTF-like gene (GAS11; growth arrest-specific gene 11) is up-regulated in growth-arrested cells and is a candidate tumor suppressor (Whitmore, S. A., Settasatian, C., Crawford, J., Lower, K. M., McCallum, B., Seshadri, R., Cornelisse, C. J., Moerland, E. W., Cleton-Jansen, A. M., Tipping, A. J., Mathew, C. G., Savnio, M., Savoia, A., Verlander, P., Auerbach, A. D., Van Berkel, C., Pronk, J. C., Doggett, N. A., and Callen, D. F. (1998) Genomics 52, 325-331), suggestive of a role in coordinating cytoskeleton activities. Consistent with this possibility, we show that the human GAS11 protein contains a 144-amino acid domain that co-localizes with microtubules when fused to the green fluorescent protein and expressed in mammalian cells. These findings suggest that TLTF represents a newly defined protein family, whose members contribute to cytoskeleton function in species as diverse as protozoa and mammals.

Identification of beta-tubulin isoforms as tumor antigens in neuroblastoma.

  • Prasannan L
  • Clin. Cancer Res.
  • 2000 Oct 29

Literature context:


Abstract:

There is currently substantial interest in the identification of human tumor antigens for diagnosis and immunotherapy of cancer. We have implemented a proteomic approach for the identification of tumor proteins that elicit a humoral response in cancer patients, which we have applied to neuroblastoma. Proteins from neuroblastoma tumors and cell lines were separated by two-dimensional PAGE and transferred to poly(vinylidene difluoride) membranes. Sera from 23 newly diagnosed patients with neuroblastoma, from 12 newly diagnosed children with other solid tumors, and from 13 normal individuals were screened for IgG and IgM autoantibodies against neuroblastoma proteins by means of Western blot analysis. Sera from 11 patients with neuroblastoma and from 1 patient with a primitive neuroectodermal tumor, but none of the other controls exhibited IgG-based reactivity against a protein constellation with an estimated Mr 50,000. NH2-terminal sequence and mass spectrometric analysis identified the major constituents of this constellation as beta-tubulin isoforms I and III. The IgG antibodies were additionally characterized to be of the subclass IgG1. Neuroblastoma patient sera that contained anti-beta-tubulin IgG antibodies also contained IgM antibodies specific against the full-length beta-tubulin molecule and against COOH-terminal beta-tubulin cleavage products. Neuroblastoma patient sera that reacted with beta-tubulin I and III isoforms in neuroblastoma tissues did not react with beta-tubulin I and III isoforms found in normal brain tissue. Our findings indicate the occurrence of beta-tubulin peptides in neuroblastoma, which are immunogenic. The occurrence of immunogenic peptides in neuroblastoma may have utility in diagnosis and in immunotherapy of this aggressive childhood tumor.

Funding information:
  • NHGRI NIH HHS - U01 HG005719-02(United States)

Taxol reduces cytosolic E-cadherin and beta-catenin levels in nasopharyngeal carcinoma cell line TW-039: cross-talk between the microtubule- and actin-based cytoskeletons.

  • Lou PJ
  • J. Cell. Biochem.
  • 2000 Sep 14

Literature context:


Abstract:

Taxol affects microtubule dynamics by promoting microtubule assembly. To obtain a better insight into possible cross-talk between the microtubule- and actin-based cytoskeletons, we studied the short-term effects of Taxol treatment on the expression of actin and the E-cadherin/catenin complex in the nasopharyngeal carcinoma cell line TW-039 using immunofluorescence, immunoprecipitation, and immunoblotting methods. Morphologic changes in actin filaments, including ventral actin clumps and perijunctional actin blebs, were seen at Taxol concentrations > or =1 microM. Levels of detergent-soluble E-cadherin fell to 53% or 58% compared to controls in cells treated, respectively, with 1 or 5 microM Taxol, while levels of detergent-soluble beta-catenin fell to 76% or 74%. Levels of the detergent-soluble pool of alpha- and gamma-catenin and the detergent-insoluble pool of the E-cadherin/catenin complex were unchanged by Taxol treatment and no significant difference was seen in the levels of adenomatous polyposis coli or glycogen synthase-3beta or tyrosine phosphorylation patterns. These results suggest that modulation of microtubule dynamics by Taxol may have effects on the expression of actin and the cytosolic E-cadherin and beta-catenin in nasopharyngeal carcinoma cells through pathways not involving the phosphorylation of beta-catenin.

Funding information:
  • NINDS NIH HHS - NS 37172(United States)

At reduced temperature, endocytic membrane traffic is blocked in multivesicular carrier endosomes in rat cardiac myocytes.

  • Punnonen EL
  • Eur. J. Cell Biol.
  • 1999 Jan 5

Literature context:


Abstract:

Temperatures around 20 degrees C are known to block degradation of endocytosed material by preventing its transport to lysosomes, accordingly reduced temperature has been widely used to define endosomes. Newer studies have revealed that the low temperature block is proximal to perinuclear late endosomes, but it is not clear whether the block is already in early endosomes, or whether the traffic proceeds to multivesicular carrier endosomes which mediate transport from early to late compartments. We have now focused on this problem using rat cardiac myocytes. First, cell fractionation on Percoll gradients showed that at reduced temperatures (22 degrees C and 26 degrees C), with prolonged chase periods, endocytosed horseradish peroxidase was able to proceed from early endosomes to later compartments but not up to lysosomes. Further, microscopic experiments with fluorescent endocytic marker FITC-dextran showed that the marker did not accumulate in the perinuclear area, as was the case at 37 degrees C, but stayed in peripheral cytoplasm at reduced temperatures, even after 16-h chase. Second, electron microscopic pulse labeling showed that, at 22 degrees C, endocytosed gold particles (BSA-gold) are transported to compartments not accessible to HRP internalised later to early endosomes. Thus, these gold particles had reached a later compartment. Morphologically these vesicles were multivesicular bodies of 0.5-1 microm in diameter. Third, we used fluorescence microscopy to study the effect of reduced temperature on transferrin uptake and recycling. At 17 degrees C and 22 degrees C, transferrin was internalized normally to peripheral (sorting) and perinuclear (recycling) vesicles. If transferrin was first taken up at 37 degrees C, and the cells were then chased at various temperatures from 37 degrees C to 17 degrees C, the recycling was slowed down but not entirely blocked at the reduced temperatures. From these results we can conclude that (1) endocytic traffic is blocked in multivesicular carrier endosomes at and below 26 degrees C, and that (2) reduced temperature slows down transport in the recycling pathway, without a complete block.

Funding information:
  • NIMH NIH HHS - 5 RC2 MH090047-01(United States)

3-Methyladenine inhibits transport from late endosomes to lysosomes in cultured rat and mouse fibroblasts.

  • Punnonen EL
  • Eur. J. Cell Biol.
  • 1994 Oct 19

Literature context:


Abstract:

The effect of 3-methyladenine on transport from endosomes to lysosomes was studied in rat embryonic and mouse 3T3 fibroblasts. Subcellular fractionation in 27% Percoll gradients showed that the pre-endocytosed (5 min pulse) horseradish peroxidase (HRP) was not transported from endosomes to dense lysosomes in cells chased in the presence of 10 mM 3-methyladenine. However, fractionation in 20% Percoll gradients, which separated early endosomes from late endosomes and lysosomes, as well as light and electron microscopic experiments, showed that HRP was transported from early endosomes to the perinuclear late endosomes. Immunoprecipitation of metabolically labeled cells was used to study the biosynthetic processing of a lysosomal proteinase, cathepsin L. The results showed that the early processing of the precursor to the intermediate form was not affected by 3-methyladenine, while the late processing of the intermediate to the mature form was retarded. In addition, immunofluorescence labeling showed that 3-methyladenine treatment caused accumulation of cathepsin L in the perinuclear area. Another lysosomal enzyme, beta-glucuronidase, was normally distributed in both perinuclear and peripheral vesicles which indicated that the localization of lysosomes was not altered. The results thus suggest that the late processing of cathepsin L was inhibited because transport from perinuclear endosomes to lysosomes was retarded. In conclusion, both endocytic pulse-chase experiments and immunoprecipitation of metabolically labeled cathepsin L indicate that 3-methyladenine inhibits transport from late endosomes to mature lysosomes in both rat and mouse fibroblasts.

Funding information:
  • NIA NIH HHS - AG038070(United States)
  • NIA NIH HHS - AG11482(United States)

Autophagic vacuoles fuse with the prelysosomal compartment in cultured rat fibroblasts.

  • Punnonen EL
  • Eur. J. Cell Biol.
  • 1993 Jun 6

Literature context:


Abstract:

We previously demonstrated both mannose 6-phosphate receptor (MPR) and cathepsin L in early autophagic vacuoles of cultured rat fibroblasts. This suggested that the enzyme may originate either from the receptor-enriched prelysosomal compartment (PLC) or from the trans-Golgi network (TGN). In the present ultrastructural study, we elucidated the roles of the PLC and TGN in lysosomal enzyme delivery to autophagic vacuoles. Firstly, we studied whether endocytic markers, cationized ferritin (CF), bovine serum albumin-gold or horseradish peroxidase (HRP), can be detected in autophagic vacuoles. Autophagy was induced by serum removal from the medium with or without leupeptin, an inhibitor of cysteine proteinases. Endocytic markers were not detected in autophagic vacuoles after short uptake which filled the early endosome, but only after longer labeling which filled the PLC. The markers were usually found in advanced autophagic vacuoles containing partially degraded cytoplasm and complex internal membranes which are the characteristic of the PLC. HRP-positive vesicles were also observed in continuity with early autophagic vacuoles containing intact cytoplasm. After uptake and transport of CF and HRP to the PLC, these markers were delivered to autophagic vacuoles even if microtubules were disrupted in vinblastine before the induction of autophagy. Secondly, we studied whether MPRs transport cathepsin L to autophagic vacuoles directly from the TGN. Two inhibitors of MPR-mediated enzyme transport, tunicamycin and chloroquine, were used. Quantitative immunocytochemistry showed that neither of these drugs prevented cathepsin L delivery to autophagic vacuoles. The results suggest that a large proportion of lysosomal enzymes is delivered to autophagic vacuoles from the PLC by a microtubule-independent manner. The first enzymes may be transported in small PLC-derived vesicles or tubules which are reached by HRP but not by CF and gold. Later, the autophaged cytoplasm is delivered to larger vacuolar parts of the PLC. Mannose 6-phosphate receptors transport no or only trace amounts of lysosomal enzymes to autophagic vacuoles directly from the TGN.

Funding information:
  • NIBIB NIH HHS - R01-EB007057(United States)

The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus.

  • Chu DT
  • Dev. Biol.
  • 1989 Nov 29

Literature context:


Abstract:

Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.

Funding information:
  • NIDCR NIH HHS - K99DE24190(United States)
  • NIMH NIH HHS - MH084020(United States)