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Mouse Anti-gamma-Tubulin Monoclonal Antibody, Unconjugated, Clone GTU-88

RRID:AB_477584

Antibody ID

AB_477584

Target Antigen

Tubulin, gamma bovine, canine, chicken/avian, hamster, human, mouse, rat, xenopus, xenopus, bovine, chicken, canine, hamster, human, rat, mouse

Proper Citation

(Sigma-Aldrich Cat# T6557, RRID:AB_477584)

Clonality

monoclonal antibody

Comments

Vendor recommendations: ELISA; Immunocytochemistry; Western Blot; Indirect ELISA, Immunocytochemistry, Western Blot

Clone ID

Clone GTU-88

Host Organism

mouse

Atf3 links loss of epithelial polarity to defects in cell differentiation and cytoarchitecture.

  • Donohoe CD
  • PLoS Genet.
  • 2018 Jul 10

Literature context:


Abstract:

Interplay between apicobasal cell polarity modules and the cytoskeleton is critical for differentiation and integrity of epithelia. However, this coordination is poorly understood at the level of gene regulation by transcription factors. Here, we establish the Drosophila activating transcription factor 3 (atf3) as a cell polarity response gene acting downstream of the membrane-associated Scribble polarity complex. Loss of the tumor suppressors Scribble or Dlg1 induces atf3 expression via aPKC but independent of Jun-N-terminal kinase (JNK) signaling. Strikingly, removal of Atf3 from Dlg1 deficient cells restores polarized cytoarchitecture, levels and distribution of endosomal trafficking machinery, and differentiation. Conversely, excess Atf3 alters microtubule network, vesicular trafficking and the partition of polarity proteins along the apicobasal axis. Genomic and genetic approaches implicate Atf3 as a regulator of cytoskeleton organization and function, and identify Lamin C as one of its bona fide target genes. By affecting structural features and cell morphology, Atf3 functions in a manner distinct from other transcription factors operating downstream of disrupted cell polarity.

Funding information:
  • NIGMS NIH HHS - R01 GM057070(United States)

IKKβ inhibition prevents fat-induced beta cell dysfunction in vitro and in vivo in rodents.

  • Ivovic A
  • Diabetologia
  • 2018 Jun 22

Literature context:


Abstract:

AIMS/HYPOTHESIS: We have previously shown that oxidative stress plays a causal role in beta cell dysfunction induced by fat. Here, we address whether the proinflammatory kinase inhibitor of (nuclear factor) κB kinase β (IKKβ), which is activated by oxidative stress, is also implicated. METHODS: Fat (oleate or olive oil) was infused intravenously in Wistar rats for 48 h with or without the IKKβ inhibitor salicylate. Thereafter, beta cell function was evaluated in vivo using hyperglycaemic clamps or ex vivo in islets isolated from fat-treated rats. We also exposed rat islets to oleate in culture, with or without salicylate and 4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-345541 (BMS, another inhibitor of IKKβ) and evaluated beta cell function in vitro. Furthermore, oleate was infused in mice treated with BMS and in beta cell-specific Ikkb-null mice. RESULTS: 48 h infusion of fat impaired beta-cell function in vivo, assessed using the disposition index (DI), in rats (saline: 1.41 ± 0.13; oleate: 0.95 ± 0.11; olive oil [OLO]: 0.87 ± 0.15; p < 0.01 for both fats vs saline) and in mice (saline: 2.51 ± 0.39; oleate: 1.20 ± 0.19; p < 0.01 vs saline) and ex vivo (i.e., insulin secretion, units are pmol insulin islet-1 h-1) in rat islets (saline: 1.51 ± 0.13; oleate: 1.03 ± 0.10; OLO: 0.91 ± 0.13; p < 0.001 for both fats vs saline) and the dysfunction was prevented by co-infusion of salicylate in rats (oleate + salicylate: 1.30 ± 0.09; OLO + salicylate: 1.33 ± 0.23) or BMS in mice (oleate + BMS: 2.25 ± 0.42) in vivo and by salicylate in rat islets ex vivo (oleate + salicylate: 1.74 ± 0.31; OLO + salicylate: 1.54 ± 0.29). In cultured islets, 48 h exposure to oleate impaired beta-cell function ([in pmol insulin islet-1 h-1] control: 0.66 ± 0.12; oleate: 0.23 ± 0.03; p < 0.01 vs saline), an effect prevented by both inhibitors (oleate + salicylate: 0.98 ± 0.08; oleate + BMS: 0.50 ± 0.02). Genetic inhibition of IKKβ also prevented fat-induced beta-cell dysfunction ex vivo ([in pmol insulin islet-1 h-1] control saline: 0.16 ± 0.02; control oleate: 0.10 ± 0.02; knockout oleate: 0.17 ± 0.04; p < 0.05 control saline vs. control oleate) and in vivo (DI: control saline: 3.86 ± 0.40; control oleate: 1.95 ± 0.29; knockout oleate: 2.96 ± 0.24; p < 0.01 control saline vs control oleate). CONCLUSIONS/INTERPRETATION: Our results demonstrate a causal role for IKKβ in fat-induced beta cell dysfunction in vitro, ex vivo and in vivo.

Targeting p38α Increases DNA Damage, Chromosome Instability, and the Anti-tumoral Response to Taxanes in Breast Cancer Cells.

  • Cánovas B
  • Cancer Cell
  • 2018 Jun 11

Literature context:


Abstract:

Breast cancer is the second leading cause of cancer-related death among women. Here we report a role for the protein kinase p38α in coordinating the DNA damage response and limiting chromosome instability during breast tumor progression, and identify the DNA repair regulator CtIP as a p38α substrate. Accordingly, decreased p38α signaling results in impaired ATR activation and homologous recombination repair, with concomitant increases in replication stress, DNA damage, and chromosome instability, leading to cancer cell death and tumor regression. Moreover, we show that pharmacological inhibition of p38α potentiates the effects of taxanes by boosting chromosome instability in murine models and patient-derived xenografts, suggesting the potential interest of combining p38α inhibitors with chemotherapeutic drugs that induce chromosome instability.

Funding information:
  • NIGMS NIH HHS - R01 GM068812(United States)

Hypoxia negatively affects senescence in osteoclasts and delays osteoclastogenesis.

  • Gorissen B
  • J. Cell. Physiol.
  • 2018 Jun 22

Literature context:


Abstract:

Cellular senescence, that is, the withdrawal from the cell cycle, combined with the acquirement of the senescence associated secretory phenotype has important roles during health and disease and is essential for tissue remodeling during embryonic development. Osteoclasts are multinucleated cells, responsible for bone resorption, and cell cycle arrest during osteoclastogenesis is well recognized. Therefore, the aim of this study was to investigate whether these cells should be considered senescent and to assess the influence of hypoxia on their potential senescence status. Osteoclastogenesis and bone resorption capacity of osteoclasts, cultured from CD14+ monocytes, were evaluated in two oxygen concentrations, normoxia (21% O2 ) and hypoxia (5% O2 ). Osteoclasts were profiled by using specific staining for proliferation and senescence markers, qPCR of a number of osteoclast and senescence-related genes and a bone resorption assay. Results show that during in vitro osteoclastogenesis, osteoclasts heterogeneously obtain a senescent phenotype. Furthermore, osteoclastogenesis was delayed at hypoxic compared to normoxic conditions, without negatively affecting the bone resorption capacity. It is concluded that osteoclasts can be considered senescent, although senescence is not uniformly present in the osteoclast population. Hypoxia negatively affects the expression of some senescence markers. Based on the direct relationship between senescence and osteoclastogenesis, it is tempting to hypothesize that contents of the so-called senescence associated secretory phenotype (SASP) not only play a functional role in matrix resorption, but also may regulate osteoclastogenesis.

Funding information:
  • NIGMS NIH HHS - R01 GM036373(United States)

Brain Somatic Mutations in MTOR Disrupt Neuronal Ciliogenesis, Leading to Focal Cortical Dyslamination.

  • Park SM
  • Neuron
  • 2018 Jun 9

Literature context:


Abstract:

Focal malformations of cortical development (FMCDs), including focal cortical dysplasia (FCD) and hemimegalencephaly (HME), are major etiologies of pediatric intractable epilepsies exhibiting cortical dyslamination. Brain somatic mutations in MTOR have recently been identified as a major genetic cause of FMCDs. However, the molecular mechanism by which these mutations lead to cortical dyslamination remains poorly understood. Here, using patient tissue, genome-edited cells, and mouse models with brain somatic mutations in MTOR, we discovered that disruption of neuronal ciliogenesis by the mutations underlies cortical dyslamination in FMCDs. We found that abnormal accumulation of OFD1 at centriolar satellites due to perturbed autophagy was responsible for the defective neuronal ciliogenesis. Additionally, we found that disrupted neuronal ciliogenesis accounted for cortical dyslamination in FMCDs by compromising Wnt signals essential for neuronal polarization. Altogether, this study describes a molecular mechanism by which brain somatic mutations in MTOR contribute to the pathogenesis of cortical dyslamination in FMCDs.

Funding information:
  • NHLBI NIH HHS - HL073284(United States)

Atypical Cadherin Dachsous1b Interacts with Ttc28 and Aurora B to Control Microtubule Dynamics in Embryonic Cleavages.

  • Chen J
  • Dev. Cell
  • 2018 May 7

Literature context:


Abstract:

Atypical cadherin Dachsous (Dchs) is a conserved regulator of planar cell polarity, morphogenesis, and tissue growth during animal development. Dchs functions in part by regulating microtubules by unknown molecular mechanisms. Here we show that maternal zygotic (MZ) dchs1b zebrafish mutants exhibit cleavage furrow progression defects and impaired midzone microtubule assembly associated with decreased microtubule turnover. Mechanistically, Dchs1b interacts via a conserved motif in its intracellular domain with the tetratricopeptide motifs of Ttc28 and regulates its subcellular distribution. Excess Ttc28 impairs cleavages and decreases microtubule turnover, while ttc28 inactivation increases turnover. Moreover, ttc28 deficiency in dchs1b mutants suppresses the microtubule dynamics and midzone microtubule assembly defects. Dchs1b also binds to Aurora B, a known regulator of cleavages and microtubules. Embryonic cleavages in MZdchs1b mutants exhibit increased, and in MZttc28 mutants decreased, sensitivity to Aurora B inhibition. Thus, Dchs1b regulates microtubule dynamics and embryonic cleavages by interacting with Ttc28 and Aurora B.

Funding information:
  • NIGMS NIH HHS - R15GM100402(United States)

Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates.

  • Sepulveda G
  • Elife
  • 2018 Apr 30

Literature context:


Abstract:

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

Funding information:
  • NIA NIH HHS - R01 AG039756(United States)
  • University of California, Davis - New Faculty Startup Funds()

Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma gondii by Limiting Its Uptake of Fatty Acids.

  • Pernas L
  • Cell Metab.
  • 2018 Apr 3

Literature context:


Abstract:

How intracellular pathogens acquire essential non-diffusible host metabolites and whether the host cell counteracts the siphoning of these nutrients by its invaders are open questions. Here we show that host mitochondria fuse during infection by the intracellular parasite Toxoplasma gondii to limit its uptake of fatty acids (FAs). A combination of genetics and imaging of FA trafficking indicates that Toxoplasma infection triggers lipophagy, the autophagy of host lipid droplets (LDs), to secure cellular FAs essential for its proliferation. Indeed, Toxoplasma FA siphoning and growth are reduced in host cells genetically deficient for autophagy or triglyceride depots. Conversely, Toxoplasma FA uptake and proliferation are increased in host cells lacking mitochondrial fusion, required for efficient mitochondrial FA oxidation, or where mitochondrial FA oxidation is pharmacologically inhibited. Thus, mitochondrial fusion can be regarded as a cellular defense mechanism against intracellular parasites, by limiting Toxoplasma access to host nutrients liberated by lipophagy.

Funding information:
  • NIAID NIH HHS - 5 R44 AI051036(United States)

Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules.

  • Martin M
  • Elife
  • 2018 Mar 16

Literature context:


Abstract:

Microtubules control different aspects of cell polarization. In cells with a radial microtubule system, a pivotal role in setting up asymmetry is attributed to the relative positioning of the centrosome and the nucleus. Here, we show that centrosome loss had no effect on the ability of endothelial cells to polarize and move in 2D and 3D environments. In contrast, non-centrosomal microtubules stabilized by the microtubule minus-end-binding protein CAMSAP2 were required for directional migration on 2D substrates and for the establishment of polarized cell morphology in soft 3D matrices. CAMSAP2 was also important for persistent endothelial cell sprouting during in vivo zebrafish vessel development. In the absence of CAMSAP2, cell polarization in 3D could be partly rescued by centrosome depletion, indicating that in these conditions the centrosome inhibited cell polarity. We propose that CAMSAP2-protected non-centrosomal microtubules are needed for establishing cell asymmetry by enabling microtubule enrichment in a single-cell protrusion.

Funding information:
  • China Scholarship Council - PhD fellowship()
  • European Research Council - Synergy 609822()
  • Fonds De La Recherche Scientifique - FNRS - FRIA fellowship()
  • H2020 Marie Skłodowska-Curie Actions - IEF fellowship()
  • Marie Sklodowska-Curie Actions - IEF fellowship()
  • NCI NIH HHS - F30 CA183497(United States)
  • Nederlandse Organisatie voor Wetenschappelijk Onderzoek - ALW Open Program grant 824.15.017()

Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7.

  • Tavano S
  • Neuron
  • 2018 Mar 21

Literature context:


Abstract:

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Funding information:
  • Intramural NIH HHS - ZIA BC010763-05(United States)

Nek9 Phosphorylation Defines a New Role for TPX2 in Eg5-Dependent Centrosome Separation before Nuclear Envelope Breakdown.

  • Eibes S
  • Curr. Biol.
  • 2018 Jan 8

Literature context:


Abstract:

Centrosomes [1, 2] play a central role during spindle assembly in most animal cells [3]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4, 5], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7, 8]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10-17]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19] and the kinase Nek9 [20], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system.

Funding information:
  • NIGMS NIH HHS - R01 GM041804(United States)

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

  • Clift D
  • Cell
  • 2017 Dec 14

Literature context:


Abstract:

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Funding information:
  • NIDCD NIH HHS - P30 DC04657(United States)

Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response.

  • Fok KL
  • Endocrinology
  • 2017 Nov 1

Literature context:


Abstract:

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells.

  • Wang JT
  • Elife
  • 2017 Sep 14

Literature context:


Abstract:

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

Funding information:
  • NIAID NIH HHS - R01 AI038382(United States)
  • NIGMS NIH HHS - F32 GM117678()
  • NIGMS NIH HHS - R01 GM052022()

Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis.

  • Krishnan S
  • Mol. Cell
  • 2017 Aug 17

Literature context:


Abstract:

Precise control of sister chromatid separation during mitosis is pivotal to maintaining genomic integrity. Yet, the regulatory mechanisms involved are not well understood. Remarkably, we discovered that linker histone H1 phosphorylated at S/T18 decorated the inter-chromatid axial DNA on mitotic chromosomes. Sister chromatid resolution during mitosis required the eviction of such H1S/T18ph by the chaperone SET, with this process being independent of and most likely downstream of arm-cohesin dissociation. SET also directed the disassembly of Shugoshins in a polo-like kinase 1-augmented manner, aiding centromere resolution. SET ablation compromised mitotic fidelity as evidenced by unresolved sister chromatids with marked accumulation of H1S/T18ph and centromeric Shugoshin. Thus, chaperone-assisted eviction of linker histones and Shugoshins is a fundamental step in mammalian mitotic progression. Our findings also elucidate the functional implications of the decades-old observation of mitotic linker histone phosphorylation, serving as a paradigm to explore the role of linker histones in bio-signaling processes.

Funding information:
  • Howard Hughes Medical Institute - R01 GM064844()
  • NCI NIH HHS - P30 CA016087()
  • NCI NIH HHS - R01 CA199652()
  • NIGMS NIH HHS - S10 RR023704()

An RNAi Screen in a Novel Model of Oriented Divisions Identifies the Actin-Capping Protein Z β as an Essential Regulator of Spindle Orientation.

  • di Pietro F
  • Curr. Biol.
  • 2017 Aug 21

Literature context:


Abstract:

Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. We developed a new cellular model of oriented cell divisions combining micropatterning and localized recruitment of Gαi and performed an RNAi screen for regulators acting downstream of Gαi. Remarkably, this screen revealed a unique subset of dynein regulators as being essential for spindle orientation, shedding light on a core regulatory aspect of oriented divisions. We further analyze the involvement of one novel regulator, the actin-capping protein CAPZB. Mechanistically, we show that CAPZB controls spindle orientation independently of its classical role in the actin cytoskeleton by regulating the assembly, stability, and motor activity of the dynein/dynactin complex at the cell cortex, as well as the dynamics of mitotic microtubules. Finally, we show that CAPZB controls planar divisions in vivo in the developing neuroepithelium. This demonstrates the power of this in cellulo model of oriented cell divisions to uncover new genes required in spindle orientation in vertebrates.

Persistent KSHV Infection Increases EBV-Associated Tumor Formation In Vivo via Enhanced EBV Lytic Gene Expression.

  • McHugh D
  • Cell Host Microbe
  • 2017 Jul 12

Literature context:


Abstract:

The human tumor viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) establish persistent infections in B cells. KSHV is linked to primary effusion lymphoma (PEL), and 90% of PELs also contain EBV. Studies on persistent KSHV infection in vivo and the role of EBV co-infection in PEL development have been hampered by the absence of small animal models. We developed mice reconstituted with human immune system components as a model for KSHV infection and find that EBV/KSHV dual infection enhanced KSHV persistence and tumorigenesis. Dual-infected cells displayed a plasma cell-like gene expression pattern similar to PELs. KSHV persisted in EBV-transformed B cells and was associated with lytic EBV gene expression, resulting in increased tumor formation. Evidence of elevated lytic EBV replication was also found in EBV/KSHV dually infected lymphoproliferative disorders in humans. Our data suggest that KSHV augments EBV-associated tumorigenesis via stimulation of lytic EBV replication.

Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy.

  • Joachim J
  • Curr. Biol.
  • 2017 Jul 24

Literature context:


Abstract:

Autophagy maintains cellular health and homeostasis during stress by delivering cytosolic material captured by autophagosomes to lysosomes for degradation. Autophagosome formation is complex: initiated by the recruitment of autophagy (Atg) proteins to the formation site, it is sustained by activation of Atg proteins to allow growth and closure of the autophagosome. How Atg proteins are translocated to the forming autophagosome is not fully understood. Transport of the ATG8 family member GABARAP from the centrosome occurs during starvation-induced autophagosome biogenesis, but how centrosomal proteins regulate GABARAP localization is unknown. We show that the centriolar satellite protein PCM1 regulates the recruitment of GABARAP to the pericentriolar material. In addition to residing on the pericentriolar material, GABARAP marks a subtype of PCM1-positive centriolar satellites. GABARAP, but not another ATG8 family member LC3B, binds directly to PCM1 through a canonical LIR motif. Loss of PCM1 results in destabilization of GABARAP, but not LC3B, through proteasomal degradation. GABARAP instability is mediated through the centriolar satellite E3 ligase Mib1, which interacts with GABARAP through its substrate-binding region and promotes K48-linked ubiquitination of GABARAP. Ubiquitination of GABARAP occurs in the N terminus, a domain associated with ATG8-family-specific functions during autophagosome formation, on residues absent in the LC3 family. Furthermore, PCM1-GABARAP-positive centriolar satellites colocalize with forming autophagosomes. PCM1 enhances GABARAP/WIPI2/p62-positive autophagosome formation and flux but has no significant effect on LC3B-positive autophagosome formation. These data suggest a mechanism for how centriolar satellites can specifically regulate an ATG8 ortholog, the centrosomal GABARAP reservoir, and centrosome-autophagosome crosstalk.

Funding information:
  • NIGMS NIH HHS - R01 HD069647()
  • Wellcome Trust - R01 GM120776()

NBS1 Phosphorylation Status Dictates Repair Choice of Dysfunctional Telomeres.

  • Rai R
  • Mol. Cell
  • 2017 Mar 2

Literature context:


Abstract:

Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2TRFH domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.

Funding information:
  • NCI NIH HHS - P30 CA016359()
  • NIA NIH HHS - R01 AG028888()

Delayed apoptosis allows islet β-cells to implement an autophagic mechanism to promote cell survival.

  • Hayes HL
  • PLoS ONE
  • 2017 Feb 17

Literature context:


Abstract:

Increased β-cell death coupled with the inability to replicate existing β-cells drives the decline in β-cell mass observed in the progression of both major forms of diabetes. Understanding endogenous mechanisms of islet cell survival could have considerable value for the development of novel strategies to limit β-cell loss and thereby promote β-cell recovery. Insulinoma cells have provided useful insight into β-cell death pathways but observations made in cell lines sometimes fail to translate to primary islets. Here, we report dramatic differences in the temporal regulation and engagement of the apoptotic program in primary rodent islets relative to the INS-1 derived 832/13 cell line. As expected, 832/13 cells rapidly induced cell stress markers in response to ER stress or DNA damage and were fully committed to apoptosis, resulting in >80% cell death within 24 h. In contrast, primary rat islets were largely refractory to cell death in response to ER stress and DNA damage, despite rapid induction of stress markers, such as XBP-1(s), CHOP, and PUMA. Gene expression profiling revealed a general suppression of pro-apoptotic machinery, such as Apaf-1 and caspase 3, and sustained levels of pro-survival factors, such as cIAP-1, cIAP-2, and XIAP, in rat islets. Furthermore, we observed sustained induction of autophagy following chronic ER stress and found that inhibition of autophagy rendered islet β-cells highly vulnerable to ER stress-induced cell death. We propose that islet β-cells dampen the apoptotic response to delay the onset of cell death, providing a temporal window in which autophagy can be activated to limit cellular damage and promote survival.

Differential Routing of Mindbomb1 via Centriolar Satellites Regulates Asymmetric Divisions of Neural Progenitors.

  • Tozer S
  • Neuron
  • 2017 Feb 8

Literature context:


Abstract:

Unequal centrosome maturation correlates with asymmetric division in multiple cell types. Nevertheless, centrosomal fate determinants have yet to be identified. Here, we show that the Notch pathway regulator Mindbomb1 co-localizes asymmetrically with centriolar satellite proteins PCM1 and AZI1 at the daughter centriole in interphase. Remarkably, while PCM1 and AZI1 remain asymmetric during mitosis, Mindbomb1 is associated with either one or both spindle poles. Asymmetric Mindbomb1 correlates with neurogenic divisions and Mindbomb1 is inherited by the prospective neuron. By contrast, in proliferative divisions, a supplementary pool of Mindbomb1 associated with the Golgi apparatus in interphase is released during mitosis and compensates for Mindbomb1 centrosomal asymmetry. Finally, we show that preventing Mindbomb1 centrosomal association induces reciprocal Notch activation between sister cells and promotes symmetric divisions. Thus, we uncover a link between differential centrosome maturation and Notch signaling and reveal an unexpected compensatory mechanism involving the Golgi apparatus in restoring symmetry in proliferative divisions.

Time-Specific Effects of Spindle Positioning on Embryonic Progenitor Pool Composition and Adult Neural Stem Cell Seeding.

  • Falk S
  • Neuron
  • 2017 Feb 22

Literature context:


Abstract:

The developmental mechanisms regulating the number of adult neural stem cells (aNSCs) are largely unknown. Here we show that the cleavage plane orientation in murine embryonic radial glia cells (RGCs) regulates the number of aNSCs in the lateral ganglionic eminence (LGE). Randomizing spindle orientation in RGCs by overexpression of Insc or a dominant-negative form of Lgn (dnLgn) reduces the frequency of self-renewing asymmetric divisions while favoring symmetric divisions generating two SNPs. Importantly, these changes during embryonic development result in reduced seeding of aNSCs. Interestingly, no effects on aNSC numbers were observed when Insc was overexpressed in postnatal RGCs or aNSCs. These data suggest a new mechanism for controlling aNSC numbers and show that the role of spindle orientation during brain development is highly time and region dependent.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

  • Kuschal C
  • Am. J. Hum. Genet.
  • 2016 Apr 7

Literature context:


Abstract:

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

Funding information:
  • NINDS NIH HHS - R01 NS023945(United States)

Registered report: Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis.

  • Fiering S
  • Elife
  • 2015 Jul 16

Literature context:


Abstract:

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replicating selected results from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012 were selected on the basis of citations and Altimetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis' by Goetz and colleagues, published in Cell in 2011 (Goetz et al., 2011). The key experiments being replicated are those reported in Figures 7C (a-d), Supplemental Figure S2A, and Supplemental Figure S7C (a-c) (Goetz et al., 2011). In these experiments, which are a subset of all the experiments reported in the original publication, Goetz and colleagues show in a subcutaneous xenograft model that stromal caveolin-1 remodels the intratumoral microenvironment, which is correlated with increased metastasis formation. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Funding information:
  • NIDCR NIH HHS - R03 DE025824(United States)

Basal bodies exhibit polarized positioning in zebrafish cone photoreceptors.

  • Ramsey M
  • J. Comp. Neurol.
  • 2013 Jun 1

Literature context:


Abstract:

The asymmetric positioning of basal bodies, and therefore cilia, is often critical for proper cilia function. This planar polarity is critical for motile cilia function but has not been extensively investigated for nonmotile cilia or for sensory cilia such as vertebrate photoreceptors. Zebrafish photoreceptors form an organized mosaic ideal for investigating cilia positioning. We report that, in the adult retina, the basal bodies of red-, green-, and blue-sensitive cone photoreceptors localized asymmetrically on the cell edge nearest the optic nerve. In contrast, no patterning was seen in the basal bodies of ultraviolet-sensitive cones or in rod photoreceptors. The asymmetric localization of basal bodies was consistent in all regions of the adult retina. Basal body patterning was unaffected in the cones of the XOPS-mCFP transgenic line, which lacks rod photoreceptors. Finally, the adult pattern was not seen in 7-days-postfertilization (dpf) larvae; basal bodies were randomly distributed in all the photoreceptor subtypes. These results establish the asymmetrical localization of basal bodies in red-, green-, and blue-sensitive cones in adult zebrafish retinas but not in larvae. This pattern suggests an active cellular mechanism regulated the positioning of basal bodies after the transition to the adult mosaic and that rods do not seem to be necessary for the patterning of cone basal bodies.

Patterns of expression of Bardet-Biedl syndrome proteins in the mammalian cochlea suggest noncentrosomal functions.

  • May-Simera HL
  • J. Comp. Neurol.
  • 2009 May 10

Literature context:


Abstract:

Bardet-Biedl syndrome is a heterogeneous disorder causing a spectrum of symptoms, including visual impairment, kidney disease, and hearing impairment. Evidence suggests that BBS gene mutations cause defective ciliogenesis and/or cilium dysfunction. Cochlear development is affected by BBS gene deletion, and adult Bbs6(-/-) and Bbs4(-/-) mice are hearing impaired. This study addresses BBS protein expression in the rodent cochlea, to gain a better understanding of its function in vivo. As predicted by in vitro studies, Bbs6 immunofluorescence was localized to the basal bodies of supporting cells and sensory hair cells prior to the onset of hearing. In adult tissue, Bbs6 expression persisted in afferent neurons, including within the dendrites that innervate hair cells, implicating Bbs6 in a sensory neuronal function. Bbs2, which interacts with Bbs6, was also localized to hair cell basal bodies and stereociliary bundles. Additionally, Bbs2 was expressed in supporting cells at their intercellular boundaries, in a spatiotemporal pattern mirroring the development of the microtubule network. Bbs4 localized to cilia and developing cytoplasmic microtubule arrays. Pcm-1, a microtubular protein that interacts with Bbs4 in vitro, showed a comparable expression. Depolymerization of microtubules in slice preparations of the living cochlea resulted in Bbs4 and Pcm-1 mislocalization. Pcm-1 was also mislocalized in Bbs4(-/-) mice. This suggests that Bbs4/Pcm-1 interactions may be important in microtubule-dependent cytoplasmic trafficking in vivo. In summary, our findings indicate that BBS proteins adopt a range of cellular distributions in vivo, not restricted to the centrosome or cilium, and so broaden the possible underlying pathomechanisms of the disease.