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Goat Anti-Rabbit IgG (H+L) Highly Cross-adsorbed Antibody, Alexa Fluor ?? 647 Conjugated

RRID:AB_141775

Antibody ID

AB_141775

Target Antigen

Rabbit IgG (H+L) rabbit

Proper Citation

(Molecular Probes Cat# A-21245, RRID:AB_141775)

Clonality

unknown

Comments

Discontinued; This product offered by Molecular Probes (Invitrogen), now part of Thermo Fisher:

Host Organism

goat

Vendor

Molecular Probes

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)

Dynamic Architecture of DNA Repair Complexes and the Synaptonemal Complex at Sites of Meiotic Recombination.

  • Woglar A
  • Cell
  • 2018 Jun 14

Literature context:


Abstract:

Meiotic double-strand breaks (DSBs) are generated and repaired in a highly regulated manner to ensure formation of crossovers (COs) while also enabling efficient non-CO repair to restore genome integrity. We use structured-illumination microscopy to investigate the dynamic architecture of DSB repair complexes at meiotic recombination sites in relationship to the synaptonemal complex (SC). DSBs resected at both ends are converted into inter-homolog repair intermediates harboring two populations of BLM helicase and RPA, flanking a single population of MutSγ. These intermediates accumulate until late pachytene, when repair proteins disappear from non-CO sites and CO-designated sites become enveloped by SC-central region proteins, acquire a second MutSγ population, and lose RPA. These and other data suggest that the SC may protect CO intermediates from being dismantled inappropriately and promote CO maturation by generating a transient CO-specific repair compartment, thereby enabling differential timing and outcome of repair at CO and non-CO sites.

Funding information:
  • NCI NIH HHS - P30 CA016672(United States)
  • NIGMS NIH HHS - R01 GM053804()
  • NIGMS NIH HHS - R01 GM067268()

Arginine Methylation by PRMT2 Controls the Functions of the Actin Nucleator Cobl.

  • Hou W
  • Dev. Cell
  • 2018 Apr 23

Literature context:


Abstract:

The complex architecture of neuronal networks in the brain requires tight control of the actin cytoskeleton. The actin nucleator Cobl is critical for neuronal morphogenesis. Here we reveal that Cobl is controlled by arginine methylation. Coprecipitations, coimmunoprecipitations, cellular reconstitutions, and in vitro reconstitutions demonstrated that Cobl associates with the protein arginine methyltransferase PRMT2 in a Src Homology 3 (SH3) domain-dependent manner and that this promotes methylation of Cobl's actin nucleating C-terminal domain. Consistently, PRMT2 phenocopied Cobl functions in both gain- and loss-of-function studies. Both PRMT2- and Cobl-promoted dendritogenesis relied on methylation. PRMT2 effects require both its catalytic domain and SH3 domain. Cobl-mediated dendritic arborization required PRMT2, complex formation with PRMT2, and PRMT2's catalytic activity. Mechanistic studies reveal that Cobl methylation is key for Cobl actin binding. Therefore, arginine methylation is a regulatory mechanism reaching beyond controlling nuclear processes. It also controls a major, cytosolic, cytoskeletal component shaping neuronal cells.

Funding information:
  • NHLBI NIH HHS - HL24415(United States)

HCN2 channels in the ventral tegmental area regulate behavioral responses to chronic stress.

  • Zhong P
  • Elife
  • 2018 Jan 2

Literature context:


Abstract:

Dopamine neurons in the ventral tegmental area (VTA) are powerful regulators of depression-related behavior. Dopamine neuron activity is altered in chronic stress-based models of depression, but the underlying mechanisms remain incompletely understood. Here, we show that mice subject to chronic mild unpredictable stress (CMS) exhibit anxiety- and depressive-like behavior, which was associated with decreased VTA dopamine neuron firing in vivo and ex vivo. Dopamine neuron firing is governed by voltage-gated ion channels, in particular hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Following CMS, HCN-mediated currents were decreased in nucleus accumbens-projecting VTA dopamine neurons. Furthermore, shRNA-mediated HCN2 knockdown in the VTA was sufficient to recapitulate CMS-induced depressive- and anxiety-like behavior in stress-naïve mice, whereas VTA HCN2 overexpression largely prevented CMS-induced behavioral deficits. Together, these results reveal a critical role for HCN2 in regulating VTA dopamine neuronal activity and depressive-related behaviors.

Funding information:
  • NICHD NIH HHS - P01 HD042137(United States)
  • NIDA NIH HHS - R01 DA035217()
  • NIGMS NIH HHS - T32 GM080202()
  • NIMH NIH HHS - F30 MH115536()
  • NIMH NIH HHS - R56 MH101146()

Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination.

  • Seemann E
  • Elife
  • 2017 Dec 5

Literature context:


Abstract:

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

Funding information:
  • NHLBI NIH HHS - HL095590(United States)

A Split-Abl Kinase for Direct Activation in Cells.

  • Diaz JE
  • Cell Chem Biol
  • 2017 Oct 19

Literature context:


Abstract:

To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase (sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N lobe. This split site leads to complete loss of activity, which can be restored upon small-molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold induction of phosphotyrosine (pTyr) modification, we employed quantitative proteomics, NeuCode SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe that this split-kinase technology will be useful for direct activation of protein kinases in cells.

Funding information:
  • NCI NIH HHS - R01 CA191018()
  • NIGMS NIH HHS - F32 GM089082()
  • NIGMS NIH HHS - P41 GM108538()
  • NIGMS NIH HHS - R01 GM097316()
  • NLM NIH HHS - T15 LM007359()

Arc Interacts with the Integral Endoplasmic Reticulum Protein, Calnexin.

  • Myrum C
  • Front Cell Neurosci
  • 2017 Oct 5

Literature context:


Abstract:

Activity-regulated cytoskeleton-associated protein, Arc, is a major regulator of long-term synaptic plasticity and memory formation. Here we reveal a novel interaction partner of Arc, a resident endoplasmic reticulum transmembrane protein, calnexin. We show an interaction between recombinantly-expressed GST-tagged Arc and endogenous calnexin in HEK293, SH-SY5Y neuroblastoma and PC12 cells. The interaction was dependent on the central linker region of the Arc protein that is also required for endocytosis of AMPA-type glutamate receptors. High-resolution proximity-ligation assays (PLAs) demonstrate molecular proximity of endogenous Arc with the cytosolic C-terminus, but not the lumenal N-terminus of calnexin. In hippocampal neuronal cultures treated with brain-derived neurotrophic factor (BDNF), Arc interacted with calnexin in the perinuclear cytoplasm and dendritic shaft. Arc also interacted with C-terminal calnexin in the adult rat dentate gyrus (DG). After induction of long-term potentiation (LTP) in the perforant path projection to the DG of adult anesthetized rats, enhanced interaction between Arc and calnexin was obtained in the dentate granule cell layer (GCL). Although Arc and calnexin are both implicated in the regulation of receptor endocytosis, no modulation of endocytosis was detected in transferrin uptake assays. Previous work showed that Arc interacts with multiple protein partners to regulate synaptic transmission and nuclear signaling. The identification of calnexin as a binding partner further supports the role of Arc as a hub protein and extends the range of Arc function to the endoplasmic reticulum, though the function of the Arc/calnexin interaction remains to be defined.

Funding information:
  • NIA NIH HHS - AG15556(United States)

Single-Cell Reconstruction of Oxytocinergic Neurons Reveals Separate Hypophysiotropic and Encephalotropic Subtypes in Larval Zebrafish.

  • Herget U
  • eNeuro
  • 2017 Oct 31

Literature context:


Abstract:

Oxytocin regulates a diverse set of processes including stress, analgesia, metabolism, and social behavior. How such diverse functions are mediated by a single hormonal system is not well understood. Different functions of oxytocin could be mediated by distinct cell groups, yet it is currently unknown whether different oxytocinergic cell types exist that specifically mediate peripheral neuroendocrine or various central neuromodulatory processes via dedicated pathways. Using the Brainbow technique to map the morphology and projections of individual oxytocinergic cells in the larval zebrafish brain, we report here the existence of two main types of oxytocinergic cells: those that innervate the pituitary and those that innervate diverse brain regions. Similar to the situation in the adult rat and the adult midshipman, but in contrast to the situation in the adult trout, these two cell types are mutually exclusive and can be distinguished based on morphological and anatomical criteria. Further, our results reveal that complex oxytocinergic innervation patterns are already established in the larval zebrafish brain.

Brain micro-inflammation at specific vessels dysregulates organ-homeostasis via the activation of a new neural circuit.

  • Arima Y
  • Elife
  • 2017 Aug 15

Literature context:


Abstract:

Impact of stress on diseases including gastrointestinal failure is well-known, but molecular mechanism is not understood. Here we show underlying molecular mechanism using EAE mice. Under stress conditions, EAE caused severe gastrointestinal failure with high-mortality. Mechanistically, autoreactive-pathogenic CD4+ T cells accumulated at specific vessels of boundary area of third-ventricle, thalamus, and dentate-gyrus to establish brain micro-inflammation via stress-gateway reflex. Importantly, induction of brain micro-inflammation at specific vessels by cytokine injection was sufficient to establish fatal gastrointestinal failure. Resulting micro-inflammation activated new neural pathway including neurons in paraventricular-nucleus, dorsomedial-nucleus-of-hypothalamus, and also vagal neurons to cause fatal gastrointestinal failure. Suppression of the brain micro-inflammation or blockage of these neural pathways inhibited the gastrointestinal failure. These results demonstrate direct link between brain micro-inflammation and fatal gastrointestinal disease via establishment of a new neural pathway under stress. They further suggest that brain micro-inflammation around specific vessels could be switch to activate new neural pathway(s) to regulate organ homeostasis.

Analyses of a Mutant Foxp3 Allele Reveal BATF as a Critical Transcription Factor in the Differentiation and Accumulation of Tissue Regulatory T Cells.

  • Hayatsu N
  • Immunity
  • 2017 Aug 15

Literature context:


Abstract:

Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate.

Concerted action of neuroepithelial basal shrinkage and active epithelial migration ensures efficient optic cup morphogenesis.

  • Sidhaye J
  • Elife
  • 2017 Apr 4

Literature context:


Abstract:

Organ formation is a multi-scale event that involves changes at the intracellular, cellular and tissue level. Organogenesis often starts with the formation of characteristically shaped organ precursors. However, the cellular mechanisms driving organ precursor formation are often not clear. Here, using zebrafish, we investigate the epithelial rearrangements responsible for the development of the hemispherical retinal neuroepithelium (RNE), a part of the optic cup. We show that in addition to basal shrinkage of RNE cells, active migration of connected epithelial cells into the RNE is a crucial player in its formation. This cellular movement is driven by progressive cell-matrix contacts and actively translocates prospective RNE cells to their correct location before they adopt neuroepithelial fate. Failure of this migration during neuroepithelium formation leads to ectopic determination of RNE cells and consequently impairs optic cup formation. Overall, this study illustrates how spatiotemporal coordination between morphogenic movements and fate determination critically influences organogenesis.

Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids.

  • Gabriel E
  • Cell Stem Cell
  • 2017 Mar 2

Literature context:


Abstract:

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.

Astrocytes Control Circadian Timekeeping in the Suprachiasmatic Nucleus via Glutamatergic Signaling.

  • Brancaccio M
  • Neuron
  • 2017 Mar 22

Literature context:


Abstract:

The suprachiasmatic nucleus (SCN) of the hypothalamus orchestrates daily rhythms of physiology and behavior in mammals. Its circadian (∼24 hr) oscillations of gene expression and electrical activity are generated intrinsically and can persist indefinitely in temporal isolation. This robust and resilient timekeeping is generally regarded as a product of the intrinsic connectivity of its neurons. Here we show that neurons constitute only one "half" of the SCN clock, the one metabolically active during circadian daytime. In contrast, SCN astrocytes are active during circadian nighttime, when they suppress the activity of SCN neurons by regulating extracellular glutamate levels. This glutamatergic gliotransmission is sensed by neurons of the dorsal SCN via specific pre-synaptic NMDA receptor assemblies containing NR2C subunits. Remarkably, somatic genetic re-programming of intracellular clocks in SCN astrocytes was capable of remodeling circadian behavioral rhythms in adult mice. Thus, SCN circuit-level timekeeping arises from interdependent and mutually supportive astrocytic-neuronal signaling.

The PERK arm of the unfolded protein response regulates satellite cell-mediated skeletal muscle regeneration.

  • Xiong G
  • Elife
  • 2017 Mar 23

Literature context:


Abstract:

Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.

Funding information:
  • NIA NIH HHS - R01 AG029623()
  • NIAMS NIH HHS - R01 AR059810()
  • NIAMS NIH HHS - R01 AR068313()

Recurrent Inhibition to the Medial Nucleus of the Trapezoid Body in the Mongolian Gerbil (Meriones Unguiculatus).

  • Dondzillo A
  • PLoS ONE
  • 2016 Aug 5

Literature context:


Abstract:

Principal neurons in the medial nucleus of the trapezoid body (MNTB) receive strong and temporally precise excitatory input from globular bushy cells in the cochlear nucleus through the calyx of Held. The extremely large synaptic currents produced by the calyx have sometimes led to the view of the MNTB as a simple relay synapse which converts incoming excitation to outgoing inhibition. However, electrophysiological and anatomical studies have shown the additional presence of inhibitory glycinergic currents that are large enough to suppress action potentials in MNTB neurons at least in some cases. The source(s) of glycinergic inhibition to MNTB are not fully understood. One major extrinsic source of glycinergic inhibitory input to MNTB is the ventral nucleus of the trapezoid body. However, it has been suggested that MNTB neurons receive additional inhibitory inputs via intrinsic connections (collaterals of glycinergic projections of MNTB neurons). While several authors have postulated their presence, these collaterals have never been examined in detail. Here we test the hypothesis that collaterals of MNTB principal cells provide glycinergic inhibition to the MNTB. We injected dye into single principal neurons in the MNTB, traced their projections, and immunohistochemically identified their synapses. We found that collaterals terminate within the MNTB and provide an additional source of inhibition to other principal cells, creating an inhibitory microcircuit within the MNTB. Only about a quarter to a third of MNTB neurons receive such collateral inputs. This microcircuit could produce side band inhibition and enhance frequency tuning of MNTB neurons, consistent with physiological observations.

The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

  • Larson ED
  • J. Neurosci.
  • 2015 Dec 2

Literature context:


Abstract:

Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response.

DYRK1A-mediated phosphorylation of GluN2A at Ser(1048) regulates the surface expression and channel activity of GluN1/GluN2A receptors.

  • Grau C
  • Front Cell Neurosci
  • 2014 Nov 4

Literature context:


Abstract:

N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser(1048) of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser(1048) hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser(1048) increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.