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RAT ANTI TUBULIN ALPHA antibody

RRID:AB_325003

Antibody ID

AB_325003

Target Antigen

TUBULIN ALPHA yeast, mammals, birds

Proper Citation

(Bio-Rad Cat# MCA77G, RRID:AB_325003)

Clonality

monoclonal antibody

Comments

manufacturer recommendations: IgG2a; IgG2a Western Blot; Immunofluorescence; ELISA; Immunohistochemistry - frozen; Radioimmunoassay; Immunohistochemistry; Immunoprecipitation; Immunohistology - Frozen, ELISA, Western Blotting, Immunoprecipitation, Radioimmunoassays, Immunofluorescence

Host Organism

rat

Vendor

Bio-Rad

A Sex Chromosome piRNA Promotes Robust Dosage Compensation and Sex Determination in C. elegans.

  • Tang W
  • Dev. Cell
  • 2018 Mar 26

Literature context:


Abstract:

In metazoans, Piwi-related Argonaute proteins engage piRNAs (Piwi-interacting small RNAs) to defend the genome against invasive nucleic acids, such as transposable elements. Yet many organisms-including worms and humans-express thousands of piRNAs that do not target transposons, suggesting that piRNA function extends beyond genome defense. Here, we show that the X chromosome-derived piRNA 21ux-1 downregulates XOL-1 (XO Lethal), a master regulator of X chromosome dosage compensation and sex determination in Caenorhabditis elegans. Mutations in 21ux-1 and several Piwi-pathway components sensitize hermaphrodites to dosage compensation and sex determination defects. We show that the piRNA pathway also targets xol-1 in C. briggsae, a nematode species related to C. elegans. Our findings reveal physiologically important piRNA-mRNA interactions, raising the possibility that piRNAs function broadly to ensure robust gene expression and germline development.

Funding information:
  • NIA NIH HHS - T32 AG021890(United States)

A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells.

  • Kleiner RE
  • Cell Chem Biol
  • 2018 Jan 18

Literature context:


Abstract:

Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics.

Funding information:
  • NIGMS NIH HHS - R01 GM098579()

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency.

  • Hurst CD
  • Cancer Cell
  • 2017 Nov 13

Literature context:


Abstract:

Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.

Hec1 Tail Phosphorylation Differentially Regulates Mammalian Kinetochore Coupling to Polymerizing and Depolymerizing Microtubules.

  • Long AF
  • Curr. Biol.
  • 2017 Jun 5

Literature context:


Abstract:

The kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1, 2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore moves along microtubules [3, 4]. Despite a growing understanding of the molecular components that mediate kinetochore binding [5-7], we do not know how kinetochores physically interact with polymerizing versus depolymerizing microtubule bundles, and whether they use the same mechanisms and regulation to do so. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8-11] in mammalian cells. Hec1's affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12-15], but its role at the interface with polymerizing versus depolymerizing microtubules remains unclear. Here we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1's role on polymerizing versus depolymerizing microtubules. We show that Hec1 tail phosphorylation tunes friction along polymerizing microtubules and yet does not compromise the kinetochore's ability to grip depolymerizing microtubules. Together, the data suggest that kinetochore regulation has differential effects on engagement with growing and shrinking microtubules. Through this mechanism, the kinetochore can modulate its grip on microtubules over mitosis and yet retain its ability to couple to microtubules powering chromosome movement.

Funding information:
  • NIGMS NIH HHS - DP2 GM119177()

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

  • Maciejowski J
  • Dev. Cell
  • 2017 Apr 24

Literature context:


Abstract:

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

Funding information:
  • NCI NIH HHS - K22 CA207458()
  • NCI NIH HHS - P30 CA008748()
  • NIGMS NIH HHS - R01 GM083988()
  • NIGMS NIH HHS - R01 GM094972()

The Apical Domain Is Required and Sufficient for the First Lineage Segregation in the Mouse Embryo.

  • Korotkevich E
  • Dev. Cell
  • 2017 Feb 6

Literature context:


Abstract:

Mammalian development begins with segregation of the extra-embryonic trophectoderm from the embryonic lineage in the blastocyst. While cell polarity and adhesion play key roles, the decisive cue driving this lineage segregation remains elusive. Here, to study symmetry breaking, we use a reduced system in which isolated blastomeres recapitulate the first lineage segregation. We find that in the 8-cell stage embryo, the apical domain recruits a spindle pole to ensure its differential distribution upon division. Daughter cells that inherit the apical domain adopt trophectoderm fate. However, the fate of apolar daughter cells depends on whether their position within the embryo facilitates apical domain formation by Cdh1-independent cell contact. Finally, we develop methods for transplanting apical domains and show that acquisition of this domain is not only required but also sufficient for the first lineage segregation. Thus, we provide mechanistic understanding that reconciles previous models for symmetry breaking in mouse development.

Development of neuromuscular organization in the ctenophore Pleurobrachia bachei.

  • Norekian TP
  • J. Comp. Neurol.
  • 2016 Jan 1

Literature context:


Abstract:

The phylogenetic position of the phylum Ctenophora and the nature of ctenphore nervous systems are highly debated topics in modern evolutionary biology. However, very little is known about the organization of ctenophore neural and muscular systems, and virtually nothing has been reported about their embryogenesis. Here we have characterized the neural and muscular development of the sea gooseberry, Pleurobrachia bachei, starting from the cleavage stages to posthatching larvae. Scanning electron microscopy and immunochemistry were used to describe the formation of the embryonic mouth, tentacles, combs, aboral organ, and putative sensory cells. The muscles started their specification at the end of the first day of Pleurobrachia development. In contrast, neurons appeared 2 days after myogenesis, just before the hatching of fully formed cydippid larvae. The first tubulin-immunoreactive neurons, a small group of four to six cells with neuronal processes, was initially recognized at the aboral pole during the third day of development. Surprisingly, this observed neurogenesis occurred after the emergence of distinct behavioral patterns in the embryos. Thus, the embryonic behavior associated with comb cilia beatings and initial muscle organization does not require morphologically defined neurons and their elongated neurites. This study provides the first description of neuromuscular development in the enigmatic ctenophores and establishes the foundation for future research using emerging genomic tools and resources.