scholarly journals Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

1990 ◽  
Vol 111 (1) ◽  
pp. 113-122 ◽  
Author(s):  
D. R. Webster
Keyword(s):  
Alpha Tubulin ◽  
Cell Biol

scholarly journals An investigation of microtubule organization and functions in living Drosophila embryos by injection of a fluorescently labeled antibody against tyrosinated alpha-tubulin.

1987 ◽  
Vol 105 (4) ◽  
pp. 1721-1730 ◽  
Author(s):  
R M Warn ◽  
L Flegg ◽  
A Warn
Keyword(s):  
Surface Layer ◽  
Monoclonal Antibodies ◽  
Normal Development ◽  
Antibody Concentration ◽  
Microtubule Organization ◽  
Alpha Tubulin ◽  
Cell Biol ◽  
Centrosome Separation ◽  
Drosophila Embryos

Rhodamine-labeled monoclonal antibodies, which react with tyrosinated alpha-tubulin (clone YL 1/2; Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) and label microtubules in vivo (Wehland, J., M. C. Willingham, and I. Sandoval, 1983, J. Cell Biol., 97:1467-1475) were microinjected into syncytial stage Drosophila embryos. At 1 mg/ml antibody concentration, the microtubule arrays of the surface caps became labeled by YL 1/2 but normal development was found to continue. The results are compared with the data from fixed material particularly with regard to interphase microtubules, centrosome separation, and spindle and midbody formation. At 5 mg/ml antibody concentration the microtubules took up larger quantities of antibodies and clumped around the nuclei. Nuclei with clumped microtubules lost their position in the surface layer and moved into the interior. As a result, the F-actin cap meshwork associated with such nuclei either failed to form or subsided. It is concluded that microtubule activity is required to maintain the nuclei in the surface layer and organize the F-actin meshwork of the caps.

scholarly journals Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.

1984 ◽  
Vol 98 (3) ◽  
pp. 1017-1025 ◽  
Author(s):  
W C Thompson ◽  
D J Asai ◽  
D H Carney
Keyword(s):  
Differential Staining ◽  
Hypotonic Medium ◽  
Cytoplasmic Microtubule ◽  
Alpha Tubulin ◽  
Apparent Correlation ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
Fibroblastic Cells ◽  
In Cells

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.

Insertions of up to 17 amino acids into a region of alpha-tubulin do not disrupt function in vivo

1987 ◽  
Vol 7 (10) ◽  
pp. 3799-3805
Author(s):  
P J Schatz ◽  
G E Georges ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Amino Acids ◽  
Variable Region ◽  
Yeast Cells ◽  
Meiotic Spindle ◽  
Nuclear Movement ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Essential Components ◽  
Altered Proteins

Microtubules in yeasts are essential components of the mitotic and meiotic spindle and are necessary for nuclear movement during cell division and mating. The yeast Saccharomyces cerevisiae has two alpha-tubulin genes, TUB1 and TUB3, either of which alone is sufficient for these processes when present in a high enough copy number. Comparisons of sequences from several species reveals the presence of a variable region near the amino terminus of alpha-tubulin proteins. We perturbed the structure of this region in TUB3 by inserting into it 3, 9, or 17 amino acids and tested the ability of these altered proteins to function as the only alpha-tubulin protein in yeast cells. We found that each of these altered proteins was sufficient on its own for mitotic growth, mating, and methods of yeast. We conclude that this region can tolerate considerable variation without losing any of the highly conserved functions of alpha-tubulin. Our results suggest that variability in this region occurs because it can be tolerated, not because it specifies an important function for the protein.

scholarly journals A beacon in the cytoplasm: Tracking translation of single mRNAs

2016 ◽  
Vol 214 (6) ◽  
pp. 649-652 ◽  
Author(s):  
Hema V. Pingali ◽  
Angela K. Hilliker
Keyword(s):  
Real Time ◽  
New Technique ◽  
Real Time Monitoring ◽  
Protein Levels ◽  
Cell Biol ◽  
A New Technique ◽  
Control Protein

Translation is carefully regulated to control protein levels and allow quick responses to changes in the environment. Certain questions about translation in vivo have been unattainable until now. In this issue, Pichon et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201605024) describe a new technique to allow real-time monitoring of translation on single mRNAs.

Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin

1990 ◽  
Vol 10 (10) ◽  
pp. 5295-5304
Author(s):  
B Weinstein ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Data Support ◽  
Cell Biol

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.

scholarly journals XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover.

1994 ◽  
Vol 127 (4) ◽  
pp. 985-993 ◽  
Author(s):  
R J Vasquez ◽  
D L Gard ◽  
L Cassimeris
Keyword(s):  
Dynamic Instability ◽  
Fold Increase ◽  
Nucleation Site ◽  
Microtubule Assembly ◽  
Shortening Velocity ◽  
Threefold Increase ◽  
Cell Biol ◽  
Dic Microscopy

We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs.

scholarly journals Redox-sensitive homodimerization of Pex11p: a proposed mechanism to regulate peroxisomal division.

1996 ◽  
Vol 135 (1) ◽  
pp. 123-137 ◽  
Author(s):  
P A Marshall ◽  
J M Dyer ◽  
M E Quick ◽  
J M Goodman
Keyword(s):  
Oleic Acid ◽  
Active Species ◽  
Monomeric Form ◽  
Wild Type ◽  
Dimer Formation ◽  
Peroxisomal Membrane ◽  
Cell Biol ◽  
Organelle Division ◽  
Inner Surface

Pex11p (formerly Pmp27) has been implicated in peroxisomal proliferation (Erdmann, R., and G. Blobel. 1995. J. Cell Biol. 128; 509-523; Marshall, P.A., Y.I. Krimkevich, R.H. Lark, J.M. Dyer, M. Veenhuis, and J.M. Goodman, 1995. J. Cell Biol. 129; 345-355). In its absence, peroxisomes in Saccharomyces cerevisiae fail to proliferate in response to oleic acid; instead, one or two large peroxisomes are formed. Conversely, overproduction of Pex11p causes an increase in peroxisomal number. In this report, we confirm the function of Pex11p in organelle proliferation by demonstrating that this protein can cause fragmentation in vivo of large peroxisomes into smaller organelles. Pex11p is on the inner surface of the peroxisomal membrane. It can form homodimers, and this species is more abundant in mature peroxisomes than in proliferating organelles. Removing one of the three cysteines in the protein inhibits homodimerization. This cysteine 3-->alanine mutation leads to an increase in number and a decrease in peroxisomal density, compared with the wild-type protein, in response to oleic acid. We propose that the active species is the "monomeric" form, and that the increasing oxidative metabolism within maturing peroxisomes causes dimer formation and inhibition of further organelle division.

scholarly journals Temporal order of RNA-processing reactions in trypanosomes: rapid trans splicing precedes polyadenylation of newly synthesized tubulin transcripts.

1993 ◽  
Vol 13 (1) ◽  
pp. 720-725 ◽  
Author(s):  
E Ullu ◽  
K R Matthews ◽  
C Tschudi
Keyword(s):  
Rna Processing ◽  
Temporal Order ◽  
Kinetic Studies ◽  
Splice Acceptor ◽  
Mrna Synthesis ◽  
Alpha Tubulin ◽  
Spliced Leader ◽  
Polycistronic Transcription ◽  
Trans Splicing

Many trypanosome genes are expressed as part of large polycistronic transcription units. This finding suggests that regulation of mRNA biogenesis may emphasize RNA-processing reactions more so than in other organisms. This study was undertaken to understand the temporal order of two RNA-processing reactions, trans splicing and polyadenylation, in the maturation of trypanosome mRNAs in vivo. Kinetic studies revealed rapid trans splicing of alpha-tubulin, beta-tubulin, and actin pre-mRNAs within 1 to 2 min after synthesis of the 3' splice site. Furthermore, following blockage of pre-mRNA synthesis, newly synthesized spliced leader RNA cannot be used for trans splicing, suggesting that trypanosomes do not accumulate substantial amounts of pre-mRNA which can provide splice acceptor sites. Thus, trans splicing is cotranscriptional. In addition, we show that trans splicing precedes polyadenylation in the processing of trypanosome tubulin pre-mRNAs.

Tubacin, An Inhibitor of HDAC6, Induces Apoptosis of Acute Lymphoblastic Leukemia Cells in Vitro and in Vivo through a Na+/K+ATPase-Dependent Pathway.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1923-1923 ◽  
Author(s):  
Agustin Rodriguez-Gonzalez ◽  
Tiffany Simms-Waldrip ◽  
Alan K. Ikeda ◽  
Tara Lin ◽  
Brett Lomenick ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Preliminary Data ◽  
Lymphoblastic Leukemia ◽  
Parp Cleavage ◽  
Sodium Glutamate ◽  
Alpha Tubulin ◽  
Acetylated Tubulin ◽  
Treat All

Abstract Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 25 to 30% of children will relapse. In adults, less than 30% of patients with ALL are cured. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying the effects of a small molecule compound known as tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6) resulting in increased acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. We found that treatment of both pre-B and T-ALL cell lines with tubacin inhibits growth at very low micromolar concentrations (Jurkat IC50=1μM, Loucy IC50=3μM, REH IC50=2μM, Nalm6 IC50=5μM). We also determined that there is a therapeutic window, since tubacin inhibits the growth of normal bone marrow progenitor cells in methylcellulose colony assays at 20μM and normal human lymphocytes cultured in IL-2 at an IC50 of 16μM. We next tested the effects of tubacin in vivo. SCID mice injected with pre-B ALL Nalm-6 cells were treated with tubacin intraperitoneally at 50 mg/kg/day. Preliminary data using bioluminescence imaging in SCID mouse models showed that tubacin inhibited leukemic progression in vivo. To understand the mechanism of tubacin in ALL cells, we examined both apoptosis and cell cycle regulation by PARP cleavage, activation of caspases, and propidium iodide staining with FACs analysis. Tubacin induced apoptosis of pre-B and T-ALL cells within 12 hours of treatment. There was no effect on cell cycle progression, Retinoblastoma protein phosphorylation, or p21 upregulation, which have been observed with other HDAC inhibitors. Unlike in myeloma cells, tubacin did not increase JunK/SAPK activation or accumulation of acetylated HSP90 in ALL cells. Tubacin treatment resulted in accumulation of acetylated alpha-tubulin after 1 hour and an increase in polyubiquitinated proteins after 7 hours. To address potential mechanisms of tubacin in ALL, we tested whether Na+/K+ ATPase could be contributing to apoptosis. Previous work has shown that treatment with L-glutamate dissociates the Na+/K+ ATPase complex from acetylated tubulin and restores ATPase enzymatic activity. We hypothesized that the accumulation of acetylated tubulin could potentially inhibit the activity of the cytosolic Na/K ATPase pump, which could be reversed by treatment with 1mM sodium glutamate. Preliminary data demonstrate that we can partially rescue the effects of tubacin on PARP cleavage with sodium glutamate. These results suggest that tubacin induces apoptosis through a novel pathway in ALL cells and provide rationale for targeting the aggresome pathway to treat ALL in the future.

scholarly journals The N-terminal domain of c-Myc associates with alpha-tubulin and microtubules in vivo and in vitro.

1995 ◽  
Vol 15 (9) ◽  
pp. 5188-5195 ◽  
Author(s):  
N Alexandrova ◽  
J Niklinski ◽  
V Bliskovsky ◽  
G A Otterson ◽  
M Blake ◽  
...  
Keyword(s):  
Potential Role ◽  
Eukaryotic Cells ◽  
Transactivation Domain ◽  
Microtubule Network ◽  
Intact Cells ◽  
Alpha Tubulin ◽  
Functional Roles ◽  
Detergent Extraction

The polymerization of alpha- and beta-tubulin into microtubules results in a complex network of microfibrils that have important structural and functional roles in all eukaryotic cells. In addition, microtubules can interact with a diverse family of polypeptides which are believed to directly promote the assembly of microtubules and to modulate their functional activity. We have demonstrated that the c-Myc oncoprotein interacts in vivo and in vitro with alpha-tubulin and with polymerized microtubules and have defined the binding site to the N-terminal region within the transactivation domain of c-Myc. In addition, we have shown that c-Myc colocalizes with microtubules and remains tightly bound to the microtubule network after detergent extraction of intact cells. These findings suggest a potential role for Myc-tubulin interaction in vivo.

Sign in / Sign up

Export Citation Format

Share Document