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T Stearns

Publications and source records attributed to T Stearns.

At least 37 records · Page 2Linked to original sources
PubMed1997

Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors.

The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

Amino Acid Sequence↗
PubMed1997

Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex.

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)-tubulin fusion protein to observe microtubules in living yeast cells. GFP-tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

Cell Division↗
PubMed1997

Synaptically coupled central nervous system neurons lack centrosomal gamma-tubulin.

In cycling cells, microtubule assembly is initiated at the centrosome and requires the centrosomal protein gamma-tubulin. Previously, it was reported that gamma-tubulin is present at the centrosome of cervical ganglion cells undergoing axonal growth, but not in the axons or dendrites. We find that although gamma-tubulin is present at the centrosomes of neurons just beginning to extend processes, it is not associated with centrosomes in hypothalamic and cortical neurons on which functional synaptic connections have formed. In contrast, another centrosomal protein, pericentrin, is associated with the centrosome at all stages. These results suggest that centrosomal microtubule nucleation is required for early stages of neurogenesis to supply sufficient microtubule polymer to support rapid axonal growth, but is not required for maintenance of axonal microtubules in synaptically coupled neurons.

Animals↗
PubMed1997

Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

Animals↗
PubMed1997

Centrosomal deployment of gamma-tubulin and pericentrin: evidence for a microtubule-nucleating domain and a minus-end docking domain in certain mouse epithelial cells.

This report provides evidence for two functionally and spatially distinct centrosomal domains in certain mouse cochlear epithelial cells. The vast majority of microtubules elongate from sites associated with the apical cell surface in these cells rather than from pericentriolar material surrounding the immediate environs of their apically situate centrioles. The distribution of gamma-tubulin and pericentrin at cell apices has been examined while microtubule nucleation is progressing because these centrosomal proteins are believed to be essential for microtubule nucleation. Antibodies to both proteins bind to pericentriolar regions but no binding has been detected at the apical cell surface-associated sites where the ends of thousands of recently nucleated microtubules are concentrated. Sparse transient microtubule populations can be detected between pericentriolar regions and surface sites while microtubule assembly advances. A procedure apparently operates in which the pericentriolar region functions as a microtubule-nucleating domain and the cell surface-associated sites operate as docking domains which capture the minus ends of microtubules that migrate to them shortly after nucleation. Docking domains may include some components of the pericentriolar material that have been relocated at the cell apex. A docking element hypothesis for centrosomal control of minus end positioning and dynamics in animal cells generally is proposed. This investigation has also shown that the concentration of gamma-tubulin and pericentrin around centrioles differs spatially and quantitatively in ways that are characteristic for the four cell types studied. Some of these characteristics can be related to differences in control of microtubule number and positioning.

Animals↗
PubMed1996

Cytoskeleton: microtubule nucleation takes shape.

The centrosomal protein gamma-tubulin is part of a ring-shaped complex that can induce microtubule polymerization. This complex may explain how the centrosome nucleates microtubule polymerization, and thereby organizes the microtubule cytoskeleton.

Animals↗
PubMed1996

Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.

gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

Animals↗
PubMed1995

Mutational analysis of Saccharomyces cerevisiae ARF1.

Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly2 or Asp26 resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp26 (homologous to Gly12 of p21ras) was essential for the binding of the activating nucleotide, guanosine 5'-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21ras or Gs alpha, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins. Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have approximately 300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.

ADP-Ribosylation Factor 1↗
PubMed1994

In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin.

The centrosome nucleates microtubule polymerization, affecting microtubule number, polarity, and structure. We use an in vitro system based on extracts of Xenopus eggs to examine the role of gamma-tubulin in centrosome assembly and function. gamma-Tubulin is present in the cytoplasm of frog eggs and vertebrate somatic cells in a large approximately 25S complex. The egg extracts assemble centrosomes around sperm centrioles. Formation of a centrosome in the extract requires both the gamma-tubulin complex and ATP and can take place in the absence of microtubules. gamma-Tubulin is not present on the sperm prior to incubation in extract, but is recruited from the cytoplasm during centrosome assembly. The gamma-tubulin complex also binds to microtubules, likely the minus end, independent of the centrosome. These results suggest that gamma-tubulin is an essential component of the link between the centrosome and the microtubule, probably playing a direct role in microtubule nucleation.

Animals↗
PubMed1993

Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4.

The essential Ras-related GTPases Ypt1 and Sec4 act at distinct stages of the secretion pathway in the yeast Saccharomyces cerevisiae: Ypt1 is required for vesicular transport from the endoplasmic reticulum to the Golgi apparatus, whereas Sec4 is required for fusion of secretory vesicles to the plasma membrane. Here we use chimaeras of the two proteins to identify a 9-residue segment of Ypt1 that, when substituted for the analogous segment of Sec4, allows the chimaera to perform the minimal functions of both proteins in vivo. This segment corresponds to loop L7 of the p21ras crystal structure. Substitution of a 24-residue Ypt1 segment, including the residues just mentioned, together with 12 residues of Ypt1 corresponding to the 'effector region' of p21ras (loop L2; refs 7,8), transforms Sec4 into a fully functional Ypt1 protein without residual Sec4 function.

Amino Acid Sequence↗
PubMed1991

Gamma-tubulin is a highly conserved component of the centrosome.

We have cloned and characterized gamma-tubulin genes from both X. laevis and S. pombe, and partial genes from maize, diatom, and a budding yeast. The proteins encoded by these genes are very similar to each other and to the original Aspergillus protein, indicating that gamma-tubulins are an ubiquitous and highly conserved subfamily of the tubulin family. A null mutation of the S. pombe gene is lethal. gamma-tubulin is a minor protein, present at less than 1% the level of alpha- and beta-tubulin, and is limited to the centrosome. In particular, gamma-tubulin is associated with the pericentriolar material, the microtubule-nucleating material of the centrosome. gamma-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it is an integral component that might play a role in microtubule organization.

Amino Acid Sequence↗
PubMed1990

Manipulating yeast genome using plasmid vectors.

The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.

Cloning, Molecular↗