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1.
We have created 41 clustered charged-to-alanine scanning mutations of the mipA, gamma-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of gamma-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that gamma-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of gamma-tubulin and these data give a good initial indication of the functionally important regions of the molecule.  相似文献   

2.
Y Zheng  M K Jung  B R Oakley 《Cell》1991,65(5):817-823
The mipA gene of A. nidulans encodes a newly discovered member of the tubulin superfamily of proteins, gamma-tubulin. In A. nidulans, gamma-tubulin is essential for nuclear division and microtubule assembly and is associated with the spindle pole body, the fungal microtubule organizing center. By low stringency hybridizations we have cloned cDNAs from D. melanogaster and H. sapiens, the predicted products of which share more than 66% amino acid identity with A. nidulans gamma-tubulin. gamma-Tubulin-specific antibodies stained centrosomes of Drosophila, human, and mouse cell lines. Staining was most intense in prophase through metaphase when microtubule assembly from centrosomes was maximal. These results demonstrate that gamma-tubulin genes are present and expressed in humans and flies; they suggest that gamma-tubulin may be a universal component of microtubule organizing centers; and they are consistent with an earlier hypothesis that gamma-tubulin is a minus-end nucleator of microtubule assembly.  相似文献   

3.
BACKGROUND: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase. RESULTS: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin. CONCLUSIONS: Mod20p appears to act by recruiting components of the gamma-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells.  相似文献   

4.
Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.  相似文献   

5.
《The Journal of cell biology》1990,111(6):2573-2586
BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.  相似文献   

6.
Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. gamma-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in gamma-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30 degrees C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant gamma-tubulin is like the wild-type protein. Prediction of gamma-tubulin structure indicates that non-alpha/beta-tubulin protein-protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the gamma-tubulin mutant and in multicopy for normal cell morphology at 30 degrees C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for gamma-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of gamma-tubulin that involves non-tubulin protein-protein interactions, presumably with a second motor, MAP, or MTOC component.  相似文献   

7.
Tubulins in Aspergillus nidulans   总被引:2,自引:0,他引:2  
The discovery and characterization of the tubulin superfamily in Aspergillus nidulans is described. Remarkably, the genes that encode alpha-, beta-, and gamma-tubulins were all identified first in A. nidulans. There are two alpha-tubulin genes, tubA and tubB, two beta-tubulin genes, benA and tubC, and one gamma-tubulin gene, mipA. Hyphal tubulin is encoded mainly by the essential genes tubA and benA. TubC is expressed during conidiation and tubB is required for the sexual cycle. Promoter swapping experiments indicate that the alpha-tubulins encoded by tubA and tubB are functionally interchangeable as are the beta-tubulins encoded by benA and tubC. BenA mutations that alter resistance to benzimidazole antimicrotubule agents are clustered and define a putative binding region for these compounds. gamma-Tubulin localizes to the spindle pole body and is essential for mitotic spindle formation. The phenotypes of mipA mutants suggest, moreover, that gamma-tubulin has essential functions in addition to microtubule nucleation.  相似文献   

8.
This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.  相似文献   

9.
NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.  相似文献   

10.
11.
The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the gamma-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.  相似文献   

12.
Emanuele MJ  Stukenberg PT 《Cell》2007,130(5):893-905
For chromosomes to congress and segregate during cell division, kinetochores must form stable attachments with spindle microtubules. We find that the centrosome protein, xCep57, localizes to kinetochores and interacts with the kinetochore proteins Zwint, Mis12, and CLIP-170. Immunodepletion of xCep57 from egg extracts yields weakened and elongated bipolar spindles which fail to align chromosomes. In the absence of xCep57, tension is lost between sister kinetochores, and spindle microtubules are no longer resistant to low doses of nocodazole. xCep57 inhibition on isolated mitotic chromosomes inhibits kinetochore-microtubule binding in vitro. xCep57 also interacts with gamma-tubulin. In xCep57 immunodepleted extracts, sperm centrosomes nucleate with normal kinetics, but are unable maintain microtubule anchorage. This characterization places xCep57 in a novel class of proteins required for stable microtubule attachments at the kinetochore and at the centrosome and suggests that the mechanism of microtubule binding at these two places is mechanistically similar.  相似文献   

13.
Winey M  Bloom K 《Genetics》2012,190(4):1197-1224
The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine.  相似文献   

14.
Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.  相似文献   

15.
16.
Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic gamma-tubulin complex, can only generate very short (<1 microm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However, SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.  相似文献   

17.
gamma-Tubulin is a member of the tubulin superfamily and plays essential roles in microtubule nucleation. While the level of other tubulins, alpha- and beta-tubulin, is strictly regulated in higher eukaryotes and overexpression of beta-tubulin is toxic in yeasts, gamma-tubulin can be overexpressed by fivefold in fission yeast without any obvious defect in growth. Extreme overexpression of gamma-tubulin in mammalian cells caused growth arrest; however, the exact level of gamma-tubulin and the critical level of gamma-tubulin necessary for growth defect were undetermined. We have constructed strains that over- or underexpress gamma-tubulin by placing the gamma-tubulin gene under the control of the inducible nmt1 promoter and its variants. Among these, the weakest promoter was able to produce enough gamma-tubulin to support normal growth when its expression was induced. A strain in which the gamma-tubulin gene was placed under the control of the strongest inducible promoter achieved 160-fold overexpression of gamma-tubulin and its growth was suppressed. Normal cytoplasmic microtubules were mostly lost in gamma-tubulin overexpressing cells and gamma-tubulin was accumulated around the periphery of nuclei. Many of the cells were arrested in mitosis. A small fraction of cells did proceed to undergo nuclear division; however, its process looked either significantly deterred or abnormal. Our results presented here suggest that excess gamma-tubulin disrupts the microtubule array and significantly deters the formation of the mitotic spindle, most likely because of random nucleation of microtubules from excess gamma-tubulin in the cytoplasm.  相似文献   

18.
To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell–nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell–nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) α-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) α-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of α-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell–nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell–nuclear division, mitochondrion separation and microbody division are dependent on microtubules.  相似文献   

19.
gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia.  相似文献   

20.
Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.  相似文献   

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