Nucleocytoplasmic transport of Alp7/TACC organizes spatiotemporal microtubule formation in fission yeast

Ran GTPase activates several target molecules to induce microtubule formation around the chromosomes and centrosomes. In fission yeast, in which the nuclear envelope does not break down during mitosis, Ran targets the centrosomal transforming acidic coiled‐coil (TACC) protein Alp7 for spindle formation. Alp7 accumulates in the nucleus only during mitosis, although its underlying mechanism remains elusive. Here, we investigate the behaviour of Alp7 and its binding partner, Alp14/TOG, throughout the cell cycle. Interestingly, Alp7 enters the nucleus during interphase but is subsequently exported to the cytoplasm by the Exportin‐dependent nuclear export machinery. The continuous nuclear export of Alp7 during interphase is essential for maintaining the array‐like cytoplasmic microtubule structure. The mitosis‐specific nuclear accumulation of Alp7 seems to be under the control of cyclin‐dependent kinase (CDK). These results indicate that the spatiotemporal regulation of microtubule formation is established by the Alp7/TACC–Alp14/TOG complex through the coordinated interplay of Ran and CDK.     

Interdependency of fission yeast Alp14/TOG and coiled coil protein Alp7 in microtubule localization and bipolar spindle formation

The Dis1/TOG family plays a pivotal role in microtubule organization. In fission yeast, Alp14 and Dis1 share an essential function in bipolar spindle formation. Here, we characterize Alp7, a novel coiled-coil protein that is required for organization of bipolar spindles. Both Alp7 and Alp14 colocalize to the spindle pole body (SPB) and mitotic spindles. Alp14 localization to these sites is fully dependent upon Alp7. Conversely, in the absence of Alp14, Alp7 localizes to the SPBs, but not mitotic spindles. Alp7 forms a complex with Alp14, where the C-terminal region of Alp14 interacts with the coiled-coil domain of Alp7. Intriguingly, this Alp14 C terminus is necessary and sufficient for mitotic spindle localization. Overproduction of either full-length or coiled-coil region of Alp7 results in abnormal V-shaped spindles and stabilization of interphase microtubules, which is induced independent of Alp14. Alp7 may be a functional homologue of animal TACC. Our results shed light on an interdependent relationship between Alp14/TOG and Alp7. We propose a two-step model that accounts for the recruitment of Alp7 and Alp14 to the SPB and microtubules.     

CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.     

Spindle-kinetochore attachment requires the combined action of Kin I-like Klp5/6 and Alp14/Dis1-MAPs in fission yeast

Fission yeast Klp5 and Klp6 belong to the microtubule-destabilizing Kin I family. In klp5 mutants, spindle checkpoint proteins Mad2 and Bub1 are recruited to mitotic kinetochores for a prolonged duration, indicating that these kinetochores are unattached. Further analysis shows that there are kinetochores to which only Bub1, but not Mad2, localizes. These kinetochores are likely to have been captured, yet lack tension. Thus Klp5 and Klp6 play a role in a spindle- kinetochore interaction at dual steps, capture and generation of tension. The TOG/XMAP215 family, Alp14 and Dis1 are known to stabilize microtubules and be required for the bivalent attachment of the kinetochore to the spindle. Despite apparent opposing activities towards microtubule stability, Klp5/Klp6 and Alp14/Dis1 share an essential function, as either dis1klp or alp14klp mutants are synthetically lethal, like alp14dis1. Defective phenotypes are similar to each other, characteristic of attachment defects and chromosome mis-segregation. Furthermore Alp14 is of significance for kinetochore localization of Klp5. We propose that Klp5/Klp6 and Alp14/Dis1 play a collaborative role in bipolar spindle formation during prometaphase through producing spindle dynamism.     

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.     

Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint.

The TOG/XMAP215-related proteins play a role in microtubule dynamics at its plus end. Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1. We show that the alp14 mutant fails to progress towards normal bipolar spindle formation. Intriguingly, Alp14 itself is a component of the Mad2-dependent spindle checkpoint cascade, as upon addition of microtubule-destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation. Live imaging of Alp14-green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles. This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6. An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles. These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules.     

The fission yeast transforming acidic coiled coil-related protein Mia1p/Alp7p is required for formation and maintenance of persistent microtubule-organizing centers at the nuclear envelope

Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner. How MTOCs occur remains an exciting question in cell biology. Here, we show that the transforming acidic coiled coil–related protein Mia1p/Alp7p functions in emergence of large MTOCs in interphase fission yeast cells. We found that Mia1p was a microtubule-binding protein that preferentially localized to the minus ends of microtubules and was associated with the sites of microtubule attachment to the nuclear envelope. Cells lacking Mia1p exhibited less microtubule bundles. Microtubules could be nucleated and bundled but were frequently released from the nucleation sites in mia1Δ cells. Mia1p was required for stability of microtubule bundles and persistent use of nucleation sites both in interphase and postanaphase array dynamics. The γ-tubulin–rich material was not organized in large perinuclear or microtubule-associated structures in mia1Δ cells. Interestingly, absence of microtubules in dividing wild-type cells prevented appearance of large γ-tubulin–rich MTOC structures in daughters. When microtubule polymerization was allowed, MTOCs were efficiently assembled de novo. We propose a model where MTOC emergence is a self-organizing process requiring the continuous association of microtubules with nucleation sites.     

The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2-cyclinB1 complex). It has previously been demonstrated that the p34cdc2-cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215-cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.     

The Centrosomal Adaptor TACC3 and the Microtubule Polymerase chTOG Interact via Defined C-terminal Subdomains in an Aurora-A Kinase-independent Manner

The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414–530) and CC2 (aa 530–630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.     

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform.     

The Xenopus XMAP215 and Its Human Homologue TOG Proteins Interact with Cyclin B1 to Target p34cdc2 to Microtubules during Mitosis

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2–cyclinB1 complex). It has previously been demonstrated that the p34cdc2–cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215–cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.     

A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae

A Saccharomyces cerevisiae gamma-tubulin-related gene, TUB4, has been characterized. The predicted amino acid sequence of the Tub4 protein (Tub4p) is 29-38% identical to members of the gamma-tubulin family. Indirect immunofluorescence experiments using a strain containing an epitope-tagged Tub4p indicate that Tub4p resides at the spindle pole body throughout the yeast cell cycle. Deletion of the TUB4 gene indicates that Tub4p is essential for yeast cell growth. Tub4p-depleted cells arrest during nuclear division; most arrested cells contain a large bud, replicated DNA, and a single nucleus. Immunofluorescence and nuclear staining experiments indicate that cells depleted of Tub4p contain defects in the organization of both cytoplasmic and nuclear microtubule arrays; such cells exhibit nuclear migration failure, defects in spindle formation, and/or aberrantly long cytoplasmic microtubule arrays. These data indicate that the S. cerevisiae gamma- tubulin protein is an important SPB component that organizes both cytoplasmic and nuclear microtubule arrays.     

Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome

Members of the transforming acidic coiled coil (TACC) protein family are emerging as important mitotic spindle assembly proteins in a variety of organisms. The molecular details of how TACC proteins function are unknown, but TACC proteins have been proposed to recruit microtubule-stabilizing proteins of the tumor overexpressed gene (TOG) family to the centrosome and to facilitate their loading onto newly emerging microtubules. Using Xenopus egg extracts and in vitro assays, we show that the Xenopus TACC protein maskin is required for centrosome function beyond recruiting the Xenopus TOG protein XMAP215. The conserved C-terminal TACC domain of maskin is both necessary and sufficient to restore centrosome function in maskin-depleted extracts, and we provide evidence that the N terminus of maskin inhibits the function of the TACC domain. Time-lapse video microscopy reveals that microtubule dynamics in Xenopus egg extracts are unaffected by maskin depletion. Our results provide direct experimental evidence of a role for maskin in centrosome function and suggest that maskin is required for microtubule anchoring at the centrosome.     

The spindle pole bodies facilitate nuclear envelope division during closed mitosis in fission yeast

Many organisms divide chromosomes within the confines of the nuclear envelope (NE) in a process known as closed mitosis. Thus, they must ensure coordination between segregation of the genetic material and division of the NE itself. Although many years of work have led to a reasonably clear understanding of mitotic spindle function in chromosome segregation, the NE division mechanism remains obscure. Here, we show that fission yeast cells overexpressing the transforming acid coiled coil (TACC)-related protein, Mia1p/Alp7p, failed to separate the spindle pole bodies (SPBs) at the onset of mitosis, but could assemble acentrosomal bipolar and antiparallel spindle structures. Most of these cells arrested in anaphase with fully extended spindles and nonsegregated chromosomes. Spindle poles that lacked the SPBs did not lead the division of the NE during spindle elongation, but deformed it, trapping the chromosomes within. When the SPBs were severed by laser microsurgery in wild-type cells, we observed analogous deformations of the NE by elongating spindle remnants, resulting in NE division failure. Analysis of dis1Δ cells that elongate spindles despite unattached kinetochores indicated that the SPBs were required for maintaining nuclear shape at anaphase onset. Strikingly, when the NE was disassembled by utilizing a temperature-sensitive allele of the Ran GEF, Pim1p, the abnormal spindles induced by Mia1p overexpression were capable of segregating sister chromatids to daughter cells, suggesting that the failure to divide the NE prevents chromosome partitioning. Our results imply that the SPBs preclude deformation of the NE during spindle elongation and thus serve as specialized structures enabling nuclear division during closed mitosis in fission yeast.     

The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a approximately 200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to "rescue" experiments. Defects in the "early" step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the "late" step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.     

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.     

Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

Acomplex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3–clathrin binding. A dileucine motif and Aurora A–phosphorylated serine 558 on TACC3 bound to the “ankle” of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3–clathrin.     

The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.     

Hrs1p/Mcp6p on the meiotic SPB organizes astral microtubule arrays for oscillatory nuclear movement

Microtubules and the motor protein dynein play pivotal roles in the movement and positioning of the nucleus and cytoplasmic organelles in a cell. In fission yeast, oscillatory movement of the nucleus termed horsetail nuclear movement (HNM) has been observed during meiotic prophase. HNM is led by an astral microtubule array emanating from the spindle pole body (SPB), a centrosome-equivalent organelle in yeasts, aided by the dynein-dynactin complex, and is proposed to facilitate the alignment of homologous chromosomes necessary for efficient meiotic recombination. Here we show that a meiosis-specific SPB component Hrs1p (also known as Mcp6p) is a key molecule to remodel microtubules into the horsetail-astral array (HAA). Deletion of Hrs1p impaired HAA formation, leading to compromised HNM. Ectopic expression of Hrs1p during the mitotic cell cycle resulted in the formation of a HAA-like astral microtubule array, which drove an oscillatory nuclear movement in interphase cells. Hrs1p interacted with components of the gamma-tubulin ring complex (gamma-TuRC) as well as with a meiotic SPB component. We propose that Hrs1p facilitates formation of the HAA, responsible for the vigorous HNM, by stabilizing connection between the SPB and minus ends of microtubules.     

Protein complexes at the microtubule organizing center regulate bipolar spindle assembly

Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G1/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22ts, encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22ts we found that stabilized microtubules or increased bundling can rescue cut7-22ts associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on γ-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to γ-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/γ-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22ts strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.