Γ-tubulin Complex Research Articles

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore.

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.

The XMAP215 Ortholog Alp14 Promotes Microtubule Nucleation in Fission Yeast

The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity invitro, it is not yet clear whether these proteins participate in MT nucleation invivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation invivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but notplus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor invivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT.

XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex.

How microtubules (MT) are generated in the cell is a major question in understanding how the cytoskeleton is assembled. For several decades, γ-tubulin has been accepted as the cell’s universal MT nucleator. Although there is evidence that γ-tubulin complexes are not the sole MT nucleators, identification of other nucleation factors has proven difficult. Here, we report that the well-characterized MT polymerase XMAP215 (chTOG/Msps/Stu2p/Alp14/Dis1 homologue) is essential for MT nucleation in Xenopus egg extracts. The concentration of XMAP215 determines the extent of MT nucleation. Even though XMAP215 and γ-tubulin ring complex (γ-TuRC) possess minimal nucleation activity individually, together these factors synergistically stimulate MT nucleation in vitro. The N-terminal TOG domains 1–5 of XMAP215 bind αβ-tubulin and promote MT polymerization, while the conserved C-terminus is required for efficient MT nucleation and directly binds γ-tubulin. In sum, XMAP215 and γ-TuRC together function as the principal nucleation module that generates MTs in cells.

Augmin Antagonizes Katanin at Microtubule Crossovers to Control the Dynamic Organization of Plant Cortical Arrays

Plant cells do not possess centrosomes, which serve as the microtubule organizing center in animal cells; how plant cell microtubule arrays are established and maintain their dynamics remain poorly understood [1]. The γ-tubulin complex and the katanin complex play central roles in the organization of plant cortical microtubules [2-6]. Previously, we reported that the augmin complex recruits the γ-tubulin complex to preexisting microtubules and initiates microtubule nucleation [7]. Moreover, we described how an intricate interaction between the katanin p60 subunit KTN1 and the p80 subunit KTN80 confers precise microtubule severing at either microtubule branching nucleation sites or crossovers [8]. Here, we observed that augmin preferentially localizes to microtubule crossovers. Live-cell observations and analyses revealed that, whereas a small portion of crossover-localized augmin complexes act to trigger nascent microtubule nucleation, the majority function in stabilizing the architecture of microtubule crossovers. Finally, genetic analyses and computational modeling confirmed that suppression of augmin activity elevates microtubule severing frequency and facilitates the formation of aligned microtubule arrays. Combined, our findings reveal an unexpected role of augmin and demonstrate that augmin antagonizes katanin-mediated microtubule severing. Furthermore, we propose a novel mechanism for how augmin determines self-organization of plant cortical microtubules by preventing microtubule severing at crossovers in addition to triggering microtubule nucleation.

Assembly and regulation of γ-tubulin complexes.

Microtubules are major constituents of the cytoskeleton in all eukaryotic cells. They are essential for chromosome segregation during cell division, for directional intracellular transport and for building specialized cellular structures such as cilia or flagella. Their assembly has to be controlled spatially and temporally. For this, the cell uses multiprotein complexes containing γ-tubulin. γ-Tubulin has been found in two different types of complexes, γ-tubulin small complexes and γ-tubulin ring complexes. Binding to adaptors and activator proteins transforms these complexes into structural templates that drive the nucleation of new microtubules in a highly controlled manner. This review discusses recent advances on the mechanisms of assembly, recruitment and activation of γ-tubulin complexes at microtubule-organizing centres.

γ-Tubulin has a conserved intrinsic property of self-polymerization into double stranded filaments and fibrillar networks

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.

The Mitotic Function of Augmin Is Dependent on Its Microtubule-Associated Protein Subunit EDE1 in Arabidopsis thaliana

The augmin complex plays an essential role in microtubule (MT)-dependent MT nucleation by recruiting the γ-tubulin complex to MT walls to generate new MTs [1]. The complex contains eight subunits (designated AUG) including AUG8, which is an MT-associated protein (MAP). When this complex is isolated from etiolated seedlings consisting of primarily interphase cells in Arabidopsis thaliana, AUG8 is an integral component [2]. EDE1 (Endosperm DEfective 1) is homologous to AUG8 [3]. Here, we demonstrate that EDE1, but not AUG8, is associated with acentrosomal spindle and phragmoplast MT arrays in patterns indistinguishable from those of the AUG1-7 subunits and the γ-tubulin complex proteins (GCPs) that exhibit biased localization toward MT minus ends. Consistent with this colocalization, EDE1 directly interacts with AUG6 invivo. Moreover, a partial loss-of-function mutation, ede1-1, compromises the localization of augmin and γ-tubulin on the spindle and phragmoplast MT arrays and leads to serious distortions in spindle MT remodeling during mitosis. However, mitosis continues even when kinetochore fibers are not obviously discernable, and cytokinesis takes place following the formation of elongated bipolar phragmoplast MT arrays in the mutant. Hence, we conclude that the mitotic function of augmin is dependent on its MAP subunit EDE1, which cannot be replaced by AUG8, and that the cell-cycle-dependent function of augmin can be differentially regulated by employing distinct MAP subunits. Our results also illustrate that plant cells can respond flexibly to serious challenges of compromised MT-dependent MT nucleation to complete mitosis and cytokinesis.

Spatial cues and not spindle pole maturation drive the asymmetry of astral microtubules between new and preexisting spindle poles.

In many asymmetrically dividing cells, the microtubule-organizing centers (MTOCs; mammalian centrosome and yeast spindle pole body [SPB]) nucleate more astral microtubules on one of the two spindle poles than the other. This differential activity generally correlates with the age of MTOCs and contributes to orienting the mitotic spindle within the cell. The asymmetry might result from the two MTOCs being in distinctive maturation states. We investigated this model in budding yeast. Using fluorophores with different maturation kinetics to label the outer plaque components of the SPB, we found that the Cnm67 protein is mobile, whereas Spc72 is not. However, these two proteins were rapidly as abundant on both SPBs, indicating that SPBs mature more rapidly than anticipated. Superresolution microscopy confirmed this finding for Spc72 and for the γ-tubulin complex. Moreover, astral microtubule number and length correlated with the subcellular localization of SPBs rather than their age. Kar9-dependent orientation of the spindle drove the differential activity of the SPBs in astral microtubule organization rather than intrinsic differences between the spindle poles. Together, our data establish that Kar9 and spatial cues, rather than the kinetics of SPB maturation, control the asymmetry of astral microtubule organization between the preexisting and new SPBs.

Characterization of gamma-tubulin filaments in mammalian cells

Overexpression of γ-tubulin leads to the formation of filaments, but nothing is known about such filaments with regard to possible presence in cells, structure and probable dynamics. Here, we used mammalian cell lines to investigate the ability of γ-tubulin to form filaments. We found that γ-tubulin produces fibers called γ-tubules in a GTP-dependent manner and that γ-tubules are made up of pericentrin and the γ-tubulin complex proteins 2, 3, 5 and 6. Furthermore, we noted that the number of cells with cytosolic γ-tubules is increased in non-dividing cells. Our experiments showed that γ-tubules are polar structures that have a low regrowth rate compared to microtubules. Also, we observed that γ-tubules were disassembled by treatment with cold, colcemid, citral dimethyl acetal, dimethyl fumarate or mutation of γ-tubulin GTPase domain, but were increased in number by treatment with taxol or by stable expression of the γ-tubulin1–333 GTPase domain. Our results demonstrate that γ-tubulin forms filaments, and such assembly is facilitated by the GTPase domain of γ-tubulin.

Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function.

Centrosomes are a functionally conserved feature of eukaryotic cells that play an important role in cell division. The conserved γ-tubulin complex organizes spindle and astral microtubules, which, in turn, separate replicated chromosomes accurately into daughter cells. Like DNA, centrosomes are duplicated once each cell cycle. Although in some cell types it is possible for cell division to occur in the absence of centrosomes, these divisions typically result in defects in chromosome number and stability. In single-celled organisms such as fungi, centrosomes [known as spindle pole bodies (SPBs)] are essential for cell division. SPBs also must be inserted into the membrane because fungi undergo a closed mitosis in which the nuclear envelope (NE) remains intact. This poorly understood process involves events similar or identical to those needed for de novo nuclear pore complex assembly. Here, we review how analysis of fungal SPBs has advanced our understanding of centrosomes and NE events.

Gamma-tubulin distribution and ultrastructural changes in root cells of soybean (Glycine max L.) seedlings under cadmium stress

Gamma-tubulin (γ-tubulin) is known to be a key component of the complexes responsible for nucleation and the provision of spatial and temporal control over the functioning of the microtubule cytoskeleton. However, the effect of heavy metal stress on the distribution of γ-tubulin containing complexes has not yet been elucidated. In this study, we present the effect of cadmium (Cd) on γ-tubulin distribution in plant cells. The localization and level of γ-tubulin was assessed in the root cells of soybean seedlings exposed to moderate (85μM) and high (170μM) Cd concentrations, using various immunological approaches. In response to Cd, the fluorescence signal for γ-tubulin was significantly weaker. This decrease was most pronounced in the neighborhood of the nuclei envelope and plasmalemma of interphase cells and in the mitotic arrays of dividing cells. This finding was in line with a biochemical analysis which revealed that Cd leads to a decrease in the level of some γ-tubulin isoforms and the appearance of low molecular γ-tubulin-related proteins. Interestingly, at the same time, exposure to Cd at high concentration resulted in the up-regulation of γ-tubulin encoding genes. The results indicate that Cd stress might provoke dysfunctionality in γ-tubulin complexes, which contribute to a disturbed MT cytoskeleton structure. Additionally, ultrastructural observations confirmed considerable Cd-dependent subcellular changes in the root cells of soybean seedlings. The most prominent of these included vacuolization of the cytoplasm, occurrence of autophagic bodies and electron dense deposits in the vacuole, changes in the nucleolus structure and callose deposition. The observed changes are discussed as a response to stress conditions.

Polo-like kinase Cdc5 regulates Spc72 recruitment to spindle pole body in the methylotrophic yeast Ogataea polymorpha.

Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha.

Wsv023 interacted with Litopenaeus vannamei γ-tubulin complex associated proteins 2, and decreased the formation of microtubules.

A previous study found the key transcription factor of Litopenaeus vannamei PERK-eIF2α pathway cyclic AMP-dependent transcription factor 4 (LvATF4) was involved in the transcriptional regulation of white spot syndrome virus (WSSV) gene wsv023. Knocked-down expression of LvATF4 reduced the viral copy number and the cumulative mortality of WSSV-infected shrimp. These results suggested that wsv023 may be critical to WSSV infection but the precise function of wsv023 was still unknown. By using co-immunoprecipitation and pull-down assays, we show that wsv023 interacts with L. vannamei gamma complex-associated protein 2 (LvGCP2), which is the core protein of the γ-tubulin small complex. Knocked-down, the wsv023 gene significantly reduced the copy number of WSSV in L. vannamei muscle, as well as the cumulative mortality of infected shrimp. And PERK-eIF2α pathway inhibition also showed reduced virus copy number and abrogated shrimp mortality. Furthermore, overexpression of wsv023 inhibited the formation of microtubules in 293T cells. Flow cytometry revealed that WSSV infection similarly decreased the formation of microtubules in L. vannamei haemocytes. These findings suggested that wsv023 plays a role in microtubule organization in host cells, which in turn may be beneficial to WSSV.

Regulation of microtubule nucleation mediated by γ-tubulin complexes.

The microtubule cytoskeleton is critically important for spatio-temporal organization of eukaryotic cells. The nucleation of new microtubules is typically restricted to microtubule organizing centers (MTOCs) and requires γ-tubulin that assembles into multisubunit complexes of various sizes. γ-Tubulin ring complexes (TuRCs) are efficient microtubule nucleators and are associated with large number of targeting, activating and modulating proteins. γ-Tubulin-dependent nucleation of microtubules occurs both from canonical MTOCs, such as spindle pole bodies and centrosomes, and additional sites such as Golgi apparatus, nuclear envelope, plasma membrane-associated sites, chromatin and surface of pre-existing microtubules. Despite many advances in structure of γ-tubulin complexes and characterization of γTuRC interacting factors, regulatory mechanisms of microtubule nucleation are not fully understood. Here, we review recent work on the factors and regulatory mechanisms that are involved in centrosomal and non-centrosomal microtubule nucleation.

Human microcephaly ASPM protein is a spindle pole-focusing factor that functions redundantly with CDK5RAP2.

Nonsense mutations in the ASPM gene have been most frequently identified among familial microcephaly patients. Depletion of the Drosophila orthologue (asp) causes spindle pole unfocusing during mitosis in multiple cell types. However, it remains unknown whether human ASPM has a similar function. Here, by performing CRISPR-based gene knockout (KO) and RNA interference combined with auxin-inducible degron, we show that ASPM functions in spindle pole organisation during mitotic metaphase redundantly with another microcephaly protein, CDK5RAP2 (also called CEP215), in human tissue culture cells. Deletion of the ASPM gene alone did not affect spindle morphology or mitotic progression. However, when the pericentriolar material protein CDK5RAP2 was depleted in ASPM KO cells, spindle poles were unfocused during prometaphase, and anaphase onset was significantly delayed. The phenotypic analysis of CDK5RAP2-depleted cells suggested that the pole-focusing function of CDK5RAP2 is independent of its known function to localise the kinesin-14 motor HSET (also known as KIFC1) or activate the γ-tubulin complex. Finally, a hypomorphic mutation identified in ASPM microcephaly patients similarly caused spindle pole unfocusing in the absence of CDK5RAP2, suggesting a possible link between spindle pole disorganisation and microcephaly.

Two coffins and a funeral: early or late caspase activation determines two types of apoptosis induced by DNA damaging agents.

Cell cytoskeleton makes profound changes during apoptosis including the organization of an Apoptotic Microtubule Network (AMN). AMN forms a cortical structure which plays an important role in preserving plasma membrane integrity during apoptosis. Here, we examined the cytoskeleton rearrangements during apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. Using fixed and living cell imaging, we showed that CPT induced two dose- and cell cycle-dependent types of apoptosis characterized by different cytoskeleton reorganizations, time-dependent caspase activation and final apoptotic cell morphology. In the one referred as "slow" (~h) or round-shaped, apoptosis was characterized by a slow contraction of the actinomyosin ring and late caspase activation. In "slow" apoptosis the γ-tubulin complexes were not disorganized and microtubules were not depolymerized at early stages. In contrast, "fast" (~min) or irregular-shaped apoptosis was characterized by early caspase activation followed by full contraction of the actinomyosin ring. In fast apoptosis γ-tubulin complexes were disorganized and microtubules were initially depolymerized. However, after actinomyosin contraction, microtubules were reformed adopting a cortical but irregular disposition near plasma membrane. In addition to distinctive cytoskeleton reorganization kinetics, round and irregular-shaped apoptosis showed different biological properties with respect to AMN maintenance, plasma membrane integrity and phagocytes response. Our results suggest that the knowledge and modulation of the type of apoptosis promoted by genotoxic agents may be important for deciding a better therapeutic option and predicting the immune response in cancer treatment.

MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template.

MOZART1/Mzt1 is required for the localization of γ-tubulin complexes to microtubule (MT)-organizing centers from yeast to human cells. Nevertheless, the molecular function of MOZART1/Mzt1 is largely unknown. Taking advantage of the minimal MT nucleation system of Candida albicans, we reconstituted the interactions of Mzt1, γ-tubulin small complex (γ-TuSC), and γ-tubulin complex receptors (γ-TuCRs) Spc72 and Spc110 in vitro. With affinity measurements, domain deletion, and swapping, we show that Spc110 and Mzt1 bind to distinct regions of the γ-TuSC. In contrast, both Mzt1 and γ-TuSC interact with the conserved CM1 motif of Spc110/Spc72. Spc110/Spc72 and Mzt1 constitute "oligomerization chaperones," cooperatively promoting and directing γ-TuSC oligomerization into MT nucleation-competent rings. Consistent with the functions of Mzt1, human MOZART1 directly interacts with the CM1-containing region of the γ-TuCR CEP215. MOZART1 depletion in human cells destabilizes the large γ-tubulin ring complex and abolishes CEP215CM1-induced ectopic MT nucleation. Together, we reveal conserved functions of MOZART1/Mzt1 through interactions with γ-tubulin complex subunits and γ-TuCRs.

Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains

Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex.

Divergent regulation of functionally distinct γ-tubulin complexes during differentiation.

Differentiation induces the formation of noncentrosomal microtubule arrays in diverse tissues. The formation of these arrays requires loss of microtubule-organizing activity (MTOC) at the centrosome, but the mechanisms regulating this transition remain largely unexplored. Here, we use the robust loss of centrosomal MTOC activity in the epidermis to identify two pools of γ-tubulin that are biochemically and functionally distinct and differentially regulated. Nucleation-competent CDK5RAP2-γ-tubulin complexes were maintained at centrosomes upon initial epidermal differentiation. In contrast, Nedd1-γ-tubulin complexes did not promote nucleation but were required for anchoring of microtubules, a previously uncharacterized activity for this complex. Cell cycle exit specifically triggered loss of Nedd1-γ-tubulin complexes, providing a mechanistic link connecting MTOC activity and differentiation. Collectively, our studies demonstrate that distinct γ-tubulin complexes regulate different microtubule behaviors at the centrosome and show that differential regulation of these complexes drives loss of centrosomal MTOC activity.

Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly.

In fission yeast, γ-tubulin ring complex (γTuRC)-specific components Gfh1(GCP4), Mod21(GCP5), and Alp16(GCP6) are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1(MOZART1), a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16(GCP6) promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1(GCP4) and Mod21(GCP5) are not required for Alp16(GCP6)-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16(GCP6) and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation.