Γ-tubulin Small Complex Research Articles

γ-Tubulin complex in Trypanosoma brucei: molecular composition, subunit interdependence and requirement for axonemal central pair protein assembly.

γ-Tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, gamma-tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex.

Overexpression and Nucleolar Localization of γ-Tubulin Small Complex Proteins GCP2 and GCP3 in Glioblastoma.

The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating γ-tubulin small complex (γTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma cells versus normal human astrocytes; these were associated with higher amounts of both γTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with γ-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that γTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype.

Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning.

Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25's association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes.

Ring closure activates yeast γTuRC for species-specific microtubule nucleation.

The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in cells. γTuRC is assembled from repeating γ-tubulin small complex (γTuSC) subunits and is thought to function as a template by presenting a γ-tubulin ring that mimics microtubule geometry. However, a previous yeast γTuRC structure showed γTuSC in an open conformation that prevents matching to microtubule symmetry. By contrast, we show here that γ-tubulin complexes are in a closed conformation when attached to microtubules. To confirm its functional importance we trapped the closed state and determined its structure, showing that the γ-tubulin ring precisely matches microtubule symmetry and providing detailed insight into γTuRC architecture. Importantly, the closed state is a stronger nucleator, suggesting this conformational switch may allosterically control γTuRC activity. Finally, we demonstrate that γTuRCs have a profound preference for tubulin from the same species.

Targeting of γ-tubulin complexes to microtubule organizing centers: conservation and divergence

Organisms with closed or open mitosis have differentially evolved various gamma-tubulin complex (γ-TuC) recruiting factors to organize diverse cellular microtubule (MT) arrays, including the mitotic spindle. γ-TuC recruiting factors not only target the γ-TuC to MT nucleation sites, but also regulate MT nucleation activity by generating the template for MT nucleation or promoting the MT nucleation activity of pre-existing γ-tubulin ring complexes (γ-TuRCs). Here we outline the current understanding of MT nucleator assembly and its regulation by γ-tubulin small complex (γ-TuSC) receptors. Moreover, we discuss the emergence of γ-TuC recruiting factors through evolution with augmented complexity and diversity and propose a hypothesis to account for the evolution of these factors in cooperative spindle assembly.

TACC3 Protein Regulates Microtubule Nucleation by Affecting γ-Tubulin Ring Complexes

Centrosome-mediated microtubule nucleation is essential for spindle assembly during mitosis. Although γ-tubulin complexes have primarily been implicated in the nucleation process, details of the underlying mechanisms remain poorly understood. Here, we demonstrated that a member of the human transforming acidic coiled-coil (TACC) protein family, TACC3, plays a critical role in microtubule nucleation at the centrosome. In mitotic cells, TACC3 knockdown substantially affected the assembly of microtubules in the astral region and impaired microtubule nucleation at the centrosomes. The TACC3 depletion-induced mitotic phenotype was rescued by expression of the TACC3 C terminus predominantly consisting of the TACC domain, suggesting that the TACC domain plays an important role in microtubule assembly. Consistently, experiments with the recombinant TACC domain of TACC3 demonstrated that this domain possesses intrinsic microtubule nucleating activity. Co-immunoprecipitation and sedimentation experiments revealed that TACC3 mediates interactions with proteins of both the γ-tubulin ring complex (γ-TuRC) and the γ-tubulin small complex (γ-TuSC). Interestingly, TACC3 depletion resulted in reduced levels of γ-TuRC and increased levels of γ-TuSC, indicating that the assembly of γ-TuRC from γ-TuSC requires TACC3. Detailed analyses suggested that TACC3 facilitates the association of γ-TuSC-specific proteins with the proteins known to be involved in the assembly of γ-TuRC. Consistent with such a role for TACC3, the suppression of TACC3 disrupted localization of γ-TuRC proteins to the centrosome. Our findings reveal that TACC3 is involved in the regulation of microtubule nucleation at the centrosome and functions in the stabilization of the γ-tubulin ring complex assembly.

Activation of the γ-Tubulin Complex by the Mto1/2 Complex

SummaryThe multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites [5–10]. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC. This was achieved through the construction of a “minimal” version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and γ-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing ∼13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of γ-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast γ-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes.

Novel Kinesin Regulators of Gamma-TuRC

Mitotic spindle assembly is a critical control point in eukaryotic chromosome segregation. In fission yeast conserved mitotic kinesin-like proteins (Klps) Kinesin-14 and Kinesin-5 are emerging as novel regulators of the γ-tubulin ring complex (γ-TuRC) MTOC. While these Klps typically participate in microtubule roles for spindle assembly, alternatively in fission yeast they localize to γ-TuRC and when co-deleted a bipolar spindle forms. Our analysis by genetics, biochemistry, cell biology and nanotechnology provides new knowledge on mitotic mechanisms in S. pombe. Previous work from our lab and others uncovered a novel functional relationship between Kinesin-14 Pkl1 and all proteins of the γ-tubulin small complex (γ-TuSC). We demonstrated that Pkl1 binds γ-TuRC through Motor and Tail interactions to directly regulate its function. A Pkl1 Tail peptide is sufficient in vitro and disrupts γ-TuRC structure by removal of γ-tubulin subunits (Cell Cycle 2013), an action that is reversible. New data from our lab indicates that the essential Kinesin-5 Cut7 is actually dispensable in the absence of Pkl1 with which it shares an important antagonistic MTOC regulatory relationship. Cut-7 associates with FPLC purified γ-TuRC and binds to γ-tubulin similarly as Pkl1. We demonstrate in vivo that Pkl1 blocks microtubule nucleation from γ-TuRC and additionally influences spindle microtubule numbers and microtubule overlap. Kinetochore-based microtubule motors Klp5 and Klp6 may facilitate spindle organization, since triple mutants of Pkl1, Cut7 and either Kinesin-6 Klp9 or Kinesin-14 Klp2 are viable. Previously we showed in vivo that human Kinesin-14 HSET, but not Drosophila Ncd, functionally replaces Pkl1 and more recently that human γ-TuSC proteins GCP2 and GCP3 replace fission yeast Alp4 and Alp6 (JCS 2013). Our research highlights conserved in vivo functions of γ-TuSC and provides a new model of spindle assembly in S. pombe.

γ-Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ-Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ-Tubulin Ring Complexes (γ-TuRCs). While the subunits that constitute γ-Tubulin Small Complexes (γ-TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ-TuRC-specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ-TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ-TuRCs on astral MTs. γ-TuRCs locate along the length of astral MTs, and depletion of γ-TuRC-specific proteins increases MT dynamics and causes the plus-end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down-regulation rescues spindle orientation defects induced by γ-TuRC depletion. Therefore, we propose a role for γ-TuRCs in regulating spindle positioning by controlling the stability of astral MTs.

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability.

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.

Kinesin-14 Pkl1 targets γ-tubulin for release from the γ-tubulin ring complex (γ-TuRC) ‬‬‬‬‬‬‬

The γ-tubulin ring complex (γ-TuRC) is a key part of microtubule-organizing centers (MTOCs) that control microtubule polarity, organization and dynamics in eukaryotes. Understanding regulatory mechanisms of γ-TuRC function is of fundamental importance, as this complex is central to many cellular processes, including chromosome segregation, fertility, neural development, T-cell cytotoxicity and respiration. The fission yeast microtubule motor kinesin-14 Pkl1 regulates mitosis by binding to the γ-tubulin small complex (γ-TuSC), a subunit of γ-TuRC. Here we investigate the binding mechanism of Pkl1 to γ-TuSC and its functional consequences using genetics, biochemistry, peptide assays and cell biology approaches in vivo and in vitro. We identify two critical elements in the Tail domain of Pkl1 that mediate γ-TuSC binding and trigger release of γ-tubulin from γ-TuRC. Such action disrupts the MTOC and results in failed mitotic spindle assembly. This study is the first demonstration that a motor protein directly affects the structural composition of the γ-TuRC, and we provide details of this mechanism that may be of broad biological importance.

Functional replacement of fission yeast γ-tubulin small complex proteins Alp4 and Alp6 by human GCP2 and GCP3

Microtubule-organizing centers such as the γ-tubulin ring complex (γ-TuRC) act as a template for polarized growth and regulation of microtubules that are essential for diverse cellular structures and processes in eukaryotes. New structural models of the budding yeast γ-tubulin small complex (γ-TuSC) of the γ-TuRC combined with functional studies done in multiple eukaryotes are revealing the first mechanistic clues into control of microtubule nucleation and organization. Cross-species studies of human and budding yeast γ-TuSC proteins in fission yeast revealed conserved and divergent structural and functional features of the γ-TuSC. We show genetically that GCP3/Spc98 function is fully conserved with Alp6 across species but that functional differences exist between GCP2/Spc97 and Alp4. By further analysis of human γ-TuSC proteins, we found that GCP3 assembles normally into the >2000 kDa fission yeast γ-TuRC and that the GCP3 gene replaces fission yeast alp6. Interestingly, human GCP2 replaces the essential alp4 gene but is unable to rescue a normally recessive G1 defect of the alp4-1891 allele that results in loss of γ-TuRC from poles in subsequent cell cycles. Biochemically, GCP2 incorporation into fission yeast γ-TuRC is limited in the presence of Alp4; instead, the bulk of GCP2 fractionates as smaller complexes. By generating a functional Alp4-GCP2 chimeric protein we determined that the GCP2 N-terminal domain limits its ability to fully displace or compete with Alp4 during γ-TuRC assembly. Our findings have broad importance for understanding the essential domains of γ-TuSC proteins in the γ-TuRC mechanism.

An extended γ-tubulin ring functions as a stable platform in microtubule nucleation

γ-Tubulin complexes are essential for microtubule (MT) nucleation. The γ-tubulin small complex (γ-TuSC) consists of two molecules of γ-tubulin and one molecule each of Spc97 and Spc98. In vitro, γ-TuSCs oligomerize into spirals of 13 γ-tubulin molecules per turn. However, the properties and numbers of γ-TuSCs at MT nucleation sites in vivo are unclear. In this paper, we show by fluorescence recovery after photobleaching analysis that γ-tubulin was stably integrated into MT nucleation sites and was further stabilized by tubulin binding. Importantly, tubulin showed a stronger interaction with the nucleation site than with the MT plus end, which probably provides the basis for MT nucleation. Quantitative analysis of γ-TuSCs on single MT minus ends argued for nucleation sites consisting of approximately seven γ-TuSCs with approximately three additional γ-tubulin molecules. Nucleation and anchoring of MTs required the same number of γ-tubulin molecules. We suggest that a spiral of seven γ-TuSCs with a slight surplus of γ-tubulin nucleates MTs in vivo.

Moving Towards Self-Assembling Machines: Harnessing Spindle Components for Biosynthetic Devices

The remarkably dynamic and complex manufacturing environment of the eukaryotic cell is currently unrivaled by manmade systems. Through dissection and harnessing of biological machines and underlying processes, and merger with synthetic materials, we hope to scale manufacturing into realms currently restricted from physical manipulation. Spindle assembly is the first critical step in chromosome segregation and a primary target for anti-cancer therapeutics. An assemblage of nanoscale components and communication networks must be integrated for spindle formation and function. We are applying nanotechnology in parallel with traditional methods of yeast genetics, timelapse microscopy, AFM and biochemistry. Dramatic reorganization of interphase microtubules into a bipolar organization for chromosome segregation requires focused nucleation from spindle pole body microtubule organizing centers (MTOCs). The γ-tubulin small complex (γ-TuSC) MTOC associates with growing microtubules and is part of a larger ring complex (γ-TuRC). The complex controls microtubule nucleation, organization and dynamics. Three conserved kinesin-like protein (Klp) families in fission yeast contribute in critical roles in spindle assembly. Kinesin-14 Pkl1 and Kinesin-5 associate with γ-TuSC at poles and provide counterbalanced roles in microtubule nucleation and organization, while Kinesin-6 acts on overlapping anti-parallel microtubules. The coupled action of the MTOC and Klps enables multiple levels of control. By purifying the γ-TuSC and γ-TuRC complexes from human and fission yeast cells using superose 6 FPLC chromatography, co-immunoprecipitation with magnetic beads and western analysis and attachment of these complexes to functionalized surfaces we are analyzing minimal components and frameworks for generating anti-parallel, bipolar and more complex microtubule array structures in the presence of multiple Klps. Our goal is to incorporate the MTOC, tubulin and multiple Klp families in different biosynthetic platforms to better understand self-assembling machines and transitional dynamic architectures thereby refining both in vivo models and in vitro advanced applications.

Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation

Microtubule nucleation in all eukaryotes involves γ-tubulin small complexes (γTuSCs) that comprise two molecules of γ-tubulin bound to γ-tubulin complex proteins (GCPs) GCP2 and GCP3. In many eukaryotes, multiple γTuSCs associate with GCP4, GCP5 and GCP6 into large γ-tubulin ring complexes (γTuRCs). Recent cryo-EM studies indicate that a scaffold similar to γTuRCs is formed by lateral association of γTuSCs, with the C-terminal regions of GCP2 and GCP3 binding γ-tubulin molecules. However, the exact role of GCPs in microtubule nucleation remains unknown. Here we report the crystal structure of human GCP4 and show that its C-terminal domain binds directly to γ-tubulin. The human GCP4 structure is the prototype for all GCPs, as it can be precisely positioned within the γTuSC envelope, revealing the nature of protein-protein interactions and conformational changes regulating nucleation activity.

Foreword: chromosomes and microtubules—the dynamic duo of mitosis

Abbreviations aMTOC Acentriolar microtubule organizing centre CPC Chromosomal passenger complex FRET Fluorescence resonance energy transfer GFP Green fluorescent protein HAUS Homologous to augmin subunits HU Hydroxyurea kMT Kinetochore microtubule MT Microtubule MAP Microtubule-associated protein MTOC Microtubule organizing centre NEB Nuclear envelope breakdown PCM Pericentriolar material SAF Spindle assembly factor γ-TuRC γ-Tubulin ring complex γ-TuSC γ-Tubulin small complex “Mitosis is complicated. Really complicated. You just won't believe how vastly, hugely, mind-bogglingly complicated it is. I mean, you may think DNA replication is pretty incomprehensible, but that's just peanuts to mitosis – listen......” (Apologies to Douglas Adams)

Microtubule stabilization in vivo by nucleation-incompetent γ-tubulin complex

Although the fission yeast Schizosaccharomyces pombe contains many of the γ-tubulin ring complex (γ-TuRC)-specific proteins of the γ-tubulin complex (γ-TuC), several questions about the organizational state and function of the fission yeast γ-TuC in vivo remain unresolved. Using 3×GFP-tagged γ-TuRC-specific proteins, we show here that γ-TuRC-specific proteins are present at all microtubule organizing centers in fission yeast and that association of γ-TuRC-specific proteins with the γ-tubulin small complex (γ-TuSC) does not depend on Mto1, which is a key regulator of the γ-TuC. Through sensitive imaging in mto1Δ mutants, in which cytoplasmic microtubule nucleation is abolished, we unexpectedly found that γ-TuC incapable of nucleating microtubules can nevertheless associate with microtubule minus-ends in vivo. The presence of γ-TuC at microtubule ends is independent of γ-TuRC-specific proteins and strongly correlates with the stability of microtubule ends. Strikingly, microtubule bundles lacking γ-TuC at microtubule ends undergo extensive treadmilling in vivo, apparently induced by geometrical constraints on plus-end growth. Our results indicate that microtubule stabilization by the γ-TuC, independently of its nucleation function, is important for maintaining the organization and dynamic behavior of microtubule arrays in vivo.

Klp Navigation of Spindle Assembly from Poles

Spindle assembly is the crucial initiating step in the mitotic mechanism. Its formation utilizes coordinated actions of Kinesin-like proteins (Klps) at poles, on microtubules and at chromosomes. We previously identified a novel conserved Kinesin-14 pole-based mechanism for spindle assembly by Klp binding to the γ-tubulin small complex (γ-TuSC) microtubule organizing center, (MTOC). Changes to the γ-TuSC MTOC in fission yeast alter microtubule dynamics to regulate bipolarity and both human and fission yeast Kinesin-14 Klps can regulate this mechanism. The Kinesin-14 Pkl1 motor domain associates with residues in helix 11 of γ-tubulin at a novel Klp binding site. The Kinesin-14 Pkl1 Tail domain is distinct from that of the well-characterized Drosophila Ncd, replacing microtubule association sites with specialized elements for spindle pole targeting and regulation of the γ-TuSC. Thus unlike Drosophila Ncd that crosslinks microtubules to stabilize mitotic spindle assembly, Kinesin-14 Pkl1 regulates assembly through the γ-TuSC. Klps can be classified into 14 families. Members of two ubiquitous families, Kinesin-14 and opposing Kinesin-5 Klps, each exhibit distinctions at multiple levels amongst members, including domain elements, functional mechanisms and spindle localization. In fission yeast, Kinesin-5 also localizes to spindle poles, consistent with the hypothesis that similar localization is needed to optimally counterbalance forces by these two Klp families. Although the stoichiometry of proteins within γ-TuSC is known and conserved in eukaryotes little is understood of the γ-TuSC mechanism. Cross-species analysis is defining shared and distinct functions and novel insights into the γ-TuSC interactome and the role of associating Klps in regulating its mechanism for spindle assembly. Our studies apply site directed mutagenesis, genetics, yeast two hybrid, biochemistry, timelapse microscopy and bioinformatic/structural approaches with novel reagents we have developed for Klp and γ-TuSC analysis.

A Developmentally Regulated Two-Step Process Generates a Noncentrosomal Microtubule Network in Drosophila Tracheal Cells

Microtubules (MTs) are essential for many cell features, such as polarity, motility, shape, and vesicle trafficking. Therefore, in a multicellular organism, their organization differs between cell types and during development; however, the control of this process remains elusive. Here, we show that during Drosophila tracheal morphogenesis, MT reorganization is coupled to relocalization of the microtubule organizing centers (MTOC) components from the centrosome to the apical cell domain from where MTs then grow. We reveal that this process is controlled by the trachealess patterning gene in a two-step mechanism. MTOC components are first released from the centrosome by the activity of the MT-severing protein Spastin, and then anchored apically through the transmembrane protein Piopio. We further show that these changes are essential for tracheal development, thus stressing the functional relevance of MT reorganization for morphogenesis.

In vivo analysis of the functions of γ-tubulin-complex proteins

To enhance our understanding of the function(s) of gamma-tubulin-complex proteins (GCPs), we identified and analyzed the functions of the Aspergillus nidulans homologs of GCP2-GCP6 (here designated GCPB-GCBF). The gamma-tubulin small complex (gamma-TuSC) components, gamma-tubulin, GCPB and GCPC, are essential for viability and mitotic spindle formation, whereas GCPD-GCPF are not essential for viability, spindle formation or sexual reproduction. GCPD-GCPF function in reducing the frequency of chromosome mis-segregation and in the assembly of large gamma-tubulin complexes. Deletion of any of the gamma-TuSC components eliminates the localization of all GCPs to the spindle pole body (SPB), whereas deletion of GCPD-GCPF does not affect localization of gamma-TuSC components. Thus, GCPD-GCPF do not tether the gamma-TuSC to the SPB, but, rather, the gamma-TuSC tethers them to the SPB. GCPD-GCPF exhibit a hierarchy of localization to the SPB. Deletion of GCPF eliminates GCPD-GCPE localization to the SPB, and deletion of GCPD eliminates GCPE (but not GCPF) localization. All GCPs localize normally in a GCPE deletion. We propose a model for the structure of the gamma-tubulin complex and its attachment to polar microtubule organizing centers.