Role In Microtubule Nucleation Research Articles

Abstract 527: Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification

Abstract Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previous demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. We also demonstrated that the minus-end directed motor protein KIFC1, a mediator of centrosome clustering, has higher expression in cells following treatment. Centrosome amplification (CA) involves aberrations in centrosome number, where cells have 3 centrosomes. Cells with extra centrosomes create a multi-polar spindle during mitosis, which presents high risk for cell death and arrest, but cancer cells with CA avoid this by clustering extra centrosomes to create a pseudo-bipolar spindle. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance. Cancer cells were treated with LD50 dose of cisplatin and released from treatment for varying lengths of time. CA in endocycling cells increased from 59.2% to 100% during the treatment recovery period while maintaining identical levels of clustering. Endocycling cancer cells have a nearly 5-fold increase in declustered centrosomes compared to proliferative cells. Centrosomes in endocycling cells are displaced from the perinuclear region and toward the cell periphery. We hypothesize that centrosome number may indicate the number of times a cell has endocycled and provide insight into the timing and mechanism of mitotic bypass. Centrosomes play a major role in microtubule nucleation, affecting cell motility and polarity, and act as signaling platforms, enhancing signaling activation and specificity. Because of this, we hypothesize that CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Citation Format: Madison Purkerson, Sarah Amend, Kenneth J. Pienta. Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 527.

A prognostic model based on the Augmin family genes for LGG patients

Gliomas are the most prevalent primary tumors in the central nervous system. Despite some breakthroughs in the treatment of glioma in recent years, survival rates remain low. Although genes of the Augmin family play a key role in microtubule nucleation, the role they play in gliomas is unclear. Transcriptome data were extracted from UCSC XENA and GTEx for low-grade glioma (LGG) and normal tissues, respectively. The protein interaction network associated with Augmin family genes was established using STRING and GeneMANIA databases. Enrichment analysis of gene-related functions and pathways was used to explore potential biological pathways and TIMER to assess immune cell infiltration. Regression analysis and Kaplan–Meier analysis were used to look at the clinical characteristics of the Augmin family genes and the association with the prognosis of patients with glioma. The results showed that the mRNA expression of Augmin family genes was significantly elevated in LGG tissues, except for HAUS7. Immunoregulation, cell cycle, apoptosis and other signaling pathways may be involved in the development and progression of LGG. Except for HAUS4 and HAUS7, the expression of all genes was positively correlated with immune cell infiltration. High expression of HAUS1, HAUS3, HAUS5, HAUS7, HAUS8 and low expression of HAUS4, HAUS6 in LGG was associated with poor prognosis. The risk models constructed based on the pivotal genes HAUS2, HAUS4 and HAUS8 were validated by nomogram and confirmed to be clinically useful for predicting the prognosis of LGG.

Microtubules, Myelin Sheaths, and Altered Behavior.

Mice are skttish creatures that innately hide from perceived threats by staying out of open areas, freezing to avoid predators, or hiding in dark spaces when an adverse stimulus is sensed. However, after inducing a global TPPP (tubulin polymerization promoting protein) gene knock-out (KO), which results in reduced myelin length and thickness (Fu et al., 2019), Nguyen et al. (2020) found that these natural behaviors become less pronounced (Fig. 1). They also report that fear conditioning is less effective in the Tppp KO mice, indicating that their ability to learn to fear a specific event is impaired. Fear impacts many brain circuits and is critical for survival, but the contribution of non-neuronal cells in fear circuits is not well understood. This work identifies a protein that influences both innate and learned fear via its role in microtubule nucleation in oligodendrocytes. Figure 1. A ) Normal branching and myelin sheath length in oligodendrocytes in WT mice is coupled with normal fear conditioning and innate fear responses. B ) Tppp KO results in highly branched oligodendrocytes with shorter, thinner myelin sheaths. These mice have dramatically reduced fear conditioning and innate fear responses. In the cell body of most cells, microtubules are nucleated from perinuclear centrosomes. These microtubule organizing centers (MTOCs) contain centrioles and a pericentriolar matrix containing g-tubulin ring complexes (γTURCs). γTURCs are the starting point for the initiation and subsequent polymerization of α/βtubulin dimers into cytoplasmic microtubules. Golgi outposts are a different type of MTOC that nucleate microtubules outside of the cell body in …

Protein tyrosine phosphatase SHP-1 modulates microtubule organization in later stages of mast cell activation

Abstract Antigen-mediated activation of mast cells initiates signaling events leading to degranulation and release of inflammatory mediators. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Important role in microtubule nucleation from centrosomes play γ-tubulin complexes. Here we report on the regulation of microtubule organization in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1). Reciprocal immunoprecipitation experiments revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Pull-down experiments with truncated forms of γ-tubulin and SHP-1 unveiled corresponding interaction domains. When compared to controls, activated BMMCs without SHP-1 had more protrusions containing microtubules, higher level of tyrosine phosphorylation, enhanced activity of Syk kinase, as well as increased expression of cytokines and prostaglandins. Intracellular calcium mobilization and degranulation also increased. Microtubule regrowth experiments in cells lacking SHP-1 revealed stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes of γ-tubulin accumulation in centrosomes. Our data suggest a novel SHP-1 dependent mechanism for the suppression of microtubule formation in later stages of mast cell activation.

Centrosome Loss Triggers a Transcriptional Program To Counter Apoptosis-Induced Oxidative Stress

Centrosomes play a critical role in mitotic spindle assembly through their role in microtubule nucleation and bipolar spindle assembly. Loss of centrosomes can impair the ability of some cells to properly conduct mitotic division, leading to chromosomal instability, cell stress, and aneuploidy. Multiple aspects of the cellular response to mitotic error associated with centrosome loss appear to involve activation of JNK signaling. To further characterize the transcriptional effects of centrosome loss, we compared gene expression profiles of wild-type and acentrosomal cells from Drosophila wing imaginal discs. We found elevation of expression of JNK target genes, which we verified at the protein level. Consistent with this, the upregulated gene set showed significant enrichment for the AP-1 consensus DNA-binding sequence. We also found significant elevation in expression of genes regulating redox balance. Based on those findings, we examined oxidative stress after centrosome loss, revealing that acentrosomal wing cells have significant increases in reactive oxygen species (ROS). We then performed a candidate genetic screen and found that one of the genes upregulated in acentrosomal cells, glucose-6-phosphate dehydrogenase, plays an important role in buffering acentrosomal cells against increased ROS and helps protect those cells from cell death. Our data and other recent studies have revealed a complex network of signaling pathways, transcriptional programs, and cellular processes that epithelial cells use to respond to stressors, like mitotic errors, to help limit cell damage and maintain normal tissue development.

Centrosome Aurora A regulates RhoGEF ECT-2 localisation and ensures a single PAR-2 polarity axis in C. elegans embryos.

Proper establishment of cell polarity is essential for development. In the one-cell C. elegans embryo, a centrosome-localised signal provides spatial information for polarity establishment. It is hypothesised that this signal causes local inhibition of the cortical actomyosin network, and breaks symmetry to direct partitioning of the PAR proteins. However, the molecular nature of the centrosomal signal that triggers cortical anisotropy in the actomyosin network to promote polarity establishment remains elusive. Here, we discover that depletion of Aurora A kinase (AIR-1 in C. elegans) causes pronounced cortical contractions on the embryo surface, and this creates more than one PAR-2 polarity axis. This function of AIR-1 appears to be independent of its role in microtubule nucleation. Importantly, upon AIR-1 depletion, centrosome positioning becomes dispensable in dictating the PAR-2 axis. Moreover, we uncovered that a Rho GEF, ECT-2, acts downstream of AIR-1 in regulating contractility and PAR-2 localisation, and, notably, AIR-1 depletion influences ECT-2 cortical localisation. Overall, this study provides a novel insight into how an evolutionarily conserved centrosome Aurora A kinase inhibits promiscuous PAR-2 domain formation to ensure singularity in the polarity establishment axis.

Roles of end-binding 1 protein and gamma-tubulin small complex in cytokinesis and flagella formation of Giardia lamblia.

Giardia lamblia is a unicellular organism with two nuclei, a median body, eight flagella, and an adhesive disk. γ‐Tubulin is a microtubule (MT)‐nucleating protein that functions in the γ‐tubulin small complex (γ‐TuSC) in budding yeast. In this study, G. lamblia γ‐tubulin (Glγ‐tubulin) was found to bind to another MT‐binding protein, namely G. lamblia end‐binding protein 1 (GlEB1), via both in vivo and in vitro assays. Hemagglutinin (HA)‐tagged Glγ‐tubulin localized to the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. The knockdown of Glγ‐tubulin expression using an anti‐Glγ‐tubulin morpholino resulted in a decreased growth rate and an increased failed cytokinesis cells of Giardia. The formation of median bodies was affected, and the central pair of MTs in flagella was frequently missing in the Giardia treated with an anti‐Glγ‐tubulin morpholino. G. lamblia γ‐tubulin complex protein 2 (GlGCP2) and GlGCP3, which are putative components of γ‐TuSC, were co‐immunoprecipitated with HA‐tagged Glγ‐tubulin in Giardia extracts. The knockdown of GlGCP2 and GlGCP3 expression also resulted in decreased formation of both the median body and flagella MTs. Knockdown of Glγ‐tubulin, GlGCP2, and GlGCP3 expression affected localization of GlEB1 in G. lamblia. In addition, decreased level of GlEB1 caused reduced formation of median body and the central pair of flagella MTs. These results indicated that Glγ‐tubulin plays a role in MT nucleation for median body formation and flagella biogenesis as a component of Glγ‐TuSC in Giardia and GlEB1 may be involved in this process.

γ-Tubulin small complex formation is essential for early zebrafish embryogenesis

The centrosomal protein γ-tubulin is part of the cytoplasmic γ-tubulin small (γ-TuSCs) and large complexes (γ-TuRCs). Both, molecular and cellular evidence indicate that γ-tubulin plays a central role in microtubule nucleation and mitotic spindle formation. However, the molecular mechanisms of complex formation and subsequent biological roles in animal development remain unclear. Here, we used γ-tubulin gene knockdown in the zebrafish early embryo model to gain insights into its activity and cellular contribution during vertebrate embryogenesis. γ-Tubulin loss-of-function impaired γ-TuSC formation, impacting the microtubule nucleation rate in vitro. Moreover, decreased γ-tubulin synthesis caused dramatic defects in nuclear dynamics and cell cycle progression, leading to developmental arrest at the mid-gastrula stage. At the subcellular level, microtubule organization and function were altered, affecting chromosome segregation and triggering cell proliferation arrest and apoptosis. Our results suggest that de novo translated γ-tubulin participates in γ-TuSC formation required for early animal development. Importantly, formation of this complex is essential for both centrosome assembly and function, and cell proliferation. Thus, γ-TuSC integrity appears to be critical for cell cycle progression, and concomitantly, for coordinating the many distinct activities carried out by the early embryo. Our findings identify a novel role for γ-TuSC in the regulation of early vertebrate embryogenesis, providing molecular and biochemical starting points for future in depth studies of γ-tubulin functionality and its specific role in development.

Non-centrosomal TPX2-Dependent Regulation of the Aurora A Kinase: Functional Implications for Healthy and Pathological Cell Division.

Aurora A has been extensively characterized as a centrosomal kinase with essential functions during cell division including centrosome maturation and separation and spindle assembly. However, Aurora A localization is not restricted to the centrosomes and compelling evidence support the existence of specific mechanisms of activation and functions for non-centrosomal Aurora A in the dividing cell. It has been now well established that spindle assembly involves an acentrosomal RanGTP-dependent pathway that triggers microtubule assembly and organization in the proximity of the chromosomes whether centrosomes are present or not. The mechanism involves the regulation of a number of NLS-containing proteins, generically called SAFS (Spindle Assembly Factors) that exert their functions upon release from karyopherins by RanGTP. One of them, the nuclear protein TPX2 interacts with and activates Aurora A upon release from importins by RanGTP. This basic mechanism triggers the activation of Aurora A in the proximity of the chromosomes potentially translating the RanGTP signaling gradient centered on the chromosome into an Aurora A phosphorylation network. Here, we will review our current knowledge on the RanGTP-dependent TPX2 activation of Aurora A away from centrosomes: from the mechanism of activation and its functional consequences on the kinase stability and regulation to its roles in spindle assembly and cell division. We will then focus on the substrates of the TPX2-activated Aurora A having a role in microtubule nucleation, stabilization, and organization. Finally, we will briefly discuss the implications of the use of Aurora A inhibitors in anti-tumor therapies in the light of its functional interaction with TPX2.

γ-Tubulin complexes in microtubule nucleation and beyond.

Tremendous progress has been made in understanding the functions of γ-tubulin and, in particular, its role in microtubule nucleation since the publication of its discovery in 1989. The structure of γ-tubulin has been determined, and the components of γ-tubulin complexes have been identified. Significant progress in understanding the structure of the γ-tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly. At the same time, data have accumulated that γ-tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation. These include roles in the regulation of plus-end microtubule dynamics, gene regulation, and mitotic and cell cycle regulation. Finally, evidence is emerging that γ-tubulin mutations or alterations of γ-tubulin expression play an important role in certain types of cancer and in other diseases.

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.

GIP/MZT1 proteins orchestrate nuclear shaping

The functional organization of the nuclear envelope (NE) is only just emerging in plants with the recent characterization of NE protein complexes and their molecular links to the actin cytoskeleton. The NE also plays a role in microtubule nucleation by recruiting γ-Tubulin Complexes (γ-TuCs) which contribute to the establishment of a robust mitotic spindle. γ-tubulin Complex Protein 3 (GCP3)-interacting proteins (GIPs) have been identified recently as integral components of γ-TuCs. GIPs have been conserved throughout evolution and are also named MZT1 (mitotic-spindle organizing protein 1). This review focuses on recent data investigating the role of GIP/MZT1 at the NE, including insights from the study of GIP partners. It also uncovers new functions for GIP/MZT1 during interphase and highlights a current view of NE-associated components which are critical for nuclear shaping during both cell division and differentiation.

Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1-6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1-3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site.

Nuclear γ‐tubulin associates with nucleoli and interacts with tumor suppressor protein C53

γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).

γ-TuRC gives microtubules pause for thought

A protein complex better known for its role in microtubule nucleation at the centrosome is also a stabilizing factor that controls cytoskeletal dynamics, say [Bouissou et al.][1] ![Figure][2] γ-TuRC (green) acts as a stabilizing factor along the length of microtubules (red). γ-Tubulin

On and Around Microtubules: An Overview

Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from alphabeta-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the beta-tubulin subunit facing the microtubule plus end conferring a structural polarity. The alpha- and beta-tubulins are highly conserved. A third member of the tubulin family, gamma-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with beta-tubulin so that microtubules consist principally of 'GDP-tubulin' stabilized at the plus end by a short 'cap'. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.

Cell Cycle-dependent Expression of γ-Tubulin in the Amicronuclear Ciliate Tetrahymena pyriformis

In ciliates, different microtubular structures are nucleated from diverse Microtubule Organizing Centers (MTOCs). gamma-Tubulin is a tubulin superfamily member that plays an essential role in microtubule nucleation at the MTOCs. However, little is known about mechanisms regulating the activity of gamma-tubulin on different MTOCs and during the cell cycle. In Tetrahymena thermophila, the alpha- and beta-tubulin expression is regulated mainly at the transcriptional level, and changes in the ratio of polymerized/unpolymerized tubulin dimers lead to an increase or decrease of alpha- and beta-tubulin transcription. This study deals with the characterization of gamma-tubulin in the amicronuclear ciliate Tetrahymena pyriformis. Sequence analysis revealed some specific substitutions in nucleotide-binding loops characteristic of the Tetrahymena genus and putative conserved phosphorylation sites located on the external surface of the gamma-tubulin molecule. gamma-Tubulin expression during the cell cycle, in the presence of microtubular poisons and after deciliation, was also characterized. We found that gamma-tubulin mRNA levels are correlated with basal body proliferation and gamma-tubulin nuclear localization. We also found that gamma-tubulin expression changes during anti-microtubular drugs treatment, but does not changes during reciliation. These findings suggest a relationship between the level of unpolymerized tubulin dimers and gamma-tubulin transcription.

Human Ninein is a Centrosomal Autoantigen Recognized by CREST Patient Sera and Plays a Regulatory Role in Microtubule Nucleation

Centrosome is the major microtubule organizing center in mammalian cells that plays a critical role in a variety of cellular events by the microtubule arrays emanating from it. Despite its significance, the molecular mechanisms underlying the structure and function of the centrosome are still not clear. Herein we describe the identification of three isotypes of human ninein by expression library screening with autoimmune sera from CREST patients. All three ninein isotypes exhibit centrosomal localization throughout the cell cycle when GFP-tagged fusion proteins are expressed transiently in mammalian cells. Construction of serial deletions of GFP-tagged ninein reveals that a stretch of three leucine zippers with a flanking sequence is required and sufficient for centrosomal targeting. Overexpression of ninein results in mislocalization of ?-tubulin, recruiting it to ectopic (non-centrosomal) ninein-containing sites which are not active in nucleating microtubules. In these cells, nucleation of microtubules from the centrosome is also inhibited. These results thus suggest a regulatory role for ninein in microtubule nucleation.

γ-Tubulin Functions in the Nucleation of a Discrete Subset of Microtubules in the Eukaryotic Flagellum

γ-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of γ-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented [1–3], its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of γ-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 γ-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 γ-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.

Distribution of gamma-tubulin in cellular compartments of higher plant cells.

Higher plants represent an important group of eukaryotes in which discrete sites for microtubule nucleation are absent in all mitotic and meiotic cells, and a concept of dispersed microtubule-nucleation sites has been generally accepted. Current models of mitotic spindle formation in the absence of centrosomes are based on chromatin-mediated microtubule organization. Moreover, even in cells equipped with centrosomes, microtubules can nucleate on cytoplasmic factors, which are so far poorly characterized (Vorobjev et al., 1997). To understand how microtubules are nucleated and organized without the centrosome, the first step is to determine the molecular composition of the dispersed cytoplasmic or chromatin-associated microtubulenucleation sites. -Tubulin, a distantly related member of the tubulin superfamily, plays a pivotal role in microtubule nucleation. It is organized into the large w2000 kDa open-ring complexes ( -TuRC), located at the centrosomes in animal cells and at the spindle pole bodies in yeast (Moritz et al., 1995). The complete genome of Arabidopsis contains two expressed genes encoding -tubulins. Using affinity purified polyclonal and monoclonal antibodies, directed against different antigenic determinants on the -tubulin molecule, we studied the subcellular distribution of -tubulin in six plant species (Arabidopsis thaliana, Vicia faba, Pisum sativum, Medicago sativa, Hordeum vulgare and Zea mays). Biochemical and immunolocalization studies revealed that -tubulin is highly abundant in different cellular compartments. -Tubulin was localized along all microtubular arrays, discrete staining was found in some interphase nuclei. The size of nuclear spots gradually increased until late G2 when they often occurred as double spots. A similar labelling pattern for -tubulin was found in prekinetochore region of isolated G2 nuclei. -Tubulin was also localized on the nuclear surface, decorating perinuclear microtubules focused to the poles. At the onset of mitosis, when nuclear envelope broke down, -tubulin was found on short kinetochore microtubule fibres organized from kinetochores. A strong signal for -tubulin was associated with kinetochore microtubules along their whole length including the close vicinity of the kinetochores. In cells recovering from anti-microtubular drugs, -tubulin was localized with the re-growing kinetochore microtubules nucleated or captured by kinetochore/centromeric regions. Similarly, on isolated chromosomes, -tubulin co-localized with -tubulin in the kinetochore/centromeric region. Flow cytometry was employed to sort isolated plant nuclei in the G1 and G2 stages of the cell cycle. They were thereafter subjected to quantitative immunoblot analysis. An increase of about 50% of -tubulin in G2 compared to G1 nuclei was found in V. faba. In contrast, when supernatants of centrifuged cell extracts from synchronized cells were analyzed by immunoblotting, there were no differences in the amount of -tubulin between cells in the G1 and G2 stages of the cell cycle (Binarova et al., 2000). Circular configurations of chromosomes with the kinetochore region in the centre and chromosome arms pointing outwards were induced transiently by treatment of root meristems of V. faba, M. sativa and A. thaliana with specific cyclin-dependent protein kinase inhibitors (Binarova et al., 1998a). Kinetochore microtubules, * Corresponding author. Tel. +42-2-4106-2130; fax: +42-4-4106-2384. Cell Biology International 27 (2003) 167–169 Cell Biology International