Centrosomal TACCtics

Although the centrosome was first described over 100 years ago, we still know relatively little of the molecular mechanisms responsible for its functions. Recently, members of a novel family of centrosomal proteins have been identified in a wide variety of organisms. The transforming acidic coiled-coil-containing (TACC) proteins all appear to play important roles in cell division and cellular organisation in both embryonic and somatic systems. These closely related molecules have been implicated in microtubule stabilisation, acentrosomal spindle assembly, translational regulation, haematopoietic development and cancer progression. In this review, I summarise what we already know of this protein family and will use the TACC proteins to illustrate the many facets that centrosomes have developed during the course of evolution.     

vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubule and the kinetochore. Our recent ultrastructural studies demonstrated a dynamic distribution of TTK, from the kinetochore to the centrosome, as cell enters into anaphase. Here, we show that a centrosomal protein TACC2 is phosphorylated in mitosis by TTK signaling pathway. TACC2 was pulled down by wild type TTK but not kinase death mutant, suggesting the potential phosphorylation-mediated interaction between these two proteins. Our immunofluorescence studies revealed that both TTK and TACC2 are located to the centrosome. Interestingly, expression of kinase death mutant of TTK eliminated the centrosomal localization of TACC2 but not other centrosomal proteins such as gamma-tubulin and NuMA, a phenotype seen in TTK-depleted cells. In these centrosomal TACC2-liberated cells, chromosomes were lagging and mis-aligned. In addition, the distance between two centrosomes was markedly reduced, suggesting that centrosomal TACC2 is required for mitotic spindle maintenance. The inter-relationship between TTK and TACC2 established here provides new avenue to study centrosome and spindle dynamics underlying cell divisional control.     

Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A-dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC-Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.     

The transforming acidic coiled coil 3 protein is essential for spindle-dependent chromosome alignment and mitotic survival

Cancer-associated centrosomal transforming acidic coiled coil (TACC) proteins are involved in mitotic spindle function. By employing gene targeting, we have recently described a nonredundant and essential role of TACC3 in regulating cell proliferation. In this study, we used an inducible RNA interference approach to characterize the molecular function of TACC3 and its role in mitotic progression and cell survival. Our data demonstrate that a TACC3 knockdown arrests G(1) checkpoint-compromised HeLa cells prior to anaphase with aberrant spindle morphology and severely misaligned chromosomes. Interestingly, TACC3-depleted cells fail to accumulate the mitotic kinase Aurora B and the checkpoint protein BubR1 to normal levels at kinetochores. Moreover, localization of the structural protein Ndc80 at outer kinetochores is reduced, indicating a defective kinetochore-microtubule attachment in TACC3-deficient cells. As a consequence of prolonged TACC3 depletion, cells undergo caspase-dependent cell death that relies on a spindle checkpoint-dependent mitotic arrest. TACC3 knockdown cells that escape from this arrest by mitotic slippage become highly polyploid and accumulate supernumerary centrosomes. Similarly, deficiency of the post-mitotic cell cycle inhibitor p21(WAF) exacerbates the effects of TACC3 depletion. Our findings therefore point to an essential role of TACC3 in spindle assembly and cellular survival and identify TACC3 as a potential therapeutic target in cancer cells.     

Centrosome maturation: Aurora lights the way to the poles

The centrosome is the main microtubule organising centre in the cell. During mitosis, centrosomes dramatically increase microtubule nucleating activity, enabling them to form a mitotic spindle. Recent studies show that Aurora A kinase promotes microtubule assembly from centrosomes through the phosphorylation of the conserved centrosomal protein TACC.     

Caenorhabditis elegans TAC-1 and ZYG-9 form a complex that is essential for long astral and spindle microtubules

TACC (transforming acidic coiled-coil) proteins were first identified by their ability to transform cell lines [1], and links between human cancer and the overexpression of TACC proteins highlight the importance of understanding the biological function of this family of proteins. Herein, we describe the characterization of a new member of the TACC family of proteins in Caenorhabditis elegans, TAC-1. In other systems, TACC proteins associate with the XMAP215 family of microtubule-stabilizing proteins; however, it is unclear whether TACC proteins have microtubule-based functions distinct from XMAP215. We depleted both the XMAP215 ortholog ZYG-9 and TAC-1 via dsRNA-mediated interference (RNAi). We found that tac-1(RNAi) resulted in microtubule-based defects that were very similar to zyg-9(RNAi). Furthermore, TAC-1 and ZYG-9 are required for long astral microtubules in general and long spindle microtubules during spindle assembly. Loss of either protein did not affect the alpha-tubulin immunofluorescence intensity near centrosomes; this finding suggests that microtubule nucleation was not compromised. Both proteins localize to centrosomes and the kinetochore/microtubule region of chromosomes in metaphase and early anaphase. Furthermore, we found that ZYG-9 and TAC-1 physically interact in vivo, and this interaction is important for the efficient localization of the ZYG-9/TAC-1 complex to centrosomes.     

D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo

We identify Drosophila TACC (D-TACC) as a novel protein that is concentrated at centrosomes and interacts with microtubules. We show that D-TACC is essential for normal spindle function in the early embryo; if D-TACC function is perturbed by mutation or antibody injection, the microtubules emanating from centrosomes in embryos are short and chromosomes often fail to segregate properly. The C-terminal region of D-TACC interacts, possibly indirectly, with microtubules, and can target a heterologous fusion protein to centrosomes and microtubules in embryos. This C-terminal region is related to the mammalian transforming, acidic, coiled-coil-containing (TACC) family of proteins. The function of the TACC proteins is unknown, but the genes encoding the known TACC proteins are all associated with genomic regions that are rearranged in certain cancers. We show that at least one of the mammalian TACC proteins appears to be associated with centrosomes and microtubules in human cells. We propose that this conserved C-terminal 'TACC domain' defines a new family of microtubule-interacting proteins.     

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.     

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.     

Involvement of poly(ADP-Ribose) polymerase 1 and poly(ADP-Ribosyl)ation in regulation of centrosome function

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.     

Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3-XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A-dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.     

The endocytic adaptor protein ARH associates with motor and centrosomal proteins and is involved in centrosome assembly and cytokinesis

Numerous proteins involved in endocytosis at the plasma membrane have been shown to be present at novel intracellular locations and to have previously unrecognized functions. ARH (autosomal recessive hypercholesterolemia) is an endocytic clathrin-associated adaptor protein that sorts members of the LDL receptor superfamily (LDLR, megalin, LRP). We report here that ARH also associates with centrosomes in several cell types. ARH interacts with centrosomal (gamma-tubulin and GPC2 and GPC3) and motor (dynein heavy and intermediate chains) proteins. ARH cofractionates with gamma-tubulin on isolated centrosomes, and gamma-tubulin and ARH interact on isolated membrane vesicles. During mitosis, ARH sequentially localizes to the nuclear membrane, kinetochores, spindle poles and the midbody. Arh(-/-) embryonic fibroblasts (MEFs) show smaller or absent centrosomes suggesting ARH plays a role in centrosome assembly. Rat-1 fibroblasts depleted of ARH by siRNA and Arh(-/-) MEFs exhibit a slower rate of growth and prolonged cytokinesis. Taken together the data suggest that the defects in centrosome assembly in ARH depleted cells may give rise to cell cycle and mitotic/cytokinesis defects. We propose that ARH participates in centrosomal and mitotic dynamics by interacting with centrosomal proteins. Whether the centrosomal and mitotic functions of ARH are related to its endocytic role remains to be established.     

In isolated human centrosomes,the associated kinases phosphorylate a specific subset of centrosomal proteins

Summary— Several studies have shown that kinases and phosphatases can interact with the centrosome during interphase and mitosis suggesting that centrosomal components might be the targets of these enzymes. The association of the cAMP-dependent protein kinase type II and the mitotic kinase p34^cdc2 with centrosomes from human lymphoblast cells has previously been shown (Keryer et al, 1993, Exp Cell Res 204, 230–240; Bailly et al, 1989, EMBO J 8, 3985–3995). In this paper we demonstrate that isolated centrosomes are able to phosphorylate a few number of centrosomal proteins (M_r 230–220000; 135000 and 50000) and also H1 histone. The phosphorylation of H1-histone is cell cycle dependent and modulated by phosphatases. The use of kinase and phosphatase inhibitors and the addition of the catalytic subunit of cAMP-dependent kinase or of cyclinB-p34^cdc2 kinase showed that both kinases phosphorylate the same centrosomal substrates. In addition two centrosomal proteins (M_r 100000 and 37000) were phosphorylated only by p34^cdc2 kinase. Although the low amount of centrosomal proteins precluded a full characterization of these substrates we discuss the identity of the major centrosomal phosphoproteins by comparison with proteins known to associate with microtubule-organizing centres or mitotic spindles. Our results raise also the intriguing possibility that the cAMP-dependent protein kinase could be regulated by the mitotic kinase at the entry of mitosis.     

Proper recruitment of gamma-tubulin and D-TACC/Msps to embryonic Drosophila centrosomes requires Centrosomin Motif 1

Centrosomes are microtubule-organizing centers and play a dominant role in assembly of the microtubule spindle apparatus at mitosis. Although the individual binding steps in centrosome maturation are largely unknown, Centrosomin (Cnn) is an essential mitotic centrosome component required for assembly of all other known pericentriolar matrix (PCM) proteins to achieve microtubule-organizing activity at mitosis in Drosophila. We have identified a conserved motif (Motif 1) near the amino terminus of Cnn that is essential for its function in vivo. Cnn Motif 1 is necessary for proper recruitment of gamma-tubulin, D-TACC (the homolog of vertebrate transforming acidic coiled-coil proteins [TACC]), and Minispindles (Msps) to embryonic centrosomes but is not required for assembly of other centrosome components including Aurora A kinase and CP60. Centrosome separation and centrosomal satellite formation are severely disrupted in Cnn Motif 1 mutant embryos. However, actin organization into pseudocleavage furrows, though aberrant, remains partially intact. These data show that Motif 1 is necessary for some but not all of the activities conferred on centrosome function by intact Cnn.     

TAC-1 and ZYG-9 form a complex that promotes microtubule assembly in C. elegans embryos

BACKGROUND: Modulation of microtubule dynamics is crucial for proper cell division. While a large body of work has made important contributions to our understanding of the mechanisms governing microtubule dynamics in vitro, much remains to be learned about how these mechanisms operate in vivo. RESULTS: We identified TAC-1 as the sole TACC (Transforming Acidic Coiled Coil) protein in C. elegans. TAC-1 consists essentially of a TACC domain, in contrast to the much larger members of this protein family in other species. We find that tac-1 is essential for pronuclear migration and spindle elongation in one-cell-stage C. elegans embryos. Using an in vivo FRAP-based assay, we establish that inactivation of tac-1 results in defective microtubule assembly. TAC-1 is present in the cytoplasm and is enriched at centrosomes in a cell cycle-dependent manner. Centrosomal localization is independent of microtubules but requires the activity of gamma-tubulin and the Aurora-A kinase AIR-1. By conducting FRAP analysis in embryos expressing GFP-TAC-1, we find that centrosomal TAC-1 exchanges rapidly with the cytoplasmic pool. Importantly, we establish that TAC-1 physically interacts with ZYG-9, a microtubule-associated protein (MAP) of the XMAP215 family, both in vitro and in vivo. Furthermore, we also uncover that TAC-1 and ZYG-9 stabilize each other in C. elegans embryos. CONCLUSIONS: Our findings identify TAC-1 as a core structural and functional member of the evolutionarily conserved TACC family of proteins and suggest that mutual stabilization between TACC and XMAP215 proteins is a key feature ensuring microtubule assembly in vivo.     

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.     

Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus

AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.     

The TACC proteins: TACC-ling microtubule dynamics and centrosome function

A major quest in cell biology is to understand the molecular mechanisms underlying the high plasticity of the microtubule network at different stages of the cell cycle, and during and after differentiation. Initial reports described the centrosomal localization of proteins possessing transforming acidic coiled-coil (TACC) domains. This discovery prompted several groups to examine the role of TACC proteins during cell division, leading to indications that they are important players in this complex process in different organisms. Here, we review the current understanding of the role of TACC proteins in the regulation of microtubule dynamics, and we highlight the complexity of centrosome function.     

Analysis of centrosome localization of BRCA1 and its activity in suppressing centrosomal aster formation

BRCA1, a product of a familial breast and ovarian cancer susceptibility gene, localizes to centrosomes and physically interacts with γ-tubulin, a key centrosomal protein for microtubule nucleation and anchoring at centrosomes. Here, we performed a rigorous analysis of centrosome localization of BRCA1, and found that BRCA1 is specifically associated with mother centrioles in unduplicated centrosomes, and daughter centrioles acquire BRCA1 prior to initiation of duplication, and thus duplicated centrosomes are both bound by BRCA1. We further found that BRCA1 suppresses centrosomal aster formation. In addition, we identified a new domain of BRCA1 critical for γ-tubulin binding, which confers not only its localization to centrosomes, but also its activity to suppress centrosomal aster formation.