Human EML4, a novel member of the EMAP family, is essential for microtubule formation

Human EML4 (EMAP-like protein 4) is a novel microtubule-associated WD-repeat protein of 120 kDa molecular weight, which is classified as belonging to the conserved family of EMAP-like proteins. Cosedimentation assays demonstrated that EML4 associates with in vitro polymerized microtubules. Correspondingly, immunofluorescence stainings and transient expression of EGFP-labeled EML4 revealed a complete colocalization of EML4 with the interphase microtubule array of HeLa cells. We present evidence that the amino-terminal portion of EML4 (amino acids 1-249) is essential for the association with microtubules. Immunoprecipitation experiments revealed that EML4 is hyperphosphorylated on serine/threonine residues during mitosis. In addition, immunofluorescence stainings demonstrated that hyperphosphorylated EML4 is associated with the mitotic spindle, suggesting that the function of EML4 is regulated by phosphorylation. siRNA-mediated knockdown of EML4 in HeLa cells led to a significant decrease in the number of cells. In no case mitotic figures could be observed in EML4 negative HeLa cells. Additionally, we observed a significant reduction of the proliferation rate and the uptake of radioactive [3H]-thymidine as a result of EML4 silencing. Most importantly, EML4 negative cells showed a completely modified microtubule network, indicating that EML4 is necessary for correct microtubule formation.     

The nuclear migration protein NUDF/LIS1 forms a complex with NUDC and BNFA at spindle pole bodies

Nuclear migration depends on microtubules, the dynein motor complex, and regulatory components like LIS1 and NUDC. We sought to identify new binding partners of the fungal LIS1 homolog NUDF to clarify its function in dynein regulation. We therefore analyzed the association between NUDF and NUDC in Aspergillus nidulans. NUDF and NUDC directly interacted in yeast two-hybrid experiments via NUDF's WD40 domain. NUDC-green fluorescent protein (NUDC-GFP) was localized to immobile dots in the cytoplasm and at the hyphal cortex, some of which were spindle pole bodies (SPBs). We showed by bimolecular fluorescence complementation microscopy that NUDC directly interacted with NUDF at SPBs at different stages of the cell cycle. Applying tandem affinity purification, we isolated the NUDF-associated protein BNFA (for binding to NUDF). BNFA was dispensable for growth and for nuclear migration. GFP-BNFA fusions localized to SPBs at different stages of the cell cycle. This localization depended on NUDF, since the loss of NUDF resulted in the cytoplasmic accumulation of BNFA. BNFA did not bind to NUDC in a yeast two-hybrid assay. These results show that the conserved NUDF and NUDC proteins play a concerted role at SPBs at different stages of the cell cycle and that NUDF recruits additional proteins specifically to the dynein complex at SPBs.     

Evolutionary analysis of WD40 super family proteins involved in spindle checkpoint and RNA export: molecular evolution of spindle checkpoint

The spindle checkpoint delays sister chromatid separation until all chromosomes have undergone bipolar spindle attachment. Previous studies have revealed BUB3, as an essential spindle checkpoint protein and its extensive sequence similarity with Rae1 (Gle2), a highly conserved member of WD40 repeat protein family throughout their length which was first shown to be involved in mRNA export. However, the recent discovery of Rae1 as an essential mitotic checkpoint protein, based on the studies from mouse and drosophila, has renewed the interest in its function during cell division. Study of evolution of proteins involved in checkpoint might throw light on evolution of eukaryotic cell cycle regulation. Here we report the evolutionary relationships between these two WD40 repeat family proteins. Amino acid sequences of BUB3 and Rae1 homologs were retrieved from various databases and phylogenetic analysis was performed with the MEGA program. Multiple sequence alignments of these two protein homologues with the ClustalX software revealed specific amino acid signatures corresponding to the protein function and also few amino acids, which are conserved in BUB3 and Rae1 indicating some common overlapping function. Data indicated a common ancestral origin of these two important proteins and further suggest that, BUB3 mediated cell cycle checkpoint might have evolved with compartmentalization of genetic material into the nucleus in eukaryotes.     

Crystal structure of the spindle assembly checkpoint protein Bub3

Bub3 is one of at least six proteins that transmit the spindle assembly checkpoint signal. These proteins delay cell cycle progression from metaphase to anaphase in response to attachment defects between kinetochores and spindle microtubules and to tension defects between sister chromatids. To explore the molecular interactions mediated by Bub3, we have determined the crystal structure of the Saccharomyces cerevisiae protein Bub3p at 2.35 A resolution. Bub3p is a seven-blade beta-propeller, although its sequence diverges from that of other WD40 family members. Several loops are substantially elongated, but extra domains or insertions are not present at the termini. In particular, two extended loops project from the top face of the propeller, forming a cleft. Amino acid residues across the top face and one aspect of the lateral surface (spanning blades 5-6) are highly conserved among Bub3 proteins. We propose that these conserved surfaces are the loci for key interactions with conserved motifs in spindle checkpoint proteins Bub1 and Mad3/BubR1. Comparison of the Bub3 sequence to the WD40 protein, Rae1, shows high sequence conservation along the same surfaces. Rae1 interaction with Bub1 is, therefore, likely to involve a similar mode of binding.     

The centrosomal protein RAS association domain family protein 1A (RASSF1A)-binding protein 1 regulates mitotic progression by recruiting RASSF1A to spindle poles

The protein RAS association domain family protein 1A (RASSF1A), which is encoded by a gene that is frequently silenced in many types of sporadic tumor, functions in mitosis as a regulator of the anaphase-promoting complex (APC). With the use of a yeast two-hybrid screen, we identified a human protein, previously designated C19ORF5, that interacts with RASSF1A. This protein, here redesignated RASSF1A-binding protein 1 (RABP1), contains two microtubule-associated protein domains, and its association with RASSF1A was confirmed in mammalian cells by immunoprecipitation and immunofluorescence analyses. RABP1 was found to be localized to the centrosome throughout the cell cycle in a manner dependent on its microtubule-associated protein domains. Ectopic expression of RABP1 induced both stabilization of mitotic cyclins and mitotic arrest at prometaphase in a RASSF1A-dependent manner. It also increased the extent of association between RASSF1A and Cdc20. Conversely depletion of RABP1 by RNA interference prevented both the localization of RASSF1A to the spindle poles as well as its binding to Cdc20, resulting in premature destruction of mitotic cyclins and acceleration of mitotic progression. These findings indicate that RABP1 is required for the recruitment of RASSF1A to the spindle poles and for its inhibition of APC-Cdc20 activity during mitosis.     

Aurora A phosphorylation of WD40-repeat protein 62 in mitotic spindle regulation

Mitotic spindle organization is regulated by centrosomal kinases that potentiate recruitment of spindle-associated proteins required for normal mitotic progress including the microcephaly protein WD40-repeat protein 62 (WDR62). WDR62 functions underlie normal brain development as autosomal recessive mutations and wdr62 loss cause microcephaly. Here we investigate the signaling interactions between WDR62 and the mitotic kinase Aurora A (AURKA) that has been recently shown to cooperate to control brain size in mice. The spindle recruitment of WDR62 is closely correlated with increased levels of AURKA following mitotic entry. We showed that depletion of TPX2 attenuated WDR62 localization at spindle poles indicating that TPX2 co-activation of AURKA is required to recruit WDR62 to the spindle. We demonstrated that AURKA activity contributed to the mitotic phosphorylation of WDR62 residues Ser49 and Thr50 and phosphorylation of WDR62 N-terminal residues was required for spindle organization and metaphase chromosome alignment. Our analysis of several MCPH-associated WDR62 mutants (V65M, R438H and V1314RfsX18) that are mislocalized in mitosis revealed that their interactions and phosphorylation by AURKA was substantially reduced consistent with the notion that AURKA is a key determinant of WDR62 spindle recruitment. Thus, our study highlights the role of AURKA signaling in the spatiotemporal control of WDR62 at spindle poles where it maintains spindle organization.     

Structural and regulatory roles of nonmotor spindle proteins

Chromosome alignment and segregation during cell division rely on a highly ordered bipolar microtubule array called the mitotic spindle. The organization of microtubules into bipolar spindles with focused poles during mitosis requires numerous microtubule-associated proteins including both motor and nonmotor proteins. Nonmotor microtubule-associated proteins display extraordinary diversity in how they contribute to mitotic spindle organization. These mechanisms include regulation of microtubule nucleation and organization, direct and indirect influences on motor function, and control of cell cycle progression. Furthermore, many nonmotor spindle proteins display altered expression in cancer cells emphasizing their important roles in cell proliferation.     

Phospho-regulation of DDA3 function in mitosis

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. Through a mass spectrometry analysis, we found that DDA3 is phosphorylated on Ser225 during mitosis. Phosphorylation of this residue is important for the mitotic function of DDA3, as the phospho-mimicking DDA3-S225D variant, but not the nonphosphorable DDA3-S225A mutant, rescues the DDA3-knockdown phenotype. We conclude that the mitotic function of DDA3 is regulated by phosphorylation on the Ser225 residue.     

The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1.     

Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization

Through mutational analysis in Drosopjila we have identified the gene multiple asters (mast), which encodes a new 165 kDa protein. mast mutant neuroblasts are highly polyploid and show severe mitotic abnormalities including the formation of mono- and multi-polar spindles organized by an irregular number of microtubule-organizing centres of abnormal size and shape. The mast gene product is evolutionarily conserved since homologues were identified from yeast to man, revealing a novel protein family. Antibodies against Mast and analysis of tissue culture cells expressing an enhanced green fluorescent protein-Mast fusion protein show that during mitosis, this protein localizes to centrosomes, the mitotic spindle, centromeres and spindle midzone. Microtubule-binding assays indicate that Mast is a microtubule-associated protein displaying strong affinity for polymerized microtubules. The defects observed in the mutant alleles and the intracellular localization of the protein suggest that Mast plays an essential role in centrosome separation and organization of the bipolar mitotic spindle.     

The 1.1-angstrom structure of the spindle checkpoint protein Bub3p reveals functional regions

Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and beta-transducin repeats), short sequence motifs appearing in clusters of 4-16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 angstrom and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure.     

Arabidopsis kinetochore fiber-associated MAP65-4 cross-links microtubules and promotes microtubule bundle elongation

The acentrosomal plant mitotic spindle is uniquely structured in that it lacks opposing centrosomes at its poles and is equipped with a connective preprophase band that regulates the spatial framework for spindle orientation and mobility. These features are supported by specialized microtubule-associated proteins and motors. Here, we show that Arabidopsis thaliana MAP65-4, a non-motor microtubule associated protein (MAP) that belongs to the evolutionarily conserved MAP65 family, specifically associates with the forming mitotic spindle during prophase and with the kinetochore fibers from prometaphase to the end of anaphase. In vitro, MAP65-4 induces microtubule (MT) bundling through the formation of cross-bridges between adjacent MTs both in polar and antipolar orientations. The association of MAP65-4 with an MT bundle is concomitant with its elongation. Furthermore, MAP65-4 modulates the MT dynamic instability parameters of individual MTs within a bundle, mainly by decreasing the frequency of catastrophes and increasing the frequency of rescue events, and thereby supports the progressive lengthening of MT bundles over time. These properties are in line with its role of initiating kinetochore fibers during prospindle formation.     

Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18

The way that microtubules reorganize from their long, stable interphase configuration to form the mitotic spindle remains a challenging and unsolved question. It is now widely recognized that microtubule polymerization during the cell cycle is regulated by a balance between microtubule-stabilizing and-destabilizing factors. Stabilizing factors include a large group of microtubule-associated proteins (MAPs; e.g. MAP4, XMAP215, XMAP230/XMAP4 and XMAP310) and the destabilizing factors are a growing family of proteins (e.g. Stathmin/Op18 and XKCM1). Recent studies have allowed a mechanistic dissection of how these stabilizing and destabilizing factors regulate microtubule dynamics and spindle assembly.     

repp86: A human protein associated in the progression of mitosis

Human repp86 becomes detectable in the nucleoplasm of cycling cells at the G(1)-S boundary, condenses at the centrosomes with the onset of mitosis, during which it progressively locates to the mitotic spindle and to the midbody, and vanishes at the completion of cytokinesis. The repp86 cDNA was cloned and sequenced. Full-length repp86 and its COOH-terminal domain cosediment with polymerized microtubules, linking repp86 to the family of microtubule-associated proteins. During prophase and metaphase, repp86 interacts on the mitotic spindle with the putative motor protein Hklp2. Thus, repp86 may function in targeting Hklp2 to the microtubule minus ends, its activity being regulated by phosphorylation of serine/threonine residues. Exogenous overexpression of repp86 provokes accumulation of cells in G(2)-M phase and subsequent polyploidization, suggesting that excess repp86 may interfere with correct nuclear division.     

A Field Guide to the Mps1 Family of Protein Kinases

Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability. The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity. The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis. Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint. More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.     

A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle

Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.     

How do Plants Organize Microtubules Without a Centrosome？

A microtubule nucleates from a γ-tubuUn complex, which consists of γ-tubulin, proteins from the SPC971SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast, the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers （MTOCs）, such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubule-dependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.     

A field guide to the Mps1 family of protein kinases

Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability. The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity. The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis. Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint. More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

The interaction of TOGp with microtubules and tubulin

TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.