Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.

Abstract. We previously demonstrated (Ookata et al., 1992(Ookata et al., , 1993 that the p34~¢2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34 ~dc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anticyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34~d~Vcyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34~cVcyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34 ~c2, cyclin B, and the COOHterminal domain of MAP4, Phu, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34 ~d~2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.p ROGaESSION through M-phase of the cell cycle is controlled by M-phase promoting factor (MPF) i, which consists of a complex of p34 cd~2 and cyclin B (for

Septins are a family of conserved proteins implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking, but their properties and modes of action are largely unknown. Here we now report findings of immunocytochemical and biochemical characterization of a mammalian septin, MSF-A. Using an antibody specific for MSF subfamily proteins, MSF-A was found to be expressed predominantly in mammary human mammary epithelial cells (HMEC). MSF-A was associated with microtubules in interphase HMEC cells as it localized with the mitotic spindle and the bundle of microtubule at midzone during mitosis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs caused incomplete cell division and resulted in the accumulation of binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to incorporation of MSF-A into septin filaments.Septins, a family of heteropolymeric filament-forming proteins, were originally discovered in yeast to be essential for budding, and have since been identified in most eukaryotic organisms, with the exception of plants (for review, see Refs.

The tobacco mitogen-activated protein kinase (MAPK) cascade, which includes MAPK NRK1/NTF6, positively regulates expansion of the cytokinetic machinery known as the phragmoplast, which is followed by the synthesis of cell plates for completion of cell division. However, molecular events lying between the MAPK and phragmoplast expansion were not known. Here, we show that NRK1/NTF6 phosphorylates the threonine residue at position 579 in NtMAP65-1a, a microtubule-associated (MT-associated) protein. Levels of phosphorylated NtMAP65-1 increase during late M phase of the cell cycle, when NRK1/NTF6 is activated. Phosphorylated NtMAP65-1 is concentrated at the equator of phragmoplast, as is NRK1/NTF6. Overexpression of mutant forms of NtMAP65-1a that cannot be phosphorylated by NRK1 delays progression of the M phase and phragmoplast expansion, also rendering phragmoplast structures resistant to an MT-depolymerizing drug. Phosphorylation of NtMAP65-1 by NRK1/NTF6 down-regulates its MT-bundling activity in vitro. These results suggest that phosphorylation of NtMAP65-1 by NRK1/NTF6 also reduces its MT-bundling activity in vivo, which enhances destabilization and turnover of MTs at the phragmoplast equator, perhaps facilitating phragmoplast expansion.[Keywords: MAP65/PRC1; MAPK; cytokinesis; microtubule; microtubule-associated proteins; phragmoplast] Supplemental material is available at http://www.genesdev.org. Cell division in eukaryotes requires dynamic changes in cytoskeletal structures that consist of microtubules (MTs) and microfilaments Jürgens 2005). During cytokinesis, the final critical step in the cell division, cells form a cytokinesis-specific apparatus (known as the central spindle in animals and the phragmoplast in plants) that plays a key role in cytokinesis (Field et al. 1999;. These structures develop from late anaphase to telophase between the two daughter nuclei, and consist of two bundles of antiparallel MTs. As cytokinesis proceeds, the central spindle becomes compacted in animal cells and the phragmoplast expands centrifugally in plant cells, and new membranes and/or cell walls are generated inside or outside the midzone of the central spindle or the phragmoplast to separate the two daughter cells from each other (Otegui et al. 2005). These dynamic processes appear to be mediated by the turnover of MTs, which involves the depolymerization of MTs and the polymerization of tubulin at the plus ends (Shelden and Wadsworth 1990;Asada et al. 1991;Hush et al. 1994;Straight and Field 2000). Recently, Austin et al. (2005) have reported that somatic-type phragmoplast MTs do not interdigitate at the cell plate mid-line and that distinct MT plus-end geometries are seen during the different stages of cytokinesis.The members of the MAP65/Ase1/PRC1 family (designated the MAP65 family herein)-including plant MAP65, yeast Ase1, and human PRC1-participate in the bundling of MTs (Chang-Jie and Sonobe 1993;Chan et al. 1999;Mollinari et al. 2002;Schuyler et al. 2003;

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We identified the first TUBB1 mutation, R318W, in a patient with congenital macrothrombocytopenia. The patient was heterozygous for Q43P, but this single-nucleotide polymorphism (SNP) did not relate to macrothrombocytopenia. Although no abnormal platelet ␤1-tubulin localization/marginal band organization was observed, the level of ␤1-tubulin was decreased by approximately 50% compared with healthy controls. Large and irregular bleb protrusions observed in megakaryocytes derived from the patient's peripheral blood CD34 ؉ cells suggested impaired megakaryocyte fragmentation and release of large platelets. In vitro transfection experiments in Chinese hamster ovary (CHO) cells demonstrated no incorporation of mutant ␤1-tubulin into microtubules, but the formation of punctuated insoluble aggregates. These results suggested that mutant protein is prone to aggregation but is unstable within megakaryocytes/platelets. Alternatively, mutant ␤1-tubulin may not be transported from the megakaryocytes into platelets. W318 ␤1-tubulin may interfere with normal platelet production, resulting in macrothrombocytopenia. IntroductionCongenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. [1][2][3] The most frequent forms include MYH9 disorders, such as May-Hegglin anomaly, and Bernard-Soulier syndrome. In approximately half of the cases the pathogenesis remains unknown; thus, a definite diagnosis is not possible. The linkage between the membrane skeleton and cytoskeletal actin filaments as well as the marginal microtubule band maintains normal platelet morphology. 4,5 Defects in these systems may result in macrothrombocytopenia. The microtubules are assembled from ␣-and ␤-tubulin heterodimers. ␤1-Tubulin expression is restricted in the megakaryocyte/platelet lineage. 6 Tubb1 knockout mice show thrombocytopenia and spherical platelets. 7 TUBB1 Q43P functional polymorphism has been reported. However, it may not be relevant to macrothrombocytopenia. 8 We identified the first TUBB1 mutation affecting microtubule assembly in congenital macrothrombocytopenia. Methods PatientThe patient was a 7-year-old boy who was incidentally found to have thrombocytopenia (platelets, 40-60 ϫ 10 9 /L). He was diagnosed with immune thrombocytopenic purpura. Peripheral blood smears showed the prominent appearance of giant platelets. Electron microscopy showed no other abnormalities ( Figure 1A,B). There were no leukocyte inclusion bodies, confirmed by myosin IIA localization. 9 The platelets aggregated normally with adenosine diphosphate (ADP), collagen, and ristocetin. Flow cytometry showed normal expression of platelet GPIb/IX. An initial bone marrow examination revealed normal megakaryocyte number and morphology. The mother of the patient also had macrothrombocytopenia. Peripheral blood samples were obtained after the mother gave informed consent in accordance with the Declaration of Helsinki for the study, which was approved by the...