Binding of E-MAP-115 to microtubules is regulated by cell cycle- dependent phosphorylation

Expression levels of E-MAP-115, a microtubule-associated protein that stabilizes microtubules, increase with epithelial cell polarization and differentiation (Masson and Kreis, 1993). Although polarizing cells contain significant amounts of this protein, they can still divide and thus all stabilized microtubules must disassemble at the onset of mitosis to allow formation of the dynamic mitotic spindle. We show here that binding of E-MAP-115 to microtubules is regulated by phosphorylation during the cell cycle. Immunolabeling of HeLa cells for E-MAP-115 indicates that the protein is absent from microtubules during early prophase and progressively reassociates with microtubules after late prophase. A fraction of E-MAP-115 from HeLa cells released from a block at the G1/S boundary runs with higher apparent molecular weight on SDS-PAGE, with a peak correlating with the maximal number of cells in early stages of mitosis. E-MAP-115 from nocodazole-arrested mitotic cells, which can be obtained in larger amounts, displays identical modifications and was used for further biochemical characterization. The level of incorporation of 32P into mitotic E-MAP-115 is about 15- fold higher than into the interphase protein. Specific threonine phosphorylation occurs in mitosis, and the amount of phosphate associated with serine also increases. Hyperphosphorylated E-MAP-115 from mitotic cells cannot bind stably to microtubules in vitro. These results suggest that phosphorylation of E-MAP-115 is a prerequisite for increasing the dynamic properties of the interphase microtubules which leads to the assembly of the mitotic spindle at the onset of mitosis. Microtubule-associated proteins are thus most likely key targets for kinases which control changes in microtubule dynamic properties at the G2- to M-phase transition.     

The N-terminal domain of DDA3 regulates the spindle-association of the microtubule depolymerase Kif2a and controls the mitotic function of DDA3

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the dynamics of the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. DDA3 interacts with the microtubule depolymerase Kif2a and controls the association of Kif2a to the mitotic spindle and the dynamic turnover of microtubules in the spindle. To understand the function and regulation of DDA3, we analyzed its domain structure and found that the C-terminal domain of DDA3 directly binds to microtubules in vitro and associates with the mitotic spindle in vivo. The N-terminal domain of DDA3 does not interact with microtubules, but acts dominant negatively over the wild-type protein. Ectopic expression of this domain prevents the endogenous DDA3 from association with the spindle and results in a high frequency of unaligned chromosomes in metaphase cells, a phenotype similar to that in metaphase cells depleted of DDA3. Mechanistically, expression of N-terminal DDA3 reduces the amount of spindle-associated Kif2a and increases the spindle microtubule density, pheno-copying those in DDA3-depleted cells. We conclude that DDA3 has a distinct domain structure. The C-terminal domain confers its ability to associate with the mitotic spindle, while the regulatory N-terminal domain controls the microtubule-binding by the C-terminal domain and determines the cellular activity of the DDA3 protein.     

Molecular components of the mitotic spindle.

Mitotic spindles constitute the machinery responsible for equidistribution of the genetic material into each daughter cell during cell division. They are transient and hence quite labile structures, changing their morphology even while performing their function. Biochemical, immunological and genetic analyses of mitotic cells have allowed us to identify a variety of molecules that are recruited to form the spindle at the onset of mitosis. Evaluation of the roles of these molecules in both the formation and in the dynamics of spindle microtubules should be important for understanding the molecular basis of mitosis and its regulation. We have recently identified a novel mitosis-specific microtubule-associated protein (MAP) using a monoclonal antibody probe raised against the mitotic spindles isolated from cultured mammalian cells. This 95/105 kDa antigen represents a unique component of the spindle distinct from any of the other MAPs reported so far. Antibody microinjection resulted in mitotic inhibition in a stage-specific and dose-dependent manner, indicating that the protein is an essential spindle component.     

Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila melanogaster

We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.     

Studies of a microtubule-associated protein using a monoclonal antibody elicited against mammalian mitotic spindles

We have devised a procedure for the identification of individual molecules which are associated with the mitotic spindle apparatus and cytoskeleton in mammalian cells. We prepared monoclonal antibody-producing hybridomas by immunizing mice with mitotic spindles isolated from cultured HeLa cells. Among several antibody-producing clones obtained, one hybridoma (22MA2) produced an antibody that recognizes a putative microtubule-associated protein which exhibits unusual distribution characteristics in cultured cells. Immunofluorescence studies showed that during mitosis the 22MA2 antigen is distributed in parallel with the spindle fibers of the mitotic apparatus, and that during interphase the antigen is always associated to a limited extent with cytoplasmic microtubules. Also, the co-distribution of the antigen with microtubules was found to be Colcemid sensitive. However, the 22MA2 antibody immunofluorescently stained the nuclei of cells in the exponential growth phase, but did not stain the nuclei of cells that had grown to confluence. This nuclear fluorescence appears to be directly related to cell density rather than nutritional (serum) factors in the growth medium. The results suggest that the antigen undergoes some change in structure or distribution in response to changes in the proliferative capacity of the cell. Biochemical analyses of cytoplasmic, nuclear, and mitotic spindle subcellular fractions show that the antigen exhibits a polypeptide molecular weight of 240,000 is found in various mammalian cells ranging from marsupial to human, and is particularly susceptible to proteolysis.     

CSAP localizes to polyglutamylated microtubules and promotes proper cilia function and zebrafish development

The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here we identify the microtubule-binding protein centriole and spindle-associated protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left-right asymmetry, defects that are qualitatively similar to those reported previously for depletion of polyglutamylation-conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations, providing specific functional capabilities to these populations.     

Identification of a minus end-specific microtubule-associated protein located at the mitotic poles in cultured mammalian cells.

A mitosis-specific centrosomal component was studied with a human autoantibody, SP-H, which immunostained mitotic poles and interphase nuclei, and a single polypeptide with an apparent molecular mass of 200 to 230 kDa in various lines of cultured cells. Early mitotic PtK1 cells treated with 10 micrograms/ml taxol contained short bundles of parallel microtubules around the nuclei and cell periphery. At the time of nuclear envelope breakdown, the nuclear staining by SP-H disappeared, and the antigen relocated at one end of the parallel microtubules. Determination of the microtubule polarity demonstrated that the peripheral bundles of microtubules were arranged with their minus ends directed to the cell periphery, and the SP-H antigen was specifically localized at this end. Parallel microtubules were further rearranged first into a fan-like shape, and then into completely radial structures as observed by De Brabander et al. (Int. Rev. Cytol. 101, 215-274 (1986)). The SP-H antigen was always detected at the minus end domain of such microtubule-containing structures during the transformation process. When microtubules were depolymerized by nocodazole treatment, the SP-H antigen appeared as discrete cytoplasmic foci, suggesting that the antigen may self-associate, forming multimeric structures. The antigen in mitotic HeLa cell extracts co-sedimented in vitro with exogenous brain microtubules. The microtubule-associated SP-H antigen was insensitive to ATP extraction, but was removed from microtubules by treatment with 0.5 M NaCl. Thus the 200 to 230 kDa centrosomal component could be a novel microtubule-associated protein with affinity for the minus end of microtubules, and it might play an essential role in the organization of spindle poles during mitosis.     

Spindle pole mechanics studied in mitotic asters: dynamic distribution of spindle forces through compliant linkages

During cell division, chromosomes must faithfully segregate to maintain genome integrity, and this dynamic mechanical process is driven by the macromolecular machinery of the mitotic spindle. However, little is known about spindle mechanics. For example, spindle microtubules are organized by numerous cross-linking proteins yet the mechanical properties of those cross-links remain unexplored. To examine the mechanical properties of microtubule cross-links we applied optical trapping to mitotic asters that form in mammalian mitotic extracts. These asters are foci of microtubules, motors, and microtubule-associated proteins that reflect many of the functional properties of spindle poles and represent centrosome-independent spindle-pole analogs. We observed bidirectional motor-driven microtubule movements, showing that microtubule linkages within asters are remarkably compliant (mean stiffness 0.025 pN/nm) and mediated by only a handful of cross-links. Depleting the motor Eg5 reduced this stiffness, indicating that Eg5 contributes to the mechanical properties of microtubule asters in a manner consistent with its localization to spindle poles in cells. We propose that compliant linkages among microtubules provide a mechanical architecture capable of accommodating microtubule movements and distributing force among microtubules without loss of pole integrity—a mechanical paradigm that may be important throughout the spindle.     

NuMA is required for the organization of microtubules into aster-like mitotic arrays

NuMA (Nuclear protein that associates with the Mitotic Apparatus) is a 235-kD intranuclear protein that accumulates at the pericentrosomal region of the mitotic spindle in vertebrate cells. To determine if NuMA plays an active role in organizing the microtubules at the polar region of the mitotic spindle, we have developed a cell free system for the assembly of mitotic asters derived from synchronized cultured cells. Mitotic asters assembled in this extract are composed of microtubules arranged in a radial array that contain NuMA concentrated at the central core. The organization of microtubules into asters in this cell free system is dependent on NuMA because immunodepletion of NuMA from the extract results in randomly dispersed microtubules instead of organized mitotic asters, and addition of the purified recombinant NuMA protein to the NuMA-depleted extract fully reconstitutes the organization of the microtubules into mitotic asters. Furthermore, we show that NuMA is phosphorylated upon mitotic aster assembly and that NuMA is only required in the late stages of aster assembly in this cell free system consistent with the temporal accumulation of NuMA at the polar ends of the mitotic spindle in vivo. These results, in combination with the phenotype observed in vivo after the prevention of NuMA from targeting onto the mitotic spindle by antibody microinjection, suggest that NuMA plays a functional role in the organization of the microtubules of the mitotic spindle.     

Distribution of cytoskeletal proteins sharing a conserved phosphorylated epitope

A group of antigens related by their reactivity with monoclonal antibodies MPM-1 and MPM-2 appear as cells enter mitosis. These antibodies bind to a phosphorylated epitope on certain proteins, and therefore the antigens are presumed to be a group of phosphoproteins. A subset of these proteins has been shown previously to be components of mitotic microtubule organizing centers in PtK1 cells. We present here evidence that the mitosis-specific appearance of these phosphoproteins is a phenomenon common to all eukaryotic cells. The MPM reactive phosphoproteins were localized to mitotic spindle poles regardless of whether the spindle formed in the cytoplasm after nuclear envelope breakdown (open mitosis) or within the nucleus (closed mitosis). This reactivity was not dependent upon the presence of centrioles at the spindle poles. Proteins that contained the phosphorylated epitope were not, however, restricted to mitotic cells. Cells of neuronal derivation and flagellated cells showed specific localization of MPM antibody to the microtubule network and basal bodies respectively. On immunoblots, the MPM antibody reacted with brain MAP-1 among a number of other phosphoproteins. The identification of microtubule-associated protein (MAP)-1 correlates with the localization of the antibody to microtubules of neuroblastoma cells. These results suggest, that different phosphoprotein molecules detected by the MPM antibody may be specific for different mitotic microtubule organizing centers, basal bodies, and other specialized cytoskeletal structures; and the presence of a related phosphorylated domain on these proteins may be important for their proper function and/or interaction with microtubules.     

Opposing motor activities are required for the organization of the mammalian mitotic spindle pole

We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end- directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.     

A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences

Microtubules and microtubule-associated proteins (MAPs) have been isolated from cultured cells of Drosophila melanogaster by a taxol-dependent polymerization procedure. The principal MAPs are a group of four polypeptides with similar electrophoretic mobilities corresponding to approximately Mr 205,000 (the 205K MAP). These proteins are resistant to precipitation by boiling. One mouse monoclonal antibody and one polyclonal rabbit antiserum specific for the Mr 205,000 MAP were produced and characterized by immunoblotting and indirect immunofluorescence. Both antibody preparations stain the Mr 205,000 molecules and an Mr 255,000 molecule in immunoblots of Drosophila cell homogenates; the rabbit antiserum also stains an Mr 150,000 triplet. Both preparations stain the microtubules of the mitotic spindle, and the rabbit antiserum stains the cytoplasmic microtubules as well. Experiments using affinity-purified rabbit antiserum demonstrate that it is the Mr 205,000 species that is located in the mitotic apparatus and on cytoplasmic microtubules. A random shear genomic library was produced in the expressing vector lambda gt11 and screened with the rabbit antiserum to isolate the DNA sequences encoding these polypeptides. Several cross-hybridizing clones were recovered, shown to encode antigenic determinants in the Mr 205,000 MAP, and characterized by hybridization to Northern blots of mRNA and Southern blots of genomic DNA. Analysis by in situ hybridization reveals that the gene encoding the 205K MAP is located in polytene region 100EF.     

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.     

Dynamic microtubules are essential for efficient chromosome capture and biorientation in S. cerevisiae

Attachment of chromosomes to the mitotic spindle has been proposed to require dynamic microtubules that randomly search three-dimensional space and become stabilized upon capture by kinetochores. In this study, we test this model by examining chromosome capture in Saccharomyces cerevisiae mutants with attenuated microtubule dynamics. Although viable, these cells are slow to progress through mitosis. Preanaphase cells contain a high proportion of chromosomes that are attached to only one spindle pole and missegregate in the absence of the spindle assembly checkpoint. Measurement of the rates of chromosome capture and biorientation demonstrate that both are severely decreased in the mutants. These results provide direct evidence that dynamic microtubules are critical for efficient chromosome capture and biorientation and support the hypothesis that microtubule search and capture plays a central role in assembly of the mitotic spindle.     

Ultrastructure and ATPase activity of the centriolar body at the mitotic stage in neoblasts of the planarianDugesia sp.

The fine structure and ATPase activity of the mitotic spindle in neoblasts of planaria were examined. In neoblasts, the cells have a large nucleus and nucleolus. Mitochondria are aggregated around the nucleus with chromatoid bodies adjacent. The cytoplasm contains little endoplasmic reticulum (ER) and few Golgi bodies but many free ribosomes, forming polysomes, can be seen throughout the cytoplasmic and spindle ground areas. In addition, centriolar bodies, atypical centrioles, can also be recognized in the cytoplasm. Cells in the G₂ stage contain a pair of electron-dense bodies, both consisting of fibrogranules but differing from each other in fine structure and, in the mitotic stage, only one fibrogranular body can be recognized at each pole. ATPase activity was detected in the centriolar bodies in the G₂ and mitotic stages and in the ground area of the cytoplasm and spindle apparatus filled by free ribosomes. The activity associated with the microtubules differed with the developmental stage.     

Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.     

NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.     

Dr. Dolittle and the making of the mitotic spindle

The intrinsic polarity of microtubules within cells is exploited each time cells divide. Kinesins, microtubule-associated motor proteins, are required to execute the dramatic events of mitosis: bipolar spindle assembly, metaphase chromosome alignment, anaphase chromosome segregation, and separation of spindle poles prior to cytokinesis. Surprisingly, kinesin-related proteins have been found to move in either "plus-ward" or "minus-ward" directions along microtubules. Evidence from genetic analyses of simple eukaryotes and in vitro activity assays supports the notion that certain subfamilies of kinesin-related proteins provide antagonistic activities necessary to balance mitotic forces. A recent study by Sharp et al.((1)) sheds further light on the subject by exploiting the genetics and cytology of the fruit fly embryo.     

How Do Kinetochores CLASP Dynamic Microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizesto the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.