A nuclear matrix-associated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D.

One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization. All isoforms primarily localized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region. Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity.

Filamentous polymers induced by overexpression of a novel centrosomal protein, Cep135.

A novel 135 kDa centrosomal component (Cep135) was identified by immunoscreening of a mammalian expression library with monoclonal antibodies raised against clam centrosomes. It is predicted to be a highly coiled-coil protein with an extensive alpha-helix, suggesting that Cep135 is a structural component of the centrosome. To evaluate how the protein is arranged in the centrosomal structure, we overexpressed Cep135 polypeptides in CHO cells by transient transfection. HA- or GFP-tagged full (amino acids 1-1144) as well as truncated (#10, 29-1144; Delta3, 29-812) polypeptides become localized at the centrosome and induce cytoplasmic dots of various size and number in CHO cells. Centrosomes are associated with massive approximately 7 nm filaments and dense particles organized in a whorl-like arrangement in which parallel-oriented dense lines appear with a regular approximately 7 nm periodicity. The same filamentous aggregates are also detected in cytoplasmic dots, indicating that overexpressed Cep135 can assemble into elaborate higher-ordered structures in and outside the centrosome. Sf9 cells infected with baculovirus containing Cep135 sequences induce filamentous polymers which are distinctive from the whorl seen in CHO cells; #10 forms highly packed spheroids, but the Delta3-containing structure looks loose. Both structures show an internal repeating unit of dense and less dense stripes. Although the distance between the outer end of two adjacent dense lines is similar between two types of polymers ( approximately 120 nm), the dense stripe of Delta3 polymers ( approximately 40 nm) is wider than #10 ( approximately 30 nm). The light band of Delta3 ( approximately 40 nm) is thus narrower than #10 ( approximately 60 nm). Since thin fibers are frequently seen to extend from one dense line to the next, the coiled-coil rod of Cep135 may span the light band. These results suggest that overexpressed Cep135 assemble into distinctive polymers in a domain-specific manner.

Function of a minus-end-directed kinesin-like motor protein in mammalian cells.

CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.

Interaction of an Overexpressed gamma-Tubulin with Microtubules In Vivo and In Vitro.

gamma-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for gamma-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus gamma-tubulin. When CHO cells were transiently transfected with the gamma-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such gamma-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that gamma-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas gamma-tubulin purified from insect Sf9 cells (), interaction between gamma-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with gamma-tubulin monomers in vitro were associated with more gold particles conjugated with gamma-tubulin than in controls where no exogenous gamma-tubulin was added. However, binding of gamma-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although gamma-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (; ), which might be ascribed to the difference in the level of protein expression in transfected cells.

The nuclear/mitotic apparatus protein NuMA is a component of the somatodendritic microtubule arrays of the neuron.

Neurons are terminally post-mitotic cells that utilize their microtubule arrays for the growth and maintenance of axons and dendrites rather than for the formation of mitotic spindles. Recent studies from our laboratory suggest that the mechanisms that organize the axonal and dendritic microtubule arrays may be variations on the same mechanisms that organize the mitotic spindle in dividing cells. In particular, we have identified molecular motor proteins that serve analogous functions in the establishment of these seemingly very different microtubule arrays. In the present study, we have sought to determine whether a non-motor protein termed NuMA is also a component of both systems. NuMA is a approximately 230 kDa structural protein that is present exclusively in the nucleus during interphase. During mitosis, NuMA forms aggregates that interact with microtubules and certain motor proteins. As a result of these interactions, NuMA is thought to draw together the minus-ends of microtubules, thereby helping to organize them into a bipolar spindle. In contrast to mitotic cells, post-mitotic neurons display NuMA both in the nucleus and in the cytoplasm. NuMA appears as multiple small particles within the somatodendritic compartment of the neuron, where its levels increase during early dendritic differentiation. A partial but not complete colocalization with minus-ends of microtubules is suggested by the distribution of the particles during development and during drug treatments that alter the microtubule array. These observations provide an initial set of clues regarding a potentially important function of NuMA in the organization of microtubules within the somatodendritic compartment of the neuron.

Inhibition of a mitotic motor compromises the formation of dendrite-like processes from neuroblastoma cells.

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.

TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells.

Analysis of a cDNA for a 125 kDa polypeptide, previously isolated from phragmoplasts of tobacco BY-2 cells as a candidate for a plus end-directed microtubule motor, revealed this polypeptide to be a novel member of the kinesin superfamily. We named this protein TKRP125 (tobacco kinesin-related polypeptide of 125 kDa). The strong similarity between TKRP125 and members of the bimC subfamily in terms of the amino acid sequence of the amino-terminal motor domain indicated that TKRP125 belonged to the bimC subfamily. An antibody against a short peptide from the motor domain of TKRP125 inhibited the GTP- or ATP-dependent translocation of phragmoplast microtubules in membrane-permeabilized BY-2 cells, suggesting a role for TKRP125 in microtubule translocation, which is considered to be involved in the formation and/or maintenance of the bipolar structure of the phragmoplast. The expression of TKRP125 was found to be cell cycle-dependent. TKRP125 was not present in cells at the G1 phase. It began to appear at the S phase and accumulated during the G2 phase. The distribution of TKRP125 changed as the arrangement of microtubules changed with the progression of the cell cycle. TKRP125 was distributed along cortical microtubules during the S phase and along microtubules in the preprophase band and perinuclear microtubules in premitotic cells. It was also present in the nucleus in premitotic cells. In cells in M phase, TKRP125 was distributed along spindle microtubules. It accumulated at the equatorial plane of the spindle as the spindle elongated. In cytokinetic cells, TKRP125 was colocalized with phragmoplast microtubules. These observations suggest the possible involvement of TKRP125 in the cell cycle-dependent changes in arrays of microtubules, including the organization of the phragmoplast, and in the movement of chromosomes in anaphase cells.

Abnormal centrosome amplification in the absence of p53.

The centrosome plays a vital role in mitotic fidelity, ensuring establishment of bipolar spindles and balanced chromosome segregation. Centrosome duplication occurs only once during the cell cycle and is therefore highly regulated. Here, it is shown that in mouse embryonic fibroblasts (MEFs) lacking the p53 tumor suppressor protein, multiple copies of functionally competent centrosomes are generated during a single cell cycle. In contrast, MEFs prepared from normal mice or mice deficient in the retinoblastoma tumor suppressor gene product do not display these abnormalities. The abnormally amplified centrosomes profoundly affect mitotic fidelity, resulting in unequal segregation of chromosomes. These observations implicate p53 in the regulation of centrosome duplication and suggest one possible mechanism by which the loss of p53 may cause genetic instability.

Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro.

Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.

Using the CHO2 monoclonal antibody raised against CHO spindles (Sellitto, C., M. Kimble, and R. Kuriyama. 1992. Cell Motil. Cytoskeleton. 22:7-24) we identified a 66-kD protein located at the interphase centrosome and mitotic spindle. Isolated cDNAs for the antigen encode a 622-amino acid polypeptide. Sequence analysis revealed the presence of 340-amino acid residues in the COOH terminus, which is homologous to the motor domain conserved among other members of the kinesin superfamily. The protein is composed of a central alpha-helical portion with globular domains at both NH2 and COOH termini, and the epitope to the monoclonal antibody resides in the central alpha-helical stalk. A series of deletion constructs were created for in vitro analysis of microtubule interactions. While the microtubule binding and bundling activities require both the presence of the COOH terminus and the alpha-helical domain, the NH2-terminal half of the antigen lacked the ability to interact with microtubules. The full-length as well as deleted proteins consisting of the COOH-terminal motor and the central alpha-helical stalk supported microtubule gliding, with velocity ranging from 1.0 to 8.4 microns/minute. The speed of microtubule movement decreased with decreasing lengths of the central stalk attached to the COOH-terminal motor. The microtubules moved with their plus end leading, indicating that the antigen is a minus end-directed motor. The CHO2 sequence shows 86% identify to HSET, a gene located at the centromeric end of the human MHC region in chromosome 6 (Ando, A., Y. Y. Kikuti, H. Kawata, N. Okamoto, T. Imai, T. Eki, K. Yokoyama, E. Soeda, T. Ikemura, K. Abe, and H. Inoko. 1994. Immunogenetics. 39:194-200), indicating that HSET might represent a human homologue of the CHO2 antigen.

Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence.

A new member of the tubulin superfamily, gamma-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa gamma-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell supernatant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin separated into two major peaks. The lagging peak contained a monomeric form of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. The released gamma-tubulin monomers were capable of binding to microtubules in vitro and biochemical quantities of active monomers were further purified using a combination of size-exclusion and ion-exchange column chromatography. The endogenous Sf9 cell gamma-tubulin migrated faster than Chlamydomonas gamma-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas gamma-tubulin was present in a monomeric form, endogenous gamma-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).

Heterogeneity and microtubule interaction of the CHO1 antigen, a mitosis-specific kinesin-like protein. Analysis of subdomains expressed in insect sf9 cells.

The CHO1 antigen is a mitosis-specific kinesin-like motor located at the interzonal region of the spindle. The human cDNA coding for the antigen contains a domain with sequence similarity to the motor domain of kinesin-like protein (Nislow et al., Nature 359, 543, 1992). Here we cloned cDNAs encoding the CHO1 antigen by immunoscreening of a CHO Uni-Zap expression library, the same species in which the original monoclonal antibody was raised. cDNAs of CHO cells encode a 953 amino acid polypeptide with a calculated molecular mass of 109 kDa. The N-terminal 73% of the antigen was 87% identical to the human clone, whereas the remaining 27% of the coding region showed only 48% homology. Insect Sf9 cells infected with baculovirus containing the full-length insert produced 105 and 95 kDa polypeptides, the same doublet identified as the original antigen in CHO cells. Truncated polypeptides corresponding to the N-terminal motor and C-terminal tail produced a 56 and 54 kDa polypeptide in Sf9 cells, respectively. Full and N-terminal proteins co-sedimented with, and caused bundling of, brain microtubules in vitro, whereas the C-terminal polypeptide did not. Cells expressing the N terminus formed one or more cytoplasmic processes. Immunofluorescence as well as electron microscopic observations revealed the presence of thick bundles of microtubules, which were closely packed, forming a marginal ring just beneath the cell membrane and a core in the processes. The diffusion coefficient and sedimentation coefficient were determined for the native CHO1 antigen by gel filtration and sucrose density gradient centrifugation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Similarities between somatic cells overexpressing the mos oncogene and oocytes during meiotic interphase.

The mos protooncogene encodes a serine/threonine kinase and is a key regulator of oocyte meiotic maturation. After acute infection of Swiss 3T3 cells with virus containing the v-mos oncogene, cells expressing high levels of v-Mos round up and detach from the monolayer (floating cells), while cells that remain attached express 10-fold lower levels of v-Mos and are transformed. The floating cells are growth arrested with their chromosomes partially condensed in the absence of histone H1 kinase activity, while mitogen-activated protein kinase activity is very high. Collectively, these properties are similar to properties observed in maturing oocytes between meiosis I and II. In v-mos-transformed cell populations, mitogen-activated protein kinase activity is also elevated, correlating with the degree of morphological transformation and the level of Mos expression. Moreover, phosphoprotein modifications specific for M are found in both the floating cells and in v-mos-transformed cells, regardless of their cell cycle stage. One explanation for both morphological transformation and the phenotypes of the floating cells is that Mos imposes a meiotic program on different stages of the somatic cell cycle. The extent of this meiotic phenotype is proportional to the level of v-Mos expression. These results suggest that both morphological transformation and the phenotypes of the floating cells induced by Mos in Swiss 3T3 cells are related to its normal activities during oocyte maturation.

Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic MAP located at the spindle pole and characterized as a homologous protein to NuMA.

Using a human autoantibody, SP-H, we identified a 200-230 kDa mitotic MAP in a variety of mammalian cell lines which shows affinity for the minus end of microtubules and also becomes associated with the spindle pole during mitosis. To examine the detailed structure and functional organization of the protein, the gene coding for the end-specific MAP was isolated and characterized by screening a human placenta lambda gt11 expression library using SP-H as a probe. Overlapping cDNA clones, which covered the entire length of the coding region of the SP-H antigen, were obtained. Polyclonal antibodies raised against fusion proteins generated from non-overlapping cDNA fragments stained the HeLa SP-H antigen in interphase and mitotic cells, and recognized a single 215 kDa band on immunoblots, as did the original SP-H antibody. Analysis of the nucleotide sequence revealed a 7,091 nucleotide sequence with an open reading frame of 6,345 nucleotides encoding a 2,115 amino acid polypeptide with a calculated molecular mass of 238,376 Da. The predicted amino acid sequence showed the protein to be composed of an alpha-helical domain, flanked by globular domains located at the amino and carboxy termini. The sequence contained five repeats of the hypothetical leucine zipper motif: one is in the N-terminal globular domain, and four are in the central alpha-helical stalk. Comparison with other sequences in the database shows that the SP-H antigen is identical to the NuMA protein reported by Yang et al. (1992) J. Cell Biol. 116, 1303-1317, but there are differences between the SP-H antigen and NuMA sequence reported by Compton et al. (1992) J. Cell Biol. 116, 1395-1408. cDNA inserts of the truncated SP-H antigen were expressed in both insect Sf9 cells and in cultured mammalian cells. The recombinant protein corresponding to the C-terminal half of the protein was restricted to the nucleus, whereas the N-terminal half of the protein was localized in the cytoplasm, suggesting the presence of a nuclear translocation signal(s) in the C-terminal domain. The C-terminal polypeptide expressed in mitotic COS cells was shown to specifically localize at the spindle pole. Microtubule-binding assays using in vitro transcribed/translated polypeptide products from different domains of the SP-H antigen further suggested that the SP-H antigen interacts with microtubules through the globular domain at the C-terminus.

A plus-end-directed motor enzyme that moves antiparallel microtubules in vitro localizes to the interzone of mitotic spindles.

Mitosis comprises a complex set of overlapping motile events, many of which involve microtubule-dependent motor enzymes. Here we describe a new member of the kinesin superfamily. The protein was originally identified as a spindle antigen by the CHO1 monoclonal antibody and shown to be required for mitotic progression. We have cloned the gene that encodes this antigen and found that its sequence contains a domain with strong sequence similarity to the motor domain of kinesin-like proteins. The product of this gene, expressed in bacteria, can cross-bridge antiparallel microtubules in vitro, and in the presence of Mg-ATP, microtubules slide over one another in a fashion reminiscent of microtubule movements during spindle elongation.

Heterogeneity of microtubule organizing center components as revealed by monoclonal antibodies to mammalian centrosomes and to nucleus-associated bodies from dictyostelium.

The molecular composition of two morphologically distinct microtubule-organizing centers (MTOCs) was compared by probing with monoclonal antibodies raised against (i) nucleus-associated bodies (NABs) isolated in a complex with nuclei from the cellular slime mold Dictyostelium discoideum and (ii) mammalian mitotic spindles isolated from Chinese hamster ovary (CHO) cells. The staining patterns observed by immunofluorescence microscopy in whole CHO cells and Dictyostelium amoebae showed that the distribution of thirteen MTOC antigens is heterogeneous. Not all antibodies recognized the MTOC in both interphase and mitosis. Most of the anti-MTOC antibodies cross-reacted with other cellular organelles such as nuclei, Golgi apparatus-like aggregates and cytoskeletal elements. Two antibodies, CHO3 and AX3, recognized phosphorylated epitopes present in both mammalian centrosomes and Dictyostelium NABs. On immunoblots, most of the antibodies showed multiple bands, often of high molecular weight, indicating that the antigenic determinants are shared among different molecules. One antibody inhibited the regrowth of microtubules onto centrosomes in vitro after addition of exogenous tubulin to detergent-lysed CHO cells on coverslips; this antibody binds to an antigen(s) that might be essential for the microtubule-nucleating activity of centrosomes. These observations demonstrate that molecular components in different MTOCs exhibit a variety of distinct subcellular localizations and functional properties, and that some antigenic molecules have been conserved among morphologically distinct MTOCs.

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.

A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression.

A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.

Immunocytochemical evidence for centrosomal phosphoproteins in mitotic sea urchin eggs.

The change in distribution of centrosomal phosphoproteins was examined in sea urchin eggs from fertilization to the first cleavage by immunofluorescence staining with the anti-phosphoprotein antibodies, MPM-1 and MPM-2. The antibodies reacted with female pronuclei in unfertilized eggs as well as centriolar complexes located at the base of sperm flagella. After insemination, male and female pronuclei fused together to form a zygotic nucleus which was visualized by staining of fertilized eggs with the antiphosphoprotein antibodies. No major change in staining pattern was detected in extracted whole eggs until mitosis. As the fertilized eggs approached mitosis, however, the antigens started to redistribute from nuclei to the perinuclear position where the mitotic centrosomes were located. Detailed immunofluorescence observation of isolated spindles revealed that the phosphoantigens were retained in isolated structures. A major 225 kd polypeptide was recognized by the antibodies, suggesting that the 225 kd protein is a phosphocomponent of centrosomes. The area recognized by the antibody in mitotic poles enlarged with the progress of mitosis, suggesting that the antigens were apparently localized in the centrosphere. Centrospheres prepared from isolated spindles by salt extraction strongly reacted with the antibodies. One or two bright dots, which may represent centrioles, were visible in the isolated centrosphere. At the end of mitosis, the antigens again appeared in the newly formed daughter nuclei. Centriole-containing cytasters and centriole-free monasters were parthenogenetically induced in unfertilized eggs (Kuriyama and Borisy, (1983) J. Cell Sci. 61: 175-189). The antibodies stained centers of both the asters whether they contained centrioles or not, indicating that the antibodies recognizes the components of the pericentriolar material.