Antibodies specific for mitotic human chromosomes

The induction of premature chromosome condensation in an interphase cell immediately following fusion with a mitotic cell suggests the presence of factors in the mitotic cell that are responsible for the transformation of an interphase nucleus into prematurely condensed chromosomes (PCC). Several lines of evidence suggest that these factors are proteins present in the cytoplasm of mitotic cells. The objective of this study was to raise antibodies to the factors responsible for PCC. Cytosol from synchronized mitotic HeLa cells was injected into rabbits in order to obtain antiserum. The IgG fraction from this antiserum reacted with 98% of mitotic HeLa cells when tested by indirect immunofluorescence. Most of the fluorescence was localized on the chromosomes. About 5% of the interphase nuclei also reacted with the antiserum, but 50% of these cells were in early G1. Antigenic reactivity was induced in the condensing interphase chromatin in 31% of the interphase nuclei found in mitotic-interphase fused cells. Rodent cells did not react with the antibody by indirect immunofluorescence. Mitotic HeLa cells were able to induce antigenic reactivity in 23 % of interphase Chinese hamster ovary (CHO) cell nuclei in fused binucleate cells, whereas the converse was not true of mitotic CHO cells. Enzyme digestion and incubation with denaturing agents suggested that antigenic reactivity depended on a DNA-non-histone protein complex.     

Nuclear and mitotically enhanced epitope.

Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.     

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.     

Localization of kinesin in cultured cells

Kinesin was isolated from bovine brain and used to elicit polyclonal antibodies in rabbits. The specificities of the resulting antibodies were evaluated by immunoblotting. Antibodies purified from these sera by their affinity for brain kinesin react with a polypeptide of approximately 120 kD in extracts from bovine brain, PtK1 cells, and mouse neuroblastoma cells. They bind to a pair of polypeptides of approximately 120 kD present in crude kinesin prepared from Xenopus eggs and with a single polypeptide of approximately 115 kD in extracts from Drosophila embryos. Antibodies raised against kinesin prepared from fruit fly embryos (by W. M. Saxton, Indiana University, Bloomington, IN) and from neural tissues of the squid (by M. P. Sheetz, Washington University, St. Louis, MO) cross react with the mammalian, the fly, and the frog polypeptides. Kinesin antigen was localized in cultured cells by indirect immunofluorescence. PtK1 cells in interphase showed dim background staining of cytoplasmic membranous components and bright staining of a small, fibrous, juxtanuclear structure. Double staining with antibodies to microtubules showed that the fibrous object was usually located near the centrosome. On the basis of shape, size, and location, we identify the kinesin-positive structure as a primary cilium. PtK1 cells in mitosis are stained at their poles during all stages of division. The structure stained is approximately spherical, but wisps of faint fluorescence also extend into the body of the spindle. Antibodies to squid or fruit fly kinesin produce identical patterns in PtK1 cells. Controls with preimmune and preabsorbed sera show that the centrosome staining is not due simply to the common tendency of rabbit antisera to stain this structure. Similar centrosome and spindle pole staining was visible when antibodies to bovine brain or squid kinesin were applied to the A6 cell line (kidney epithelial cells from Xenopus laevis). Some possible functions of kinesin localized at the spindle poles are discussed.     

Clathrin in mitotic spindles

Subconfluent cultures ofMadin-Darby canine kidney (MDCK) and CV-1 cells were immunostained withtwo monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrinheavy chain and with polyclonal antiserum against a conserved region ofall mammalian clathrin light chains. In interphase MDCK and CV-1 cells,staining by all three antibodies resulted in the characteristicintracellular punctate vesicular and perinuclear staining pattern. Inmitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in thespindle. Staining of the mitotic spindle was evident in mitotic cellsfrom prometaphase to telophase and in spindles in mitotic cellsreleased from a thymidine-nocodazole block. In CV-1 cells, staining ofclathrin in the mitotic spindle was not affected by brefeldin A. OnWestern blots, clathrin was detected, but not enriched, in isolatedspindles. The immunodetection of clathrin in the mitotic spindle maysuggest a novel role for clathrin in mitosis. Alternatively, therecruitment of clathrin to the spindle may suggest a novel regulatorymechanism for localization of clathrin in mitotic cells.

     

Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed. Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 micrometers in diameter. Preabsorption of the antisera was attended using microtubule protein, purified tubulin, actin, and microtubule-associated proteins. None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region. In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle.     

Detection of cross-reactive determinants shared by human monoclonal IgM reacting with myelin-associated glycoprotein

Monoclonal IgM from patients with peripheral neuropathy frequently have anti-myelin-associated glycoprotein (MAG) activity. We investigated the idiotypes of 10 monoclonal anti-MAG by using rabbit polyclonal antisera. Three groups of anti-idiotypic antisera could be distinguished. Four sera reacted only with the immunizing protein. Two sera reacted with a single other anti-MAG IgM in addition to the immunizing one. Immunoenzymatic studies showed that these two couples of anti-MAG IgM reacted identically with 100% cross-inhibition, indicating that the whole set of idiotypes identified by the rabbit antiserum was present on both IgM antibodies. The four other anti-idiotypic sera reacted with the homologous IgM, as well as with most of the other anti-MAG IgM. In contrast to the previous antisera the binding of these four sera to the homologous IgM could not be inhibited by other cross-reacting anti-MAG IgM. However, when a heterologous IgM was used for coating, these antisera with one exception showed complete cross-inhibition. The absence of inhibition by purified MAG of the patient of the anti-idiotypic antisera sera suggests that these antibodies are most likely to be directed against framework determinants rather than against combining site epitopes.     

The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells

A homolog of the fission yeast cdc2-encoded protein kinase (p34) is a component of M phase promoting factor in Xenopus oocytes. The homologous kinase in human HeLa cells is maximally active during mitosis, suggesting a mitotic role in mammalian somatic cells. This has been directly investigated by microinjection of anti-p34 antibodies into serum-stimulated rat fibroblasts. DNA synthesis was unaffected but cell division was quantitatively blocked in injected cells. Injection of antibodies against p13suc1, a component of the p34 kinase complex, did not block mitosis but caused mitotic abnormalities resulting in cells containing multiple micronuclei in the subsequent interphase. p34 localized in the nucleus during interphase. During mitosis, a fraction tightly associated with centrosomes. p13 was more evenly distributed between the nucleus and cytoplasm. These observations demonstrate that cdc2 is a nuclear and centrosomal protein that is required for mitosis in mammalian cells.     

Spindle-pole organization during early mouse development

Spindle-pole organization during early mouse development was examined using a variety of immunological reagents that recognize centrosomal components. Spindle poles of unfertilized eggs and blastocysts were found to react positively with two antisera (centrin and NRS-01), whereas poles of activated eggs and early cleavage-stage embryos were negative when treated with the same sera. In contrast, a third antiserum (5051) showed positive spindle-pole staining throughout the preimplantation stages of development. Two monoclonal antibodies (MPM-1 and MPM-2) that are known to react with mitotic phosphoproteins were also used in this study. Both antibodies stained the cytoplasm of mitotic cells with extremely high intensity. In addition, MPM-2 was found to stain spindle poles. These results suggest that organizational changes in the spindle pole are occurring during early mouse development. Embryos homozygous for a recessive lethal mutation known as oligosyndactyly (Os) were also treated with the reagents described above. This mutation results in a metaphase arrest at the blastocyst stage with intact spindles being present. Spindle poles were observed in Os homozygous mutants stained with centrin, NRS-01, and 5051. However, when Os mutants were stained with the MPM monoclonal antibodies, about half of the mitotic cells completely lacked the dramatic cytoplasmic staining. This observation is in contrast to that observed for wild-type embryos, where greater than 95% of mitotic cells showed positive cytoplasmic staining.     

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.     

Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells

Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re- initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events.     

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.     

The Golgi protein GM130 regulates centrosome morphology and function

The Golgi apparatus (GA) of mammalian cells is positioned in the vicinity of the centrosome, the major microtubule organizing center of the cell. The significance of this physical proximity for organelle function and cell cycle progression is only beginning to being understood. We have identified a novel function for the GA protein, GM130, in the regulation of centrosome morphology, position and function during interphase. RNA interference-mediated depletion of GM130 from five human cell lines revealed abnormal interphase centrosomes that were mispositioned and defective with respect to microtubule organization and cell migration. When GM130-depleted cells entered mitosis, they formed multipolar spindles, arrested in metaphase, and died. We also detected aberrant centrosomes during interphase and multipolar spindles during mitosis in ldlG cells, which do not contain detectable GM130. Although GA proteins have been described to regulate mitotic centrosomes and spindle formation, this is the first report of a role for a GA protein in the regulation of centrosomes during interphase.     

Hsp70 protects mitotic cells against heat-induced centrosome damage and division abnormalities

The effect of heat shock on centrosomes has been mainly studied in interphase cells. Centrosomes play a key role in proper segregation of DNA during mitosis. However, the direct effect and consequences of heat shock on mitotic cells and a possible cellular defense system against proteotoxic stress during mitosis have not been described in detail. Here, we show that mild heat shock, applied during mitosis, causes loss of dynamitin/p50 antibody staining from centrosomes and kinetochores. In addition, it induces division errors in most cells and in the remaining cells progression through mitosis is delayed. Expression of heat shock protein (Hsp)70 protects against most heat-induced division abnormalities. On heat shock, Hsp70 is rapidly recruited to mitotic centrosomes and normal progression through mitosis is observed immediately after release of Hsp70 from centrosomes. In addition, Hsp70 expression coincides with restoration of dynamitin/p50 antibody staining at centrosomes but not at kinetochores. Our data show that during mitosis, centrosomes are particularly affected resulting in abnormal mitosis. Hsp70 is sufficient to protect against most division abnormalities, demonstrating the involvement of Hsp70 in a repair mechanism of heat-damaged mitotic centrosomes.     

Production and characterization of monoclonal antibodies to N-acetyl-aspartyl-glutamate

N-acetyl-aspartyl-glutamate (NAAG) is a putative neuromodulator/neurotransmitter in the mammalian nervous system. Immunohistochemical studies with polyclonal NAAG antisera have revealed immunoreactive neurons and processes in several brain regions. However, these antisera crossreact to some degree with N-acetyl-aspartate (NAA), which is present in mM concentrations in brain, prompting the development of monoclonal antibodies (MAb) more specific for NAAG. By fusing spleen lymphocytes obtained from BALB/c mice pre-immunized with NAAG covalently linked to bovine serum albumin by carbodiimide with SP2/0-Ag 14 mouse myeloma cells, we produced three IgG2a (kappa) MAb which specifically reacted with NAAG. These MAb exhibited negligible crossreactivity with NAA or with structurally similar peptides, as shown by solid-phase radioimmunoassay. Antibody activity was absorbed out selectively by both NAAG-thyroglobulin conjugate and free NAAG. These MAb stained many nuclei of the medulla-pons and midbrain, mitral cells in the olfactory bulb, pyramidal neurons in sensorimotor cortex, locus ceruleus, and several cholinergic cranial nuclei. The staining pattern strongly correlated with NAAG levels determined by HPLC. Monoclonal antibodies significantly enhanced sensitivity of staining, allowing visualization of dorsal horn neurons in spinal cord, which were not readily detectable with polyclonal antiserum. Availability of these MAb now facilitates further clarification of the role of NAAG in the brain.     

Tau-like Proteins Associated with Centrosomes in Cultured Cells

The subcellular association of tau-like proteins with centrosomes in cultured cell lines and its effects in nucleating microtubule assembly were analyzed using biochemical and immunocytochemical approaches. Tau proteins, major components of microtubules, appear to be tightly associated with actin filaments in a variety of cell lines, while in pathological conditions of neurons, they are part of paired helical filaments found in Alzheimer's disease. Different studies suggest that, in addition to tau interactions with the components of the cytoskeletal network, tau polypeptides appear to be associated with highly structured cellular elements, in both interphase and mitotic cells. An in-depth analysis of tau subcellular distribution us- ing different polyclonal and monoclonal antibodies showed colocalization of tau-like components with centrosomes in interphase cells of the human Huh-7 hepatoma, in SW-13 adenocarcinoma, and in normal human fibroblasts. Tau associated with centrosomes in mitotic Huh-7 cells was also identified. However, antibodies against the tau binding repeats did not stain centrosomes. A set of different tau isoforms was also identified by Western blot analysis on isolated centrosomal preparations from Huh-7 cells, obtained by differential centrifugation through sucrose gradients. Microtubule nucleationin vitroover isolated centrosomes was inhibited by both the polyclonal antibody against native tau and an antibody to the N-terminal tau sequence, as revealed by immunofluorescence analysis and assembly kinetics experiments. The antibody TRS1.2 against the fragment containing the first binding repeat on tau did not affect nucleation. These studies allowed us to characterize tau association with the isolated centrosomal preparation and its involvement in microtubule assembly nucleated over centrosomes, thus suggesting possible structural and functional roles for these interactions.     

Characterization of antisera to glutamate and aspartate

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.     

Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase)

Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.     

NuMA: an unusually long coiled-coil related protein in the mammalian nucleus

A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.     

p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study

Immunofluorescent staining of mitotic centrosomes and spindles by anti-p53 antibodies was observed in the embryonic chick epiblast by epifluorescence microscopy and in three human cancer cell lines, an SV40-immortalized cell line, and a normal human fibroblast culture by confocal microscopy. In the chick epiblast, the centrosomes stained from early prophase through to the formation of the G1 nuclei and the spindle fibers stained from prophase through to telophase. In the human cells, the staining was observed from late prophase to telophase. The epiblast was stained by the anti-p53 antibodies DO-1, Ab-6, and Bp53-12. The human cells were also stained by these antibodies as well as by other anti-p53 antibodies. Preabsorption of DO-1 and Bp53-12 with purified tubulin did not diminish the immunostaining, showing that the antibodies were not reacting with tubulin in the mitotic centrosomes and spindles. The immunostaining in the chick epiblast was very clearly localized to the mitotic centrosomes and spindles, revealing a cytoplasmic location for p53 during mitosis and accounting for earlier reports of an association between p53, tubulin, and centrosomes. The localization of p53 to the spindle supports an involvement of p53 in spindle function.