Specific attachment of nuclear-mitotic apparatus protein to metaphase chromosomes and mitotic spindle poles: possible function in nuclear reassembly

NuMA protein is the largest, abundant, primate-specific chromosomal protein. The protein was purified from HeLa cells and monospecific monoclonal antibodies were prepared that react exclusively with NuMA protein in immunoblot analysis. These antibodies were used to define the intracellular location and properties of NuMA protein. Using indirect immunofluorescence, NuMA protein was detected only in the nucleus of interphase cells and on the chromosomes in mitotic cells. One class of monoclonal antibody called the 2E4-type antibody, caused NuMA protein (or a complex of proteins including NuMA) to be released from its binding site on metaphase or anaphase chromosomes. The separation of NuMA protein from chromosomes was observed either with the immunofluorescence assay or in electrophoretic analyses of proteins released from isolated metaphase chromosomes after reaction with 2E4 antibody. The immunofluorescence studies also showed that after release of the NuMA protein from chromosomes of metaphase or anaphase cells, the protein bound specifically to the polar region of the mitotic spindle. It was shown that exogenously added NuMA antigen/antibody complex bound only to the mitotic spindle poles of permeabilized primate cells and not to the spindle poles of other mammalian cells, thus demonstrating the specificity of the spindle-pole interaction. The antibody mediated transfer of NuMA from chromosomes to poles was blocked when the chromosomes were treated with cross-linking fixatives. Results suggest that the NuMA protein has specific attachment sites on both metaphase chromosomes and mitotic spindle poles (the site where post-mitotic nuclear assembly occurs). A model is proposed suggesting that a protein having such dual binding sites could function during nuclear reassembly to link mitotic chromosomes into the reforming nucleus.     

A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.     

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.     

The Poly-E motif in Titin's PEVK region undergoes pH dependent conformational changes

The muscle protein titin plays a crucial role in passive elasticity and the disordered PEVK region within titin is central to that function. The PEVK region is so named due to its high proline, glutamate, valine and lysine content and the high charge density in this region results in a lack of organized structure within this domain. The PEVK region is highly extensible but the molecular interactions that contribute to the elastic nature of the PEVK still remain poorly described. The PEVK region is formed by two unique sequence motifs. The PPAK motif is a 26 to 28 amino acid sequence that contains a mixture of charged and hydrophobic residues and is the primary building block for the PEVK region. Poly-E sequence motifs vary in length and contain clusters of 3–4 glutamic acids distributed throughout the motif. In this study, we derived two 28-residue peptides from the human titin protein sequence and measured their structural characteristics over a range of pHs. Our results demonstrate that the poly-E peptide undergoes a shift from a more rigid and elongated state to a more collapsed state as pH decreases with the midpoint of this transition being at pH ~5.5. Interestingly, a similar conformational shift is not observed in the PPAK peptide. These results suggest that the poly-E motif might provide a nucleating site for the PEVK when the muscle is not in an extended state.     

Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus

AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.     

The elasticity of individual titin PEVK exons measured by single molecule atomic force microscopy

The I-band region of the giant muscle protein titin contains a large domain enriched for the amino acids proline, glutamate, valine, and lysine and is denoted the PEVK domain. The PEVK domain of titin encodes a random coil shown to be an important factor in the passive elasticity of titin. Muscle-specific splicing of 116 PEVK exons encodes this domain. It has been proposed that proline contents determine the elasticity of the PEVK polypeptide, where the individual exons code for "flexibility cassettes." To test this hypothesis, we have measured the elasticity of three distinct polypeptides encoded by individual PEVK exons (161, 120 and 184) that varied greatly in their proline contents (7, 14, and 37% respectively) and total PEVK contents (55, 70, and 87%). We used single molecule atomic force microscopy techniques to measure the persistence length, p, of the engineered PEVK proteins. Surprisingly, all three exons 161, 120, and 184 coded for proteins with similar values of persistence length, p = 0.92 +/- 0.38, 0.89 +/- 0.42, and 0.98 +/- 0.4 nm, respectively. We conclude that the PEVK exons encode polypeptides of similar elastic properties, unrelated to their total PEVK contents. Hence, alternative splicing solely adjusts the length of the PEVK domain of titin.     

Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells

In animals and yeast, the small GTP-binding protein Ran has multiple functions - it is involved in mediating (i) the directional passage of proteins and RNA through the nuclear pores in interphase cells; and (ii) the formation of spindle asters, the polymerization of microtubules, and the re-assembly of the nuclear envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation by the RanGTPase protein RanGAP. Here we report the complementation of the yeast RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding RanGAP-like proteins. Confocal laser microscopy of Arabidopsis plants overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that the fusion protein is localized to patchy areas at the nuclear envelope of interphase cells. In contrast, the cellular distribution of RanGAPs in synchronized tobacco cells undergoing mitosis is characteristically different. Double-immunofluorescence shows that RanGAPs are co-localized with spindle microtubules during anaphase, with the microtubular phragmoplast and the surface of the daughter nuclei during telophase. Co-assembly of RanGAPs with tubulin correlates with these in vivo observations. The detected localization pattern is consistent with the postulated function of plant RanGAPs in the regulation of nuclear transport during interphase, and suggests a role for these proteins in the organization of the microtubular mitotic structures.     

A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies.

Six monoclonal antibodies identify a 210 kDa polypeptide which shows a cell cycle specific redistribution from the nucleus to the mitotic spindle. In interphase cells this polypeptide was localized in the nucleus and behaved during differential cell extraction as a component of the nuclear matrix. It accumulated in the centrosome region at prophase, in the pole regions of the mitotic spindle at metaphase and in crescents at the poles in anaphase, and reassociated with the nuclei as they reformed in telophase. Due to its staining pattern we call the protein the Spindle Pole-Nucleus (SPN) antigen. The localization of SPN antigen during mitosis was dependent on the integrity of the spindle since treatment of cells with nocodazole resulted in the dispersal of SPN antigen into many small foci which acted as microtubule organizing centres when the drug was removed. The SPN antigen was present in nuclei and mitotic spindles of all human and mammalian cell lines and tissues so far tested. When microinjected into the cytoplasm or nuclei of HeLa cells, one antibody caused a block in mitosis. Total cell number remained constant or decreased slightly after 24 h. At this time, about half the cells were arrested in a prometaphase-like state and revealed aberrant spindles. Many other cells were multinucleate. These results show that the SPN antigen is a protein associated with mitotic spindle microtubules which has to function correctly for the cell to complete mitosis.     

Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy

The formation of mitotic centrosomes is a complex process in which a number of cellular proteins translocate to mitotic poles and play a critical role in the organization of the mitotic apparatus. The 238-kDa nuclear mitotic apparatus protein NuMA is one of the important proteins that plays a significant role in this process. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps of phosphorylations. The role of NuMA in the interphase nucleus is not well known but it is clear that NuMA responds to external signals (such as hormones) that induce cell division, or heat shock that induces apoptosis. In order to determine the function of NuMA it is important to study its localization. Here we report on nuclear organization of NuMA during the cell cycle in estrogen responsive MCF-7 breast cancer cells and in androgen responsive LNCaP prostate cancer cells using immunoelectron microscopy, and on correlation to MPM-2 monoclonal phosphoprotein antibody. These results show that NuMA is present in speckled and punctate form associated with distinct material corresponding to a speckled or punctate immunofluorescence appearance in the nucleus while MPM-2 is uniformly dispersed in the nucleus. At prophase NuMA disperses in the cytoplasm and associates with microtubules while MPM-2 is uniformly distributed in the cytoplasm. During metaphase or anaphase anti-NuMA labeling is associated with spindle fibers. During telophase NuMA relocates to electron-dense areas around chromatin and finally to the reconstituted nuclei. These results demonstrate NuMA organization in MCF-7 and LNCaP cells in the log phase of cell culture growth.     

Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells

A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinity-purified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis. Immunofluorescence examination of CHO, 3T3, NRK, PTK-2, and HeLa cells resulted in a distinct pattern of mitotic spindle fluorescence. The localization pattern of this 52-kD CBP directly parallels that of CaM in the spindle apparatus throughout the various stages of mitosis. Interestingly, there was no association of this 52-kD CBP with cytoplasmic microtubules. As is the case with CaM, the localization pattern of the 52-kD CBP in interphase cells is diffuse within the cytoplasm and is not associated with any discrete, cellular structures. This 52-kD CBP appears to represent the first mitotic spindle-specific calmodulin-binding protein identified and represents an initial step toward the ultimate determination of CaM function in the mitotic spindle apparatus.     

CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.     

Targeting of RCC1 to chromosomes is required for proper mitotic spindle assembly in human cells

Ran GTPase is involved in several aspects of nuclear structure and function, including nucleocytoplasmic transport and nuclear envelope formation. Experiments using Xenopus egg extracts have shown that generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 also plays roles in mitotic spindle assembly. Here, we have examined the localization and function of RCC1 in mitotic human cells. We show that RCC1, either the endogenous protein or that expressed as a fusion with green fluorescent protein (GFP), is localized predominantly to chromosomes in mitotic cells. This localization requires an N-terminal lysine-rich region that also contains a nuclear localization signal and is enhanced by interaction with Ran. Either mislocalization of GFP-RCC1 by removal of the N-terminal region or the expression of dominant Ran mutants that perturb the GTP/GDP cycle causes defects in mitotic spindle morphology, including misalignment of chromosomes and abnormal numbers of spindle poles. These results indicate that the generation of Ran-GTP in the vicinity of chromosomes by RCC1 is important for the fidelity of mitotic spindle assembly in human cells. Defects in this system may result in abnormal chromosome segregation and genomic instability, which are characteristic of many cancer cells.     

TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.     

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.

     

Detection of the 125-kDa nuclear protein mitotin in centrosomes, the poles of the mitotic spindle, and the midbody.

Mitotin is a nuclear protein detectable in all proliferating cells investigated so far, including human and plant cells. In interphase cells the protein is localized mainly in the nucleoplasm. In G2/M phase it displays a characteristic redistribution and a marked increase which initiated the name mitotin. This study presents the precise localization of mitotin in cytoplasmic structures in two cell types, the potoroo rat kangaroo PtK2 cell and the human lung cancer EPLC 65 cell. In addition to its nuclear localization the antigen is detectable in centrosomes, in the poles of the mitotic spindle, and along spindle fibers. During the last mitotic stages, cytokinesis and reconstitution of nuclei, mitotin displays a rapid decrease and another redistribution. A significant amount of the antigen is retained in the bridge connecting the dividing cells, the midbody.     

Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly : Properties of purified NuMA protein

Monoclonal antibodies and human autoimmune sera specific for the nuclear mitotic apparatus protein (NuMA protein) were applied to study the structure of this protein and its intracellular distribution. The NuMA protein was purified using immuno-affinity columns. Studies on this large (250 kD) nuclear protein indicated that it is a highly asymmetric phosphoprotein. It is present in all mammalian cells examined and in those of some non-mammals. Immunofluorescence studies on fixed cells demonstrated that its intracellular distribution is essentially the same in all species at all stages of the cell cycle. Immunoblot (western blot) analysis showed that the size of the NuMA protein varies slightly in different species. At the onset of mitosis the NuMA protein redistributes from the nucleus to two centrosomal structures that later will become part of the mitotic spindle pole. This occurs at the time of nuclear breakdown and eventually leads to an accumulation of the NuMA protein at the polar region of the mitotic spindle. After anaphase the protein redistributes from the spindle polar region into the reforming nucleus and concentrates initially at the site where nuclear lamins and perichomatin have been reported to assemble. Living cells microinjected with fluorescent anti-NuMA antibodies were studied to examine parameters that effect the redistribution of the NuMA protein in vivo. These experiments indicate that microtubule assembly is essential for the NuMA protein to accumulate in the polar region.     

Live imaging reveals that the Drosophila actin-binding ERM protein, moesin, co-localizes with the mitotic spindle

Members of the vertebrate ezrin-radixin-moesin (ERM) protein family crosslink the actin cytoskeleton and the cell membrane and are, therefore, considered cytoplasmic regulators of cell adhesion, cell movement and membrane trafficking. Here we demonstrate that besides its cytoplasmic functions Drosophila moesin, the only ERM protein in Drosophila melanogaster, exhibits a dynamic cell cycle-dependent nuclear localization. In a small fraction of cells and at a low level, moesin can be detected in interphase nuclei in regions complementary to the chromatin; its level rapidly increases during prophase and it co-localizes with the actin network surrounding the mitotic spindles throughout mitosis. We also found that the predicted single nuclear localization signal in moesin is not necessary for the nuclear accumulation of the protein. FRAP experiments confirmed this finding and further revealed that the mitotic localization of moesin is highly dynamic. Immuno-histochemical staining for moesin demonstrated the existence of spindle association in wild-type embryos. The biological relevance of this phenomenon is indicated by the mitotic phenotypes detected in S2 cells treated with moesin RNAi, and awaits future exploration.     

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.