Academic literature on the topic 'Nuclear mitotic apparatus'

The formation and maintenance of the bipolar mitotic spindle apparatus require a complex and balanced interplay of several mechanisms, including the stabilization and separation of polar microtubules and the action of various microtubule motors. Nonmicrotubule elements are also present throughout the spindle apparatus and have been proposed to provide a structural support for the spindle. The Nuclear-Mitotic Apparatus protein (NuMA) is an abundant 240 kD protein that is present in the nucleus of interphase cells and concentrates in the polar regions of the spindle apparatus during mitosis. Sequence analysis indicates that NuMA possesses an unusually long alpha-helical central region characteristic of many filament forming proteins. In this report we demonstrate that microinjection of anti-NuMA antibodies into interphase and prophase cells results in a failure to form a mitotic spindle apparatus. Furthermore, injection of metaphase cells results in the collapse of the spindle apparatus into a monopolar microtubule array. These results identify for the first time a nontubulin component important for both the establishment and stabilization of the mitotic spindle apparatus in multicellular organisms. We suggest that nonmicrotubule structural components may be important for these processes.

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.

Intermediate filaments (IF) appear to be attached to the nuclear envelope in various mammalian cell types. The nucleus of mouse keratinocytes is enveloped by a cagelike network of keratin-containing bundles of IF (IFB). This network appears to be continuous with the cytoplasmic IFB system that extends to the cell surface. Electron microscopy reveals that the IFB appear to terminate at the level of the nuclear envelope, frequently in association with nuclear pore complexes (Jones, J. C .R., A. E. Goldman, P. Steinert, S. Yuspa, and R. D. Goldman, 1982, Cell Motility, 2:197-213). Based on these observations of nuclear-IF associations, it is of interest to determine the fate and organizational states of IF during mitosis, a period in the cell cycle when the nuclear envelope disassembles. Immunofluorescence microscopy using a monoclonal keratin antibody and electron microscopy of thin and thick sections of mitotic mouse keratinocytes revealed that the IFB system remained intact as the cells entered mitosis and surrounded the developing mitotic spindle. IFB were close to chromosomes and often associated with chromosome arms. In contrast, in HeLa, a human epithelial cell, keratin-containing IFB appear to dissemble as cells enter mitosis (Franke, W. W., E. Schmid, C. Grund, and B. Geiger, 1982, Cell, 30:103-113). The keratin IFB in mitotic HeLa cells appeared to form amorphous nonfilamentous bodies as determined by electron microscopy. However, in HeLa, another IF system composed primarily of a 55,000-mol-wt protein (frequently termed vimentin) appears to remain morphologically intact throughout mitosis in close association with the mitotic apparatus (Celis, J.E., P.M. Larsen, S.J. Fey, and A. Celis, 1983, J. Cell Biol., 97:1429-34). We propose that the mitotic apparatus in both mouse epidermal cells and in HeLa cells is supported and centered within the cell by IFB networks.

A protein of 62 kD is a substrate of a calcium/calmodulin-dependent protein kinase, and both proteins copurify with isolated mitotic apparatuses (Dinsmore, J. H., and R. D. Sloboda. 1988. Cell. 53:769-780). Phosphorylation of the 62-kD protein increases after fertilization; maximum incorporation of phosphate occurs during late metaphase and anaphase and correlates directly with microtubule disassembly as determined by in vitro experiments with isolated mitotic apparatuses. Because 62-kD protein phosphorylation occurs in a pattern similar to the accumulation of the mitotic cyclin proteins, experiments were performed to determine the relationship between cyclin and the 62-kD protein. Continuous labeling of marine embryos with [35S]methionine, as well as immunoblots of marine embryo proteins using specific antibodies, were used to identify both cyclin and the 62-kD protein. These results clearly demonstrate that the 62-kD protein is distinct from cyclin and, unlike cyclin, is a constant member of the cellular protein pool during the first two cell cycles in sea urchin and surf clam embryos. Similar results were obtained using immunofluorescence microscopy of intact eggs and embryos. In addition, immunogold electron microscopy reveals that the 62-kD protein associates with the microtubules of the mitotic apparatus in dividing cells. Interestingly, the protein changes its subcellular distribution with respect to microtubules during the cell cycle. Specifically, during mitosis the 62-kD protein associates with the mitotic apparatus; before nuclear envelope breakdown, however, the 62-kD protein is confined to the nucleus. After anaphase, the 62-kD protein returns to the nucleus, where it resides until nuclear envelope disassembly of the next cell cycle.

We have recently shown that the nuclear mitotic apparatus protein (NuMA) is composed of at least three isoforms that differ mainly at the carboxy terminus, and are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (J. Cell Sci. (1993) 104, 249–260). Transient expression of human NuMA-1 isoform (T33/p230) in Chinese hamster ovary polyoma (CHOP) cells showed that NuMA-1 was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. However, expression of two other isoforms (NuMA-m and -s) revealed a distinct subcellular localization. NuMA-m (U4/p195) and NuMA-s (U6/p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, however, NuMA-m and -s moved to the mitotic spindle pole. Analysis of a series of linker scanning-mutants and NuMA/beta-galactosidase chimeric proteins showed that residues 1972–2007 of NuMA-1 constitute a novel nuclear localization signal (NLS) and residues 1538–2115 are necessary and sufficient for spindle association. Further analysis of the NLS by site-specific mutagenesis indicated that Lys1988 is essential for nuclear targeting, whereas Arg1984 is not. These results have allowed us tentatively to assign specific biological activities to distinct structural domains of the NuMA polypeptide.

The mAb RK7, previously shown to recognize keratin 19, was also found to cross-react with a biologically unrelated 102 kDa protein, which becomes associated with the poles of the mitotic apparatus. This newly identified protein, called cytocentrin, is a stable cellular component, may be at least in part phosphorylated, and displays a cell cycle-dependent cellular localization. In interphase cells, it is diffusely distributed in the cytosol and shows no affinity for cytoplasmic microtubules. It becomes localized to the centrosome in early prophase, prior to nuclear envelope breakdown, separation of replicated centrosomes, and nucleation of mitotic apparatus microtubules. During metaphase, cytocentrin is located predominately at the mitotic poles, often appearing as an aggregate of small globular sub-components; it also associates with some polar microtubules. In late anaphase/early telophase cytocentrin dissociates entirely from the mitotic apparatus and becomes temporarily localized with microtubules in the midbody, from which it disappears by late telophase. In taxol-treated cells cytocentrin was associated with the center of the miniasters but also showed affinity for some cytoplasmic microtubules. Studies employing G2-synchronized cells and nocodazole demonstrated that cytocentrin can become associated with mitotic centrosomes independently of tubulin polymerization and that microtubules regrow from antigen-containing foci. We interpret these results to suggest that cytocentrin is a cytoplasmic protein that becomes specifically activated or modified at the onset of mitosis so that it can affiliate with the mitotic poles where it may provide a link between the pericentriolar material and other components of the mitotic apparatus.

ABSTRACT We previously observed that high-risk human papillomavirus type 16 (HPV16) E7 expression leads to the delocalization of dynein from mitotic spindles (C. L. Nguyen, M. E. McLaughlin-Drubin, and K. Munger, Cancer Res. 68:8715-8722, 2008). Here, we show that HPV16 E7 associates with nuclear mitotic apparatus protein 1 (NuMA) and that NuMA binding and the ability to induce dynein delocalization map to similar carboxyl-terminal sequences of E7. Additionally, we show that the delocalization of dynein from mitotic spindles by HPV16 E7 and the interaction between HPV16 E7 and NuMA correlate with the induction of defects in chromosome alignment during prometaphase even in cells with normal centrosome numbers. Furthermore, low-risk HPV6b and HPV11 E7s also associate with NuMA and also induce a similar mitotic defect. It is possible that the disruption of mitotic events by HPV E7, via targeting of the NuMA/dynein complex and potentially other NuMA-containing complexes, contributes to viral maintenance and propagation potentially through abrogating the differentiation program of the infected epithelium. Furthermore, in concert with activities specific to high-risk HPV E6 and E7, such as the inactivation of the p53 and pRB tumor suppressors, respectively, the disruption of the NuMA/dynein network may result in mitotic errors that would make an infected cell more prone to the accumulation of aneuploidy even in the absence of supernumerary centrosomes.

The overall objective of this study was to identify novel proteins of the nuclear matrix in order to contribute to a better understanding of nuclear structure and organization. To accomplish this, a monoclonal antibody specific for the nuclear matrix was used to screen a human λgt11 expression library. Several cDNAs were isolated, cloned, sequenced, and shown to represent NuMA, the nuclear mitotic spindle apparatus protein. Further characterization of the gene and RNA was undertaken in an effort to obtain information about NuMA. The NuMA gene was present at a single site on human chromosome 11q13. Northern and PCR analysis of NuMA mRNA showed a major 7.2 kb transcript and minor forms of 8.0 and 3.0 kb. The minor forms were shown to be alternatively spliced although their functional significance is not yet understood. Immunofluorescence microscopy demonstrated that NuMA oscillates between the nucleus and the microtubule spindle apparatus during the mitotic cell cycle. NuMA appeared as a 200-275 kDa protein detectable in all mammalian cells except human neutrophils. To determine whether NuMA's changes in intracellular distribution correlated with post-translational modifications, the protein's phosphorylation state was examined through the cell cycle using highly synchronized cells. NuMA was a phosphoprotein in interphase and underwent additional phosphorylation events in mitosis. The mitotic phosphorylation events occurred with similar timing to lamin B (G2/M transition) and were concomitant with NuMA's release from the nucleus and its association with the mitotic spindle. However, the mitotic phosphorylation occurred in the absence of spindle formation. Dephosphorylation of NuMA did not correlate with reassociation with the nuclear matrix but occurred in two distinct steps after nuclear reformation. Based on the timing of these events, phosphorylation may playa role in nuclear processes. In conclusion, the work in this dissertation identified NuMA, a nuclear matrix protein and showed that it is phosphorylated during the cell cycle and may be important for nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.

In animal cells, the duplicated genetic material is aligned on a microtubule-based structure known as the mitotic spindle during mitosis. At the mitotic exit, the mitotic spindle elongates, and the sister chromatids get separated. The separation of sister chromatids is followed by the cleavage furrow formation and its ingression, which eventually partition the cytoplasmic constituents and genetic material into newly formed daughter cells. How the chromosome separation is coordinated with cleavage furrow formation is incompletely understood. Also, when animal cells enter mitosis, the chromatin gets highly condensed, and the transcription is chiefly paused. However, when cells exit mitosis, the chromatin should get decondensed in a tightly regulated manner to ensure proper landscaping of chromosome territories, which makes it competent enough for DNA-based processes like replication and transcription. The accurate functioning of these processes is critical for the development and for stem cell divisions. In this study, we have linked the function of an evolutionarily conserved protein, nuclear mitotic apparatus (NuMA), in cleavage furrow formation and chromatin decondensation at the mitotic exit. In the first part of my thesis, we have tried to characterize the function of chromatin-localized NuMA in regulating chromatin decondensation. In the second part, we have attempted to provide insight into how spatial localization of NuMA at the plasma membrane coordinates chromosome separation with cleavage furrow formation. 1). NuMA regulates chromatin decondensation at the mitotic exit and nuclear shape in interphase cells NuMA is a highly abundant (~10^6 copies) protein of interphase nuclei. Few studies hint that nuclear NuMA may have a role in chromatin organization, and it is hypothesized to be a part of the nuclear structural framework. In this regard, the loss of NuMA's function based on antibody-based microinjections was associated with nuclear shape defects. However, since the depletion of NuMA is linked with multiple mitotic abnormalities, it remained unclear whether the nuclear shape defects seen upon NuMA depletion is an indirect effect due to impairment of NuMA's mitotic function or a direct outcome of the absence of NuMA in the nucleus. Further, whether NuMA is bound to chromatin in the nucleus was also unknown. Even if NuMA is bound to chromatin, what mechanisms ensure its release upon mitotic entry was unknown. In this work, by utilizing fluorescence recovery after photobleaching (FRAP) and biochemical analysis, we report that NuMA is transiently bound to chromatin in the nucleus. We show that NuMA, which is bound to DNA, is released in late prophase upon nuclear envelope breakdown (NEBD) by the action of Cdk1-CyclinB kinase. Importantly, we identify evolutionarily conserved sequences rich in basic amino acids, arginine, and lysine, at the C-terminus of NuMA that aid in its direct interaction with DNA. In the absence of such interaction, NuMA becomes significantly mobile in the nucleus. Notably, the expression of the DNA-binding deficient mutant of NuMA delays chromatin decondensation at the mitotic exit. Furthermore, we discovered that DNA binding deficient NuMA polymerizes into high-order structures such as fibrillar networks, which perturbs nuclear shape. The DNA-binding property of NuMA prevents the formation of these higher-order structures and thus helps in maintaining the proper nuclear architecture. Overall, this study links the chromatin binding ability of NuMA with the proper chromatin decondensation at mitotic exit and maintenance of nuclear shape in interphase, independent of its mitotic role. 2). Polarized membrane distribution of NuMA/dynein and Ect2/Cyk4/Mklp1 regulate cleavage furrow formation Animal cells partition their genetic material and cellular constituents through cytokinesis. The initiation of cytokinesis is regulated by the activation of small GTPase RhoA that helps in myosin II activation and actin polymerization at the equatorial membrane, resulting in cleavage furrow formation. RhoA is spatiotemporally regulated by a heterotetrameric complex known as centralspindlin consisting of a dimer of kinesin-6 member Mklp1 and a dimer of RhoGAP Cyk4. The centralspindlin complex localizes at the spindle midzone and promotes the localization of its downstream effectors RhoGEF Ect2 which directly activates RhoA and regulates cytokinesis. However, how a precise RhoA zone at the equatorial membrane is established and maintained remained unclear. In anaphase, the mitotic protein NuMA is enriched at the polar membrane via its direct interaction with membrane phospholipids, PtIns(4)P and PtIns(4,5)P2 and is vital for proper spindle elongation by cortically anchoring the dynein/dynactin complex. However, despite the presence of PtIns(4)P and PtIns(4,5)P2 throughout the membrane, the NuMA/dynein complexes are restricted to the polar membrane and are excluded from the equatorial membrane, which is mutually exclusively occupied by RhoA. The mechanism of equatorial membrane exclusion of NuMA/dynein complex and its biological relevance remained unknown. In this work, we uncovered that Ect2, Cyk4, and Mklp1 are critical in restricting NuMA/dynein to the polar cortical region. In the absence of Ect2, Cyk4, or Mklp1, NuMA/dynein complex occupies the equatorial cortex, which impacts proper spindle elongation. Further, we show that Ect2 is in complex with Cyk4 and Mklp1 in anaphase cells. We establish that the membrane localization, but not the spindle midzone localization of the Ect2/Cyk4/Mklp1 complex, is critical for NuMA/dynein exclusion and, thus, for proper spindle elongation. Conversely, we show that polar membrane localization of the NuMA/dynein complex confines RhoA to a narrow zone at the equatorial membrane, which ensures cleavage furrow formation and cytokinesis. Overall our work provides insight into the mechanism that restricts NuMA/dynein and Ect2/Cyk4/Mklp1 to mutually exclusive membrane surfaces, which ensures proper chromatin segregation and cleavage furrow formation in animal cells. This coordination is critical for an error-free cell division program.

博士
國防醫學院
生命科學研究所
89
I. Genomic Organization and Promoter Analysis of the Mouse AIE1 Protein Kinase Gene We previously described two novel testis-specific protein kinases, AIE1 (mouse) and AIE2 (human), that share high amino acid (a.a.) identities with the kinase domains of fly Aurora, yeast Ipl1 and frog Eg2 (Tseng et al., 1998). Here we report the entire intron/exon organizations of the AIE1 gene and analyze the expression patterns of AIE1 mRNA during testis development. The cis-acting elements in the promoter region, and the transcription factors that may affect AIE1 expression, are also investigated. By screening a mouse genomic library, I have obtained the entire 18.5 kb AIE1 genomic sequence. The mouse AIE1 gene spans ~8 kilobases (kb) and contains seven exons. The results from primer extension assay indicated that the AIE1 gene has two transcription initiation sites: PE1 (minor form, -34 bp from 103d cDNA clone) and PE2 (major form, -18 bp from 103d cDNA clone). The sequences of the exon-intron boundaries of the AIE1 gene conform to the consensus sequences (GT/AG) of splicing donor and acceptor sites of most eukaryotic genes. Comparative sequence analysis revealed that the gene structure is highly conserved between mouse AIE1 and human AIE2. However, much less homology was found in the sequence outside the kinase domain. I have also mapped the AIE1 gene to mouse chromosome 7A2-A3 by fluorescent in situ hybridization (FISH). RT-PCR analysis has demonstrated that AIE1 mRNAs are predominantly expressed in testis, kidney, and sperm; but are not present in egg and in the embryonic tissues in early developmental stage. Northern blot analysis showed that AIE1 mRNA is expressed at a low level at day 14 and reaches its plateau at day 19 in the developing postnatal testis. RNA in situ hybridization indicated that the expression of AIE1 transcripts were restricted to meiotically active germ cells with highest levels detected in late pachytene spermatocytes. These findings suggest that AIE1 may play a role in spermatogenesis, particularly during the stages of meiosis. I have transfected a series of 5''-end deletion constructs of AIE1 promoter that fused with a luciferase reporter gene (AIE1p-Luc) into a mouse testis cell line to define the minimum region for AIE1 expression. My results showed that the promoter region (nt. -659 ~ -583) is essential for PE1 transcription initiation, while the region (nt. -442 ~ -343) is important for for PE2 transcription initiation. I also demonstrated that the Sp1/hsp70 binding site is necessary for PE2 transcription initiation and the CRE-like element (nt. -1093 ~ -1103) is essential for AIE1 expression. The influence of other transcription factors or protein kinases are analyzed by cotransfected with various deleted AIE1p-Luc constructs. My preliminary results showed that (1) PKA can avtivate, but MEKK-1 inhibit the AIE1p activity; (2) the l-TZFP is a repressor, but the s-TZFP is an activator. Furthermore, several specific transcription factors, such as TZFP (testis zinc finger protein) and CRE binding protein, that bind to the promoter region of AIE1 gene have been identified by the electrophoresis mobility shift assay (EMSA). The competition assay demonstrated the binding specificity. These results will help us to understand the control mechanism of AIE1 gene expression. II. The Expression and Functional Analysis of the Nuclear Mitotic Apparatus Protein (NuMA) During Early Mouse Embryonic Development The nuclear mitotic apparatus protein (NuMA) was first described by Lydersen and Pettijohn (1980) as a predominantly nuclear protein that is present in the interphase nucleus and is concentrated at the spindle pole of mitotic cells. Recently, cDNA clones that cover the entire coding region of human NuMA have been isolated and sequenced (Compton et al., 1992; Yang et al., 1992). Structure analysis reveals that NuMA is composed of a long a-helical central core flanked by two globular domains. We have recently shown that human NuMA is composed of at least three isoforms that differ mainly at the carboxy terminus. Multiple NuMA isoforms are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (Tang et al., 1993). The NuMA-l (T33 / p230) was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. NuMA-m (U4 / p195) and NuMA-s (U6 / p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, NuMA-m and -s moved to the mitotic spindle pole (Tang et al., 1994). In the mouse oocytes, the centrioles were reported to appear first in blastocysts (Magnuson and Epstein, 1984). Therefore, early mouse embryos offer an opportunity to elucidate the mechanisms of microtubule nucleation and spindle morphogenesis in the acentriolar centrosome. To investigate the expression patterns of different NuMA proteins or mRNAs during early mouse embryogenesis, the second part of my thesis is to clone and sequence the mouse homologue of human NuMA cDNA. By screening a mouse spleen cDNA library, I obtained the cDNA clones that cover the entire NuMA coding region. I also used the primer extension assay to define the transcription initiation site of NuMA mRNA. Furthermore, I have characterized the expression pattern of different NuMA isoforms in mouse tissues and during early stage of mouse embryonic development by RT-PCR method. My results showed that NuMA-l is present in all mouse tissues and in the 1.5 d.p.c. to 4.5 d.p.c. embryos. Interestingly, NuMA-m is expressed in all examined tissues except sperm, while NuMA-s is not detectable. Furthermore, the +42 bp NuMA isoform was detected in all tissues, while the -42 bp NuMA isoform was detected in testis and kidney only. To analyze the functional effect of NuMA on embryonic development, I injected mouse embryos with either antisense oligonucleotides (complementary to NuMA mRNA) or polyclonal antibodies against NuMA. Our results showed that injection of anti-NuMA antibody did significantly interfer the cell division during embryo-genesis. It suggested that NuMA may play a role in the cell division during early embryonic development.