Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor Cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.     

Interphase nucleo-cytoplasmic shuttling and localization of SIRT2 during mitosis

The human NAD+-dependent protein deacetylase SIRT2 resides predominantly in the cytoplasm where it functions as a tubulin deacetylase. Here we report that SIRT2 maintains a largely cytoplasmic localization during interphase by active nuclear export in a Crm1-dependent manner. We identified a functional, leptomycin B-sensitive, nuclear export signal sequence within SIRT2. During the cell cycle, SIRT2 becomes enriched in the nucleus and is associated with mitotic structures, beginning with the centrosome during prophase, the mitotic spindle during metaphase, and the midbody during cytokinesis. Cells overexpressing wild-type or a catalytically inactive SIRT2 exhibit an increase in multinucleated cells. The findings suggest a novel mechanism of regulating SIRT2 function by nucleo-cytoplasmic shuttling, as well as a role for SIRT2 in the nucleus during interphase and throughout mitosis.     

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.     

Nucleocytoplasmic Shuttling of the TACC Protein Mia1p/Alp7p Is Required for Remodeling of Microtubule Arrays during the Cell Cycle

Microtubule arrays are remodeled as cells proceed through the cell cycle. It is important to understand how remodeling is regulated in time and space. In fission yeast, the conserved microtubule associated TACC/TOG complex plays an important role in organizing microtubules throughout the cell cycle. Here we show that this complex undergoes nucleocytoplasmic shuttling through the nuclear import and export signals located in the TACC protein Mia1p/Alp7p. When the Crm1p-dependent nuclear export signal of Mia1p is disabled, Mia1p accumulates in the nucleus while its partner protein Alp14p/TOG is restricted to the cytoplasm. This leads to defects in assembly of both interphase arrays and the mitotic spindle. Artificial targeting of Alp14p to the nucleus partially rescues the mitotic spindle defects caused by lack of Mia1p nuclear export. Interestingly, the nuclear export sequence of Mia1p appears to overlap with the Alp14p binding site. We propose that intricate regulation of the subcellular distribution of TACC/TOG complexes drives microtubule array remodeling as cells progress through the cell cycle.     

Distribution of c-myc, c-myb, and Ki-67 antigens in interphase and mitotic human cells evidenced by immunofluorescence staining technique

Using specific antibodies and the immunofluorescence staining technique we found a similar subcellular distribution pattern of the cellular proto-oncogene proteins c-myc and c-myb in interphase and mitotic HL60 and Molt4 cells. Antibodies against c-myc as well as those against c-myb protein gave rise to a nuclear staining excluding the nucleoli. In mitotic cells both proteins are apparently not associated with the chromatin of the condensed chromosomes, but appear diffusely distributed throughout the cytoplasm. In contrast, immunostaining using the proliferation marker antibody Ki-67 yielded in both cell lines several prominent specks in the nucleus and a weak finely dispersed staining throughout the nucleoplasm. No fluorescence was detectable in the cytoplasm. In dividing cells Ki-67 immunofluorescence was found to be associated with the surface of the chromosomes. The functional significance of the different localizations of the proteins is discussed in light of what is currently known about nuclear antigens.     

GTP酶Ran在细胞核运输、核分裂与重建中的作用

小分子的单体G蛋白Ran具有鸟苷三磷酸酶活性,其结合形式Ran-GTP作为区分间期细胞的核质和胞质的一个分子标记,并参与调控核质运输、指导纺锤体形成以及引导核膜解体与装配。现就Ran在真核细胞核质运输、有丝分裂纺锤体组装与核膜动力学中的功能作一综述。     

Spindle formation in Aspergillus is coupled to tubulin movement into the nucleus

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/alpha-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1 transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.     

Nucleoporins: leaving the nuclear pore complex for a successful mitosis

The nuclear envelope (NE) separates the cytoplasm and the cell nucleus of interphase eukaryotic cells and nuclear pore complexes (NPCs) mediate the macromolecular exchange between these two compartments. The NE and the NPCs of vertebrate cells disassemble during prophase and the nuclear pore proteins (nucleoporins) are distributed within the mitotic cytoplasm. For an increasing number of them active mitotic functions have been assigned over the past few years. Nucleoporins are participating in spindle assembly, kinetochore organisation, and the spindle assembly checkpoint, all processes that control chromosome segregation and are important for maintenance of genome integrity. But nucleoporins are also engaged in early and late mitotic events, such as centrosome positioning and cytokinesis. Here we will highlight recent progress in deciphering the roles for nucleoporins in the distinct steps of mitosis.     

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.     

Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining

We have studied the distribution of myosin and tubulin molecules inside the same tissue culture cells by using two antibodies labeled with contrasting fluorochromes. Antimyosin raised against human platelet myosin was labeled with rhodamine. Antitubulin raised against sea urchin vinblastine-induced tubulin crystals was labeled with fluorescein. The two antibodies stained entirely different structures inside the same flat interphase cell: antimyosin bound to stress fibers and antitubulin bound to thin, wavy fibers thought to be individual microtubules. Compact interphase cells stained diffusely with both antibodies. From prophase through early anaphase both antibodies stained the mitotic spindle, although the fluorescence contrast between the spindle and the cytoplasm was much higher with antitubulin than with antimyosin. From anaphase through telophase, strong antimyosin staining occurred in the cleavage furrow, while antitubulin stained the region between the separated chromosomes. This study established the feasibility of high-resolution fluorescent antibody localization of pairs of motility proteins in the cytoplasm of single cells, an approach which will make it possible to map out the sites of the various contractile protein interactions in situ.     

Fine structure of the Golgi complex during mitosis of cartilaginous cells in vitro

Chondrocytes were isolated enzymatically from guinea-pig epiphyses and grown in vitro. The fate of the Golgi complex during mitosis in relation to changes in the cytoplasmic microtubules was then studied by transmission electron microscopy. Interphase cells were observed to be polarized, with the Golgi complex occupying a well-defined juxtanuclear area of the cell's cytoplasmic pole. During prophase the cytoplasmic microtubules were largely lost, the nucleus moved to the center of the cell and the Golgi complex dissolved into single dictyosomes spread diffusely throughout the cytoplasm. The distribution of other organelles also changed to a more random pattern. In telophase, i.e. after the completion of nuclear division, the mitotic spindle decomposed and cytoplasmic microtubules reappeared. Furthermore, the organization of the Golgi complex and other organelles returned to that characteristic of interphase cells. Previous studies on cells treated with colchicine have indicated that the polarized distribution of cell organelles is dependent on the presence of intact cytoplasmic micro-tubules. It is suggested that the disappearance of such tubules observed here to be coupled with the disorganization of cell interphase structure fulfills the double function of providing free tubulin units from which to build the mitotic spindle and ensuring an approximately equal distribution of dictyosomes and other organelles to the daughter cells during cytokinesis.     

Cell cycle-dependent change of proteasome distribution during embryonic development of the ascidian Halocynthia roretzi.

The proteasome is a multicatalytic proteinase complex composed of nonidentical subunits. By immunocytochemical analysis using monoclonal antibody raised against the egg proteasome, we demonstrate that the proteasome undergoes changes in its subcellular distribution, depending on the cell division cycle during embryonic development of the ascidian Halocynthia roretzi. During interphase, the proteasome is localized in the nucleus, i.e., in the nucleoplasm and along the nuclear membrane. The proteasome disappears from the nucleoplasm in prophase and from the nuclear envelope in prometaphase. During early metaphase, the proteasome is detectable in the chromosomes and, at late stages of metaphase, the immunoreactivity also occurs in the peripheral region of each spindle pole and at the mitotic spindle. In anaphase, however, the staining disappears in the mitotic apparatus. In telophase, the proteasome is again localized in the newly formed nucleus. In addition to the localization in the nucleus and around the mitotic apparatus, the proteasome shows cytoplasmic localization throughout the cell division cycle. Such a change of subcellular distribution of the proteasome is clearly demonstrated in the synchronously dividing blastomeres and also is believed to occur in the postcleavage embryos. These observations suggest that the proteasome may play a key role in the progression of cell division cycle.     

Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.     

Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities [published erratum appears in J Cell Biol 1997 Jul 14;138(1):215]

There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. We compared the enzymatic activities as well as the subcellular localizations of these isoforms in Xenopus cells. MHC-A and MHC-B were purified from cells by immunoprecipitation with isoform-specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A was 3.3-fold faster than that by MHC-B. Likewise, the Vmax of the actin-activated Mg(2+)-ATPase activity of MHC-A was 2.6- fold greater than that of MHC-B. Immunofluorescence microscopy demonstrated distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium was dramatically enriched for MHC-B suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A was absent from the cell periphery and was arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also had distinct localizations throughout mitosis. During prophase, the MHC-B redistributed to the nuclear membrane, and then resumed its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localized to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalized to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions.     

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.     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

Actin organization during the cell cycle in meristematic plant cells. Actin is present in the cytokinetic phragmoplast

The distribution and organisation of F-actin during the cell cycle of meristematic root-tip cells of Allium was investigated using a rhodamine-labelled phalloidin to stain F-actin in isolated cell preparations. Such preparations could, in addition, be stained for tubulin by immunofluorescence, enabling a comparison between F-actin and microtubule distributions in the same cell. In interphase, an extensive array of actin-filament bundles was present in the cytoplasm of elongating cells, the bundles generally following the long axis of the cell and passing in close proximity to the nucleus. In contrast, the interphase microtubule array occupied the cortex of the cell and was oriented at right angles to the actin bundles. In smaller, isodiametric cells, microfilament arrays were present but less well developed. During cell division, phalloidin-specific staining was seen in the cytokinetic phragmoplast, and co-distributed with microtubules at all stages of cell plate formation; however, neither the pre-prophase band nor the mitotic spindle were stained with phalloidin. Co-distribution of F-actin and microtubules only occurs, therefore, at cytokinesis. The relationship between microfilaments and microtubules is discussed, together with the possible role of actin in the phragmoplast.     

Nuclear targeting of adenovirus type 2 requires CRM1-mediated nuclear export

Incoming adenovirus type 2 (Ad2) and Ad5 shuttle bidirectionally along microtubules, biased to the microtubule-organizing center by the dynein/dynactin motor complex. It is unknown how the particles reach the nuclear pore complex, where capsids disassemble and viral DNA enters the nucleus. Here, we identified a novel link between nuclear export and microtubule-mediated transport. Two distinct inhibitors of the nuclear export factor CRM1, leptomycin B (LMB) and ratjadone A (RJA) or CRM1-siRNAs blocked adenovirus infection, arrested cytoplasmic transport of viral particles at the microtubule-organizing center or in the cytoplasm and prevented capsid disassembly and nuclear import of the viral genome. In mitotic cells where CRM1 is in the cytoplasm, adenovirus particles were not associated with microtubules but upon LMB treatment, they enriched at the spindle poles implying that CRM1 inhibited microtubule association of adenovirus. We propose that CRM1, a nuclear factor exported by CRM1 or a protein complex containing CRM1 is part of a sensor mechanism triggering the unloading of the incoming adenovirus particles from microtubules proximal to the nucleus of interphase cells.     

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.