Plasmodium induced by SU6656, an Src family kinase inhibitor,is accompanied by a contractile ring defect

We have shown that SU6656, a potent Src family kinase inhibitor, has the ability to induce multinucleation at a high frequency in diverse cells: rat skin fibroblasts, bone marrow adherent cells, 5F9A mesenchymal stem cell‐like clones, 2C5 tracheal epithelial cells and MDCK epithelial cells from dog kidney. To gain insight into the mechanism of multinucleation, we observed the process by time‐lapse and confocal microscopy. These multinuclei generally seem to exist independently in one cell without any connections with each other. By time‐lapse microscopy, multinucleated cells were found to be formed through the mechanism of plasmodium: karyokinesis without cytokinesis. The observation of EGFP‐actin transfected cells by time‐lapse confocal laser scanning microscopy suggested that plasmodium occurred with deficient contractile ring formation. Although we examined the differentiation of these cells, the multinucleated cells could not be categorized into any type of cell in vivo known to exhibit multinuclei. Copyright © 2011 John Wiley & Sons, Ltd.     

Cellular features of the extra-embryonic endoderm during elongation in the ovine conceptus

The extraembryonic endoderm of the elongating ovine conceptus was analyzed by scanning and transmission electron microscopy and by whole mount actin staining and immunofluorescence. Morphological and functional differences between the visceral endoderm (VE), the founding cell layer, and the parietal endoderm (PE) are presented. During the elongation process, the PE differentiated to fusiform multinucleated cells aligned parallel to the elongation axis of the conceptus, whereas the VE cells retained the aspect of typical epithelial cells. The multinucleated PE cells however, expressed cellular and nuclear markers typical of endodermal and polarized epithelial cells. The proteins of the extracellular matrix, laminin, and fibronectin, were specifically expressed in the PE. The presence of pairs of nuclei linked by mid-bodies positively stained with tubulin antibodies, indicated that the syncytial differentiation of the PE was due to karyokinesis which was not followed by cytokinesis rather than by cell fusion.     

Mechanisms of cellular multinucleation as affected by cytochalasin B in a culture of transformed fibroblasts

The multinucleation of cytochalasin B was studied by means of phase contract microscopy and time-lapse cinematography. Several types of alteration of normal cytokineses resulted in the formation of multinucleated cells. The main way of multinucleation consists in the complete development of a cleavage furrow with the formation of daughter cells and an intercellular bridge that is followed by the fusion of these cells due to extending the entercellular bridge. These results suggest that the terminal stage of cytokinesis is the most sensitive to cytochalasin B.     

Simultaneous visualization of the actin plate and new cell partition membrane during cytokinesis in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)

In many brown algae, cytokinesis is accomplished through the centrifugal expansion of the membrane structure formed by the fusion of Golgi vesicles and flat cisternae. In contrast, it has been reported that cytokinesis in Sphacelaria rigidula progresses centripetally by adding Golgi vesicles and flat cisternae to cleaving furrows of the plasma membrane. The reason why this cytokinetic pattern was observed only in Sphacelaria species is unknown. In either cytokinesis pattern, a plate-like actin structure (the actin plate) coincides with the cytokinetic plane between the daughter nuclei. However, it is unclear how the actin plate is related to cytokinesis progression. In this study, we re-examined cytokinesis in the apical cells of S. rigidula using transmission electron microscopy. Double staining of the actin plate and the developing membrane was followed by fluorescence microscopy analysis to determine the relationship between these two formations. The results showed that cytokinesis in S. rigidula, as in many brown algae, was completed by centrifugal growth of the new cell partition membrane. A furrow of the plasma membrane was observed at the beginning of cytokinesis; however, further invagination did not occur. The actin plate arose at the center of the cytokinetic plane before membrane fusion and extended parallel to the expansion of the new cell partition membrane. When cytokinesis was slow due to insufficient Golgi vesicle supply to the cytokinetic plane in the cells under brefeldin A treatment, the extension of the actin plate was also suspended. In this study, the spatiotemporal relationship between the occurrence and expansion of the actin plate and the new cell partition membrane was revealed. These observations indicate that the actin plate might promote membrane fusion or lead to the growth of a new cell partition membrane.     

Participation of the adhesive disc during karyokinesis in Giardia lamblia

Evidence is presented for a potential involvement of the adhesive disc on the nucleus division in Giardia lamblia. The trophozoite mitotic nucleus was studied by transmission electron microscopy, freeze-fracture, freeze-substitution and also by immunofluorescence microscopy using anti-tubulin antibodies specific to spindle microtubules and Panotic staining. Prior to cell division the nucleus elongated and a displaced disc fragment, established contact with the nucleus. A progressive nucleus indentation was coincident with the concomitant presence of a disc fragment at the constricted region. One nucleus each time progressively divided until the karyokinesis was finished and two daughter-nuclei were observed. After the first karyokinesis a second karyokinesis takes place following the same procedure. When Giardia gets the four nuclei, cytokinesis occurs. Duplicated basal bodies were seen in between the first and the second karyokinesis. Immunofluorescence microscopy, using a panel of anti-tubulin antibodies, and electron microscopy of cells processed using microtubule stabilizer buffers, or cells fast-frozen and freeze-substituted, did not reveal the presence of a typical spindle. We propose that Giardia lamblia presents an uncommon mitotic behavior where the adhesive disc, a microtubular structure, seems to participate in the karyokinesis process.     

Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherin

Rearrangement of cell-cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E-cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E-cadherin-rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E-cadherin-immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E-cadherin-mediated cell adhesion vary with the state of differentiation.     

Ultrastructure of an anaplastic giant cell tumor of the thyroid

A case of anaplastic, multinucleated giant cell tumor of the thyroid was studied by light and electron microscopy. The coexistence of anaplastic sarcomatous tumor and well differentiated follicular carcinoma, and the presence of desmosomes among the mononuclear cells suggested that this tumor originates in thyroid follicular cells. The multinucleated giant cells, which characterize this thyroid tumor, appeared to be formed by fusion of follicular carcinoma cells and mononuclear epithelial cells, and not by nuclear division without cytoplasmic division.     

Nuclear envelope fission is linked to cytokinesis in budding yeast

We have investigated the relationship between nuclear envelope fission and cytokinesis during mitotic cell division in budding yeast. By carrying out time-lapse and optical sectioning video microscopy analysis of cells that express green fluorescent protein (GFP)-tagged nuclear envelope and actomyosin ring components, we found that nuclear division is temporally coupled to cytokinesis. Light and electron microscopy analysis also showed that nuclear envelope fission and the division of the nucleoplasm are severely delayed in cytokinesis mutants, resulting in discoupling between the nuclear division cycle and the budding cycle. These results suggest that homotypic membrane fusion may be activated by components or the mechanical action of cytokinetic structures and presents a mechanism for the equal partitioning of the nucleus and the temporal coordination of this event with chromosome segregation during mitosis.     

Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.     

INCOMPLETE CYTOKINESIS IN FUNARIA STOMATA

The development of the one-celled condition in Funaria (Musci) stomata was investigated using light and electron microscopy. The guard cell parent cell is unusual in that it undergoes karyokinesis but incomplete cytokinesis. The septal wall, and the cell plate from which it forms, have incurved edges in contact with the polar cytoplasm. No evidence was found to support Haberlandt's claim that the stomate is initially two celled but undergoes wall resorption. Preprophase microtubule bands appear to be present in nonstomatal epidermal cells with normal cytokinesis, but the possibility is raised that they are absent in guard cell parent cells.     

Sensitivity of cytokinesis to hydrophobic interactions. Chemical induction of bi-and multinucleated cells

We have tested whether cytokinesis is as sensitive to hydrophobic interactions as karyokinesis, and evaluated the usefulness of the frequency of binucleated cells as end-point. Treating cultured cells for 2 or 24 h, with different lipophilic alcohols and chlorinated hydrocarbons made this possible. Colcemid and cytochalasin B were applied as positive controls for inhibition of karyokinesis and cytokinesis, respectively. Several-fold increases of binucleated cells could be seen with cytochalasin B after 2 h of treatment, while there was no increase with colcemid, which instead blocked cells in prometaphase/metaphase. The solvent acted primarily through hydrophobic interactions. For each solvent, the blocking of cells in prometaphase/metaphase and a minor increase in binucleated cells, were seen at approximately the same concentration; the binucleated cells probably emanated from cells in anaphase/telophase at the start of treatment. We conclude that the spindle function and cleavage show similar sensitivity to hydrophobic interactions. After prolonged treatment, allowing escape from the metaphase block, the solvents induced binucleated and multinucleated cells. By forming the quotient between multinucleated (MULTI) and binucleated (BIN) cells one could distinguish between effects primarily on the spindle or cytokinesis, respectively. All solvents, and a combination of colcemid and cytochalasin B, showed quotients intermediate between those observed with colcemid (high MULTI/BIN) and cytochalasin B (low MULTI/BIN), respectively. Both protocols revealed the same relationship between lowest active concentration and lipophilicity for the solvents, implying that concentration, not dose were of prime importance. The specific inhibitors acted at low concentrations in relation to lipophilicity, clearly demonstrating their chemical mechanisms. This approach can be used for rapid screening of potential aneugens, distinguishing between routes, and when lipophilicity is known, also reveal the principal mechanism of action, i.e. physico-chemical or chemical.     

Presence of a defect in karyokinesis during megakaryocyte endomitosis

Megakaryocyte is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, a process corresponding to a late failure of cytokinesis with a backward movement of the daughter cells. Generally, a pure defect in cytokinesis produces a multinucleated cell, but megakaryocytes are characterized by a single polylobulated nucleus with a 2^N ploidy. Here, we show the existence of a defect in karyokinesis during the endomitotic process. From late telophase until the reversal of cytokinesis, some dipolar mitosis/endomitosis and most multipolar endomitosis present a thin DNA link between the segregated chromosomes surrounded by an incomplete nuclear membrane formation, which implies that sister chromatid separation is not complete. This observation may explain why polyploid megakaryocytes display a single polylobulated nucleus along with an increase in ploidy.     

Morphological characterization of actively fusing L6 myoblasts

We have characterized morphologically the surface of L6 myoblasts at the time of active cell fusion using transmission electron microscopy. Two subclones of the L6 line were used in these studies: the L6Cl55 line that fuses to form multinucleated syncytia and the NF44 non-fusing variant. Ultrastructural analysis revealed an electron-opaque material at localized points of cell-cell apposition in actively fusing L6Cl55 cells. This material may be transported by and secreted from smooth-surfaced cytoplasmic vesicles with an electron-dense core. In contrast to L6Cl55 cells, the electron-dense plaques were seen infrequently in cultures of the NF44 non-fusing variant. This previously unidentified substance may be associated with cell-cell recognition or adhesion, both necessary prerequisites for myoblast membrane fusion. Alternatively, the electron-dense plaques may be directly involved in the fusion event.     

The development of Chlamydia trachomatis inclusions within the host eukaryotic cell during interphase and mitosis

The dynamic nature of Chlamydia trachomatis inclusions was studied by video and 35 mm time-lapse photomicrography of live cells, and by immunolocalization of inclusions in fixed cells. A serotype E isolate was used to infect the MCCoy cell line and endometrial epithelia. Then resulting inclusions were observed over 4 d. They appeared as slowly expanding fluid-filled membrane vesicles whose growth varied considerably, and which were subject to great physical distortion by the host cell during interphase and mitosis. When this distortion became extreme the inclusion was observed to divide. However, as inclusions were mobile within the cytoplasm and thus able to come into contact with each other, there was a net tendency for the opposite process of inclusion fusion to occur when cells contained more than one inclusion. The proportion of infected cells decreased with time as a result of host cell proliferation, despite transmission of inclusions to progeny at the time of mitosis. Inclusion growth physically disrupted karyokinesis and cytokinesis so that host cell division became distorted or blocked on the second or third day of infection. Cell death eventually occurred by a very rapid lysis event.     

Dynamics of cell adhesion and motility in living cells is altered by a single amino acid change in E-cadherin fused to enhanced green fluorescent protein

E-Cadherin regulates epithelial cell adhesion and is critical for the maintenance of tissue integrity. In sporadic diffuse-type gastric carcinoma, mutations of the E-cadherin gene are frequently observed that predominantly affect putative calcium binding motifs located in the linker region between the second and third extracellular domains. A single amino acid change (D370A) as found in a gastric carcinoma patient reduces cell adhesion and up-regulates cell motility. To study the effect of this mutation on the dynamics of cell adhesion and motility in living cells, enhanced green fluorescent protein (EGFP) was C-terminally fused to E-cadherin. The resulting mutant E-cadherin-EGFP fusion protein with a point mutation in exon 8 (p8-EcadEGFP) and a wild-type E-cadherin-EGFP fusion construct (wt-EcadEGFP) were expressed in human MDA-MB-435S cells. Fluorescent images were acquired by time-lapse laser scanning microscopy and E-cadherin was visualized during contact formation and in moving cells. Spatial and temporal localization of p8- and wt-EcadEGFP differed significantly. While wt-EcadEGFP was mainly localized at lateral membranes of contacting cells and formed E-cadherin puncta and plaques, p8-EcadEGFP-expressing cells frequently formed transient cell-cell contacts. During random cell migration, p8-EcadEGFP was found in lamellipodia. In contrast, wt-EcadEGFP localized at lateral cell-cell contact sites in low or non-motile cells. Inhibition of the epidermal growth factor (EGF) receptor, which plays a major role in lamellipodia formation and cell migration, reduced the motility of p8-EcadEGFP-expressing cells and caused lateral membrane staining of p8-EcadEGFP. Conversely, EGF induced cell motility and caused formation of lamellipodia that were E-cadherin positive. In conclusion, our data show that mutant E-cadherin significantly alters the dynamics of cell adhesion and motility in living cells and interferes with the formation of stable cell-cell contacts.     

Cytokinesis of neuroepithelial cells can divide their basal process before anaphase

Neuroepithelial (NE) cells, the primary stem and progenitor cells of the vertebrate central nervous system, are highly polarized and elongated. They retain a basal process extending to the basal lamina, while undergoing mitosis at the apical side of the ventricular zone. By studying NE cells in the embryonic mouse, chick and zebrafish central nervous system using confocal microscopy, electron microscopy and time-lapse imaging, we show here that the basal process of these cells can split during M phase. Splitting occurred in the basal-to-apical direction and was followed by inheritance of the processes by either one or both daughter cells. A cluster of anillin, an essential component of the cytokinesis machinery, appeared at the distal end of the basal process in prophase and was found to colocalize with F-actin at bifurcation sites, in both proliferative and neurogenic NE cells. GFP-anillin in the basal process moved apically to the cell body prior to anaphase onset, followed by basal-to-apical ingression of the cleavage furrow in telophase. The splitting of the basal process of M-phase NE cells has implications for cleavage plane orientation and the relationship between mitosis and cytokinesis.     

Annexin 11 is required for midbody formation and completion of the terminal phase of cytokinesis

Annexins are Ca(2+)-binding, membrane-fusogenic proteins with diverse but poorly understood functions. Here, we show that during cell cycle progression annexin 11 translocates from the nucleus to the spindle poles in metaphase and to the spindle midzone in anaphase. Annexin 11 is recruited to the midbody in late telophase, where it forms part of the detergent-resistant matrix that also contains CHO1. To investigate the significance of these observations, we used RNA interference to deplete cells of annexin 11. A combination of confocal and video time-lapse microscopy revealed that cells lacking annexin 11 fail to establish a functional midbody. Instead, daughter cells remain connected by intercellular bridges that contain bundled microtubules and cytoplasmic organelles but exclude normal midbody components such as MKLP1 and Aurora B. Annexin 11-depleted cells failed to complete cytokinesis and died by apoptosis. These findings demonstrate an essential role for annexin 11 in the terminal phase of cytokinesis.     

Cellular events during sexual development from amoeba to plasmodium in the slime mould Physarum polycephalum

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.     

Ontogenesis of Tannin Coenocytes in Sambucus racemosa L. I. Development of the Coenocytes from Mononucleate Tannin Cells

Tannin coenocytes in shoots of Sambucus racemosa L. developfrom mono-nucleate tannin cells which can be distinguished amongthe cells of the first internode, and which keep on growing.After karyokinesis without cytokinesis bi-nucleate tannin cellsoccur which yield a synchronous karyokinesis leading to 4 nucleiin the tube. The number of nuclei in the coenocyte is 2ⁿ wheren equals the number of karyokineses that have occurred. Sometimesthe number of nuclei is different and one nucleus is bigeerthan the rest indicating that a previous fusion of nuclei hasoccurred. The distribution of nuclei in the coenocyte supportsthe possibility of fusion of chromosome sets at the moment ofnuclear envelope dispersion. Sambucus racemosa L., coenocytes, synchronous karyokinesis, development     

Localization, dynamics, and function of survivin revealed by expression of functional survivinDsRed fusion proteins in the living cell

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.