The distribution of TPX2 in dividing leaf cells of the fern Asplenium nidus

Plant cell division requires the dynamic organisation of several microtubule arrays. The mechanisms of regulation of the above arrays are under rigorous research. Among several factors that are involved in plant microtubule dynamics, the Targeting Protein for Xklp2 (TPX2) has been found to play a role in spindle organisation, in combination with Aurora kinases, in dividing cells of angiosperms. Microtubule organisation in dividing cells of ferns exhibits certain peculiarities. Accordingly, the presence and distribution of a TPX2 homologue might be helpful in understanding the patterns and regulatory mechanisms of microtubule arrays in this plant group. In this study, a putative TPX2 homologue was identified using Western blotting in the fern Asplenium nidus. It was found, using immunostaining and CLSM, that it is co‐localised with perinuclear preprophase microtubules and the prophase spindle, and follows the microtubule pattern during metaphase/anaphase and telophase. During cytokinesis, while in angiosperms TPX2 is degraded, in A. nidus the TPX2 signal persists, co‐localising with the phragmoplast. In early post‐cytokinetic cells, a TPX2 signal is present on the nuclear surface facing the daughter cell wall and, thereafter it is co‐localised with the fern‐specific microtubule aggregation that lines the new wall, which is possibly involved in cortical microtubule assembly.     

Fine filaments observed within the cytoplasm surrounding the leading edge of the septum in telophase cells of Spirogyra (Zygnematales, Chlorophyta)

Electron microscope observation revealed the presence of many fine filaments within the cytoplasm surrounding the leading edge of the septum in telophase cells of Spirogyra verruculosa Jao. These filaments, about 7 nm each in diameter, ran parallel to one another along the leading edge of the septum and, sometimes, they appeared to be gathered into at least two bundles. These filament distribution patterns coincided well with those of the fluorescence of rhodamine-labeled phalloidin in the vicinity of the septum in telophase cells. The present results suggest that the fine filaments observed within the cytoplasm surrounding the leading edge of the septum may be actin filaments.     

Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression

When centrosomes are destroyed during prophase by laser microsurgery, vertebrate somatic cells form bipolar acentrosomal mitotic spindles (Khodjakov, A., R.W. Cole, B.R. Oakley, and C.L. Rieder. 2000. Curr. Biol. 10:59-67), but the fate of these cells is unknown. Here, we show that, although these cells lack the radial arrays of astral microtubules normally associated with each spindle pole, they undergo a normal anaphase and usually produce two acentrosomal daughter cells. Relative to controls, however, these cells exhibit a significantly higher (30-50%) failure rate in cytokinesis. This failure correlates with the inability of the spindle to properly reposition itself as the cell changes shape. Also, we destroyed just one centrosome during metaphase and followed the fate of the resultant acentrosomal and centrosomal daughter cells. Within 72 h, 100% of the centrosome-containing cells had either entered DNA synthesis or divided. By contrast, during this period, none of the acentrosomal cells had entered S phase. These data reveal that the primary role of the centrosome in somatic cells is not to form the spindle but instead to ensure cytokinesis and subsequent cell cycle progression.     

Cell-cycle dependent anti-FGF-2 staining of chicken cardiac myocytes: Movement from chromosomal to cleavage furrow- and midbody-associated sites

Fibroblast growth factor-2 (FGF-2) promotes cardiac myocyte proliferation and has been detected in extracellular as well as cytoplasmic and nuclear compartments. As a first step in examining the participation of intracellular FGF-2 in cardiac myocyte cell cycle we have investigated its localization in proliferative chicken cells during interphase and the various stages of mitosis in culture. We have used a previously characterized and affinity-purified anti-FGF-2 antibody preparation which recognizes the 19-22 kDa variants of chick FGF-2. By immunofluorescence, bright, punctate anti-FGF-2 labelling was observed in 26% of interphase nuclei from myocytes derived from 5 day embryonic heart ventricles; these nuclei were positive for anti-bromodeoxyuridine staining indicating that they are at the S- or G2 phase of the cell cycle. In prophase and metaphase, bright anti-FGF-2 staining was detected in apparent association with chromosomes. During anaphase, however, anti-FGF-2 staining dissociated from chromosomal locations distinctly remaining in strand-like structures in the area of ensuing cleavage furrow formation. In late telophase and cytokinesis, strong staining persisted in the area of the midbody and reappeared in a small fraction of newly formed daughter nuclei. Absorption of the antibody preparation with immobilized FGF-2 eliminated all staining. This dynamic pattern of anti-FGF-2 staining suggests that chick FGF-2 or immunologically related protein(s) not only increase in DNA-synthesizing nuclei but they may play a role in subsequent stages of mitosis and cytokinesis.     

Autonomous activation of histone H1 kinase, cortical activity and microtubule organization in one- and two-cell mouse embryos

The activation of M-phase promoting factor (MPF) in one-cell mouse embryo is independent from the nucleus. Other autonomous phenomena include the cortical activity observed at the end of the first cell cycle and the reorganization of the microtubule network. Here, we observed that the autonomous control of MPF activation is present also in two-cell mouse embryos (H1 kinase activity being higher in the first than in the second cell cycle). Moreover, the disappearance of the cortical activity in anucleated halves is observed when MPF activation takes place. The rounding up of the cytoplast and the mitotic reorganization of the microtubule network correlates with the maximum activity of H1 kinase in anucleated halves from one-cell embryos. In anucleated halves of two-cell stage blastomeres neither the cortical activity nor the microtubule reorganization were observed. The degree of activation of histone H1 kinase, and, as a consequence, the cortical activity and the microtubule reorganization, does not depend on the distribution of cyclin B. Finally, the level of cyclin B synthesis is similar in anucleated and nucleated halves derived from both one- and two-cell embryos.     

Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, we show that FIP3 binds to Cyk-4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid-body. We have mapped the FIP3-binding region on Cyk-4 and show that it overlaps with the ECT2-binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.     

Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine.

A major feature of epithelial cell polarity is regulated positioning of the mitotic spindle within the cell. Spindles in cells of symmetrically expanding tissues are predicted to align parallel to the tissue plane. Direct measurement of this alignment has been difficult in mammalian tissues. Here, we analyzed the position of spindles in intact mouse intestinal epithelium using microtubule immunofluorescence and three-dimensional confocal imaging. Mitotic cells were identified in the proliferative zone of intestinal crypts. Spindle angle relative to the apical cell surface was determined either by direct measurement from confocal images or with a computational algorithm. Angles averaged within 10 degrees of parallel to the apical surface in metaphase and anaphase cells, consistent with robust planar spindle positioning, whereas spindles in prometaphase cells showed much greater angle variability. Interestingly, cytokinetic furrows appeared to extend from the basal cell surface toward the apical surface. This type of image analysis may be useful for studying the regulation of spindle position during tissue remodeling and tumor formation.     

Cell cycle function of a rice B2-type cyclin interacting with a B-type cyclin-dependent kinase

Cyclin-dependent kinases (CDKs) are involved in the control of cell cycle progression. Plant A-type CDKs are functional homologs of yeast Cdc2/Cdc28 and are expressed throughout the cell cycle. In contrast, B-type CDK (CDKB) is a family of mitotic CDKs expressed during the S/M phase, and its precise function remains unknown. Here, we identified two B2-type cyclins, CycB2;1 and CycB2;2, as a specific partner of rice CDKB2;1. The CDKB2;1-CycB2 complexes produced in insect cells showed a significant level of kinase activity in vitro, suggesting that CycB2 binds to and activates CDKB2. We then expressed green fluorescent protein (GFP)-fused CDKB2;1 and CycB2;2 in tobacco BY2 cells to investigate their subcellular localization during mitosis. Surprisingly, the fluorescence signal of CDKB2;1-GFP was tightly associated with chromosome alignment as well as with spindle structure during the metaphase. During the telophase, the signal was localized to the spindle midzone and the separating sister chromosomes, and then to the phragmoplast. On the other hand, the CycB2;2-GFP fluorescence signal was detected in nuclei during the interphase and prophase, moved to the metaphase chromosomes, and then disappeared completely after the cells passed through the metaphase. Co-localization of CDKB2;1-GFP and CycB2;2-GFP on chromosomes aligned at the center of the metaphase cells suggests that the CDKB2-CycB2 complex may function in retaining chromosomes at the metaphase plate. Overexpression of CycB2;2 in rice plants resulted in acceleration of root growth without any increase in cell size, indicating that CycB2;2 promoted cell division probably through association with CDKB2 in the root meristem.     

Overexpression of an Aurora-C kinase-deficient mutant disrupts the Aurora-B/INCENP complex and induces polyploidy

Aurora kinases are emerging as key regulators of centrosome function, chromosome segregation and cytokinesis. We previously isolated Aurora-C (Aie1), a third type of Aurora kinase, in a screen for kinases expressed in mouse sperm and eggs. Currently, we know very little about the precise localization and function of Aurora-C. Immunofluorescence analysis of ectopically expressed GFP-Aurora-C has revealed that Aurora-C is a new member of the chromosomal passenger proteins localizing first to the centromeres and then to the central spindles during cytokinesis. In order to study the potential role of Aurora-C, we examined the effects of a kinase-deficient (KD) mutant (AurC-KD) in HeLa Tet-Off cells under tetracycline control. Our results showed that overexpression of AurC-KD causes defects in cell division and induces polyploidy and apoptosis. Interestingly, AurC-KD overexpression also inhibits centromere/kinetochore localization of Aurora-B, Bub1, and BubR1, reduces histone H3 phosphorylation, and disrupts the association of INCENP with Aurora-B. Together, our results showed that Aurora-C is a chromosomal passenger protein, which may serve as a key regulator in cell division.     

Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150^Glued subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.     

Mitotic activity and cell elongation in geostimulated roots of Zea mays

Mitotic activity was investigated in the primary meristem of horizontally oriented excised root tips of Zea mays during the first six hours of their georeaction. The only statistically significant change that could be detected in the meristem was a decrease of the length of its upper half. No significant difference in mitotic activity was found between the upper and lower halves of roots kept continuously horizontal for 6 h. Cell proliferation thus seems relatively insensitive to changes in the redistribution of endogenous growth regulators that are believed to occur within the meristem during the onset of geotropism. In the zone of bending proximal to the meristem cell length was significantly greater in the upper half than in either the lower half or in the equivalent position in vertical control roots. Thus, cell elongation seems to be promoted in the upper half of the horizontal root. Thus, The differences in cell length were not accompanied by any change in the proportion of nuclei synthesising DNA in these elongating, non-meristematic cells.     

Specific organization of intracytoplasmic filaments in the dog testicular interstitial cell

Summary In the dog testicular interstitial cells the cytoplasmic filaments are occasionally arranged in large bundles piled closely in an extensive area adjacent to the Golgi region in the cytoplasm. Some of the large bundles show conspicuous circular or spiral configurations which are composed of elaborate arrangements of both circular and longitudinal filaments and accompany tubules of agranular endoplasmic reticulum running parallel to the longitudinal filaments.This paper is dedicated to Prof. Dr. Jun-ichiro Satoh on his 60 birthday. The authors wish to thank the members in the Department of Pathophysiology (Head: Prof. Dr. K. Yamashita), Atomic Disease Institut, Nagasaki University School of Medicine, for their technical assistance.     

An anillin homologue,Mid2p,acts during fission yeast cytokinesis to organize the septin ring and promote cell separation

Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.     

The nuclear envelope in the plant cell cycle: structure, function and regulation

Background

Higher plants are, like animals, organisms in which successful completion of the cell cycle requires the breakdown and reformation of the nuclear envelope in a highly controlled manner. Interestingly, however, while the structures and processes appear similar, there are remarkable differences in protein composition and function between plants and animals.

Scope

Recent characterization of integral and associated components of the plant nuclear envelope has been instrumental in understanding its functions and behaviour. It is clear that protein interactions at the nuclear envelope are central to many processes in interphase and dividing cells and that the nuclear envelope has a key role in structural and regulatory events.

Conclusion

Dissecting the mechanisms of nuclear envelope breakdown and reformation in plants is necessary before a better understanding of the functions of nuclear envelope components during the cell cycle can be gained.     

Applied AC and DC magnetic fields cause alterations in the mitotic cycle of early sea urchin embryos

This study demonstrates that exposure to 60 Hz magnetic fields (3.4–8.8 mT) and magnetic fields over the range DC-600 kHz (2.5–6.5 mT) can alter the early embryonic development of sea urchin embryos by inducing alterations in the timing of the cell cycle. Batches of fertilized eggs were exposed to the fields produced by a coil system. Samples of the continuous cultures were taken and scored for cell division. The times of both the first and second cell divisions were advanced by ELF AC fields and by static fields. The magnitude of the 60 Hz effect appears proportional to the field strength over the range tested. The relationship to field frequency was nonlinear and complex. For certain frequencies above the ELF range, the exposure resulted in a delay of the onset of mitosis. The advance of mitosis was also dependent on the duration of exposure and on the timing of exposure relative to fertilization. © 1995 Wiley-Liss, Inc.     

A mechanism for ParB-dependent waves of ParA,a protein related to DNA segregation during cell division in prokaryotes

Prokaryotic plasmids encode partitioning (par) loci involved in segregation of DNA to daughter cells at cell division. A functional fusion protein consisting of Walker-type ParA ATPase and green fluorescent protein (Gfp) oscillates back and forth within nucleoid regions with a wave period of about 20 minutes. A model is discussed which is based on cooperative non-specific binding of ParA to the nucleoid, and local ParB initiated generation of ParA oligomer degradation products, which act autocatalytically on the degradation reaction. The model yields self-initiated spontaneous pattern formation, based on Turing's mechanism, and these patterns are destroyed by the degradation products, only to initiate a new pattern at the opposite nucleoid region. A recurrent wave thus emerges. This may be a particular example of a more general class of pattern forming mechanisms, based on protein oligomerization upon a template (membranes, DNA a.o.) with resulting enhanced NTPase function in the oligomer state, which may bring the oligomer into an unstable internal state. An effector initializes destabilization of the oligomer to yield degradation products, which act as seeds for further degradation in an autocatalytic process. We discuss this mechanism in relation to recent models for MinDE oscillations in E.coli and to microtubule degradation in mitosis. The study points to an ancestral role for the presented pattern types in generating bipolarity in prokaryotes and eukaryotes.     

Fluid transport and dimensions of epithelial cells and intercellular spaces in frog gallbladder

Summary Morphologic findings of widely dilated intercellular spaces in fluid transporting epithelia have been claimed as evidence for the existence of an epithelial compartment in which the coupling between solute and water fluxes takes place. The validity of using epithelial geometry in sectioned material as an argument can be questioned. The present report describes the morphological appearance of frog gallbladder epithelium — normal and ouabain-treated — in the living state in vitro and after fixation, dehydration and embedding. Gallbladder segments were photographed in the living state and at the end of each step of the preparative procedure. Direct observations of whole-mounted gallbladder segments were carried out, taking advantage of the possibility of optical sectioning and high resolution by Nomarski-microscopy. The same specimens were then sectioned and examined by conventional light and electron microscopy. The observations were quantitated and showed that the epithelial cells of normal and ouabain-treated gallbladders experienced an average linear shrinkage down to 70% of their length in Ringer's solution, which corresponds to a volume shrinkage down to 35%. Moreover, dilated lateral intercellular spaces appeared during the dehydration and embedding procedure in normal but only very moderately or not at all in ouabain-treated gallbladder specimens.