Asymmetrical localization and segregation of Paracentrotus lividus Bep4 maternal protein.

Asymmetric divisions that produce two distinct cells play a fundamental role in generating different cell types during development. Here we investigate the role of the cortex region and mitotic apparatus in asymmetrical localization and segregation of Bep4 protein in Paracentrotus lividus egg. By centrifugation of eggs with or without drugs we established an involvement of the cortex region in localization of Bep4 protein, confirmed by immunohistochemistry of isolated cortex. Association with the mitotic apparatus during cell division permits selective partitioning of Bep4 protein into the daughter cells. Direct association with spindle was also demonstrated both by Western blot and immunohistochemistry after isolation of the mitotic apparatus.     

Common themes in centriole and centrosome movements

Centrioles are found in nearly all eukaryotic cells and are required for growth and maintenance of the radial array of microtubules, the mitotic spindle, and cilia and flagella. Different types of microtubule structures are often required at different places in a given cell; centrioles must move around to nucleate these varied structures. Here, we draw together recent data on diverse centriole movements to decipher common themes in how centrioles move. Par proteins establish and maintain the required cellular asymmetry. The actin cytoskeleton facilitates movement of multiple basal bodies. Microtubule forces acting on the cell cortex, and nuclear-cytoskeletal links, are important for positioning individual centrosomes, and during cell division. Knowledge of these common mechanisms can inform the study of centriole movements across biology.     

Multi‐step down‐regulation of the secretory pathway in mitosis: A fresh perspective on protein trafficking

The secretory pathway delivers proteins synthesized at the rough endoplasmic reticulum (RER) to various subcellular locations via the Golgi apparatus. Currently, efforts are focused on understanding the molecular machineries driving individual processes at the RER and Golgi that package, modify and transport proteins. However, studies are routinely performed using non‐dividing cells. This obscures the critical issue of how the secretory pathway is affected by cell division. Indeed, several studies have indicated that protein trafficking is down‐regulated during mitosis. Moreover, the RER and Golgi apparatus exhibit gross reorganization in mitosis. Here I provide a relatively neglected perspective of how the mitotic cyclin‐dependent kinase (CDK1) could regulate various stages of the secretory pathway. I highlight several aspects of the mitotic control of protein trafficking that remain unresolved and suggest that further studies on how the mitotic CDK1 influences the secretory pathway are necessary to obtain a deeper understanding of protein transport.     

Reorganization of chromatin in Xenopus egg extracts: electron microscopic studies.

The structural basis of mitotic condensation of chromosomes is one of the problems of cell biology yet to be elucidated. A variety of approaches have been used to study this problem and a large number of hypotheses have been proposed to explain the different levels of compaction of chromatin. Xenopus egg extracts, now widely used to study various aspects of cell biology, provide a valuable tool to study mitotic condensation of chromosomes. No detailed study has however yet been reported on the submicroscopic organization of condensed chromosomes in vitro in egg extracts. We present here the results of our electron microscopic studies on the organization of condensed chromosomes in vitro, using demembranated sperm nuclei and mitotic (CSF-arrested) extracts of Xenopus laevis eggs, clarified by high speed centrifugation. Upon introduction of sperm nuclei in egg extracts, the nuclei swell and the chromatin undergoes a rapid decondensation; at this stage the chromatin is formed of 10 nm fibrils. After longer incubation, the chromatin condenses, and by 2 h chromosomal structures can be visualized by staining with DAPI or Hoechst 33258. Our results on the organization of chromosomes in different stages of condensation are discussed in relation to the different hypotheses proposed to explain the process of mitotic condensation of chromosomes. Finally, this study demonstrates the feasibility of high-resolution analysis of the process of chromosome condensation.     

Cytokinesis: relative alignment of the cell division apparatus and the mitotic spindle

The cell division apparatus is assembled at different stages of the cell cycle in different eukaryotic organisms. Mechanisms exist in all organisms, however, to ensure that the cell division apparatus and the mitotic spindle are aligned perpendicular to each other. Such an alignment ensures that each daughter cell receives a nucleus and that the cell division apparatus does not cleave and destroy the genetic material. The interaction(s) of astral microtubules with the cell cortex appears to play an important role in establishing perpendicularity between chromosome segregation and cell division machinery.     

Perturbing mitosis for anti‐cancer therapy: is cell death the only answer?

Interfering with mitosis for cancer treatment is an old concept that has proven highly successful in the clinics. Microtubule poisons are used to treat patients with different types of blood or solid cancer since more than 20 years, but how these drugs achieve clinical response is still unclear. Arresting cells in mitosis can promote their demise, at least in a petri dish. Yet, at the molecular level, this type of cell death is poorly defined and cancer cells often find ways to escape. The signaling pathways activated can lead to mitotic slippage, cell death, or senescence. Therefore, any attempt to unravel the mechanistic action of microtubule poisons will have to investigate aspects of cell cycle control, cell death initiation in mitosis and after slippage, at single‐cell resolution. Here, we discuss possible mechanisms and signaling pathways controlling cell death in mitosis or after escape from mitotic arrest, as well as secondary consequences of mitotic errors, particularly sterile inflammation, and finally address the question how clinical efficacy of anti‐mitotic drugs may come about and could be improved.     

Transitions regulating the timing of cytokinesis in embryonic cells

Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells. In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit. Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition. Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not. Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA. Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint.     

Why does a cleavage plane develop parallel to the spindle axis in conical sand dollar eggs? A key question for clarifying the mechanism of contractile ring positioning

Three types of models have been proposed about how the mitotic apparatus determines the position of the cleavage furrow in animal cells. In the first and second types, the contractile ring appears in a cortical region that least and most astral microtubules reach, respectively. The third type is that the spindle midzone positions the contractile ring. In the previous study, a new model was proposed through analyses of cytokinesis in sand dollar and sea urchin eggs. Gradients of the surface density of microtubule plus ends are assumed to drive membrane proteins whose accumulation causes the formation of contractile-ring microfilaments. In the present study, the validity of each model is examined by simulating the furrow formation in conical sand dollar eggs with the mitotic apparatus oriented perpendicular to the cone axis. The new model predicts that unilateral furrows with cleavage planes roughly parallel to the spindle axis appear between the mitotic apparatus and the vertex besides the normally positioned furrow. The predictions are consistent with the observations by Rappaport & Rappaport (1994, Dev. Biol.164, 258-266). The other three types of models do not predict the formation of the ectopic furrows. Furthermore, it is pointed out that only the new model has the ability to explain the geometrical relationship between the mitotic apparatus and the contractile ring under various experimental conditions. These results strongly suggest the real existence of the membrane proteins postulated in the model.     

Golgi apparatus partitioning during cell division

This review discusses the mitotic segregation of the Golgi apparatus. The results from classical biochemical and morphological studies have suggested that in mammalian cells this organelle remains distinct during mitosis, although highly fragmented through the formation of mitotic Golgi clusters of small tubules and vesicles. Shedding of free Golgi-derived vesicles would consume Golgi clusters and disperse this organelle throughout the cytoplasm. Vesicles could be partitioned in a stochastic and passive way between the two daughter cells and act as a template for the reassembly of this key organelle. This model has recently been modified by results obtained using GFP- or HRP-tagged Golgi resident enzymes, live cell imaging and electron microscopy. Results obtained with these techniques show that the mitotic Golgi clusters are stable entities throughout mitosis that partition in a microtubule spindle-dependent fashion. Furthermore, a newer model proposes that at the onset of mitosis, the Golgi apparatus completely loses its identity and is reabsorbed into the endoplasmic reticulum. This suggests that the partitioning of the Golgi apparatus is entirely dependent on the partitioning of the endoplasmic reticulum. We critically discuss both models and summarize what is known about the molecular mechanisms underlying the Golgi disassembly and reassembly during and after mitosis. We will also review how the study of the Golgi apparatus during mitosis in other organisms can answer current questions and perhaps reveal novel mechanisms.     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Regulated assembly of the mitotic spindle: a perspective from two ends

Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes attached to a dynamic array of microtubules. The microtubule array is nucleated by centrosomes and organized by associated structural and motor proteins. Mechanical linkages between sister chromatids and microtubules are critical for spindle assembly and chromosome segregation. Defects in either chromosome or centrosome segregation can lead to aneuploidy and are correlated with cancer progression. In this review, we discuss current models of how centrosomes and chromosomes organize the spindle for their equal distribution to each daughter cell.     

Ultrastructure of the mitotic apparatus of metaphase cells in a pig embryo kidney tissue culture after stopping the action of 2-mercaptoethanol

The ultrastructure of the metaphase mitotic apparatus has been studied in the KEPV cells during 6 hours after the removal of 2-mercaptoethanol (0.001 M). Starting from the analysis of chromosome disposition, the structures of the kinetochore regions and of the mitotic spindle poles, and the degree of integrity of the mitotic spindle microtubules, six types of metaphase cells were revealed. A comparison of the results of the present paper with those of the earlier studies enabled us to present the dynamics of the metaphase mitotic apparatus reconstruction. Four basic stages are revealed in this process. At the first stage, the K-metaphase centrioles form diplosomes again, the number and extent of kinetochore microtubules increase too. At the second stage, the metaphase plate forms, but interpolar and astral microtubules are absent. At the third stage, the structure of the kinetochore regions becomes normal. Thus, the metaphase plate may have formed before the orientation of kinetochores to the poles took place. At the fourth stage, the interpolar and astral microtubules appear; the mitotic spindle reestablishes completely. It is supposed that the formation and functioning of the mitotic apparatus is not confined to the interaction of microtubules of different types.     

Chromosome shaping by two condensins

It remains a big challenge in modern cell biology to determine the mechanism by which a discrete set of rod-shaped chromosomes is assembled from an amorphous mass of interphase chromatin. Recent studies start to shed new lights on how this process is actively supported by a class of multiprotein complexes called condensins. In vertebrate cells, two different condensin complexes make distinct mechanistic contributions to determining the shape and integrity of mitotic chromosomes.     

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.     

The mitochondrial cycle of Arabidopsis shoot apical meristem and leaf primordium meristematic cells is defined by a perinuclear tentaculate/cage-like mitochondrion

Plant cells exhibit a high rate of mitochondrial DNA (mtDNA) recombination. This implies that before cytokinesis, the different mitochondrial compartments must fuse to allow for mtDNA intermixing. When and how the conditions for mtDNA intermixing are established are largely unknown. We have investigated the cell cycle-dependent changes in mitochondrial architecture in different Arabidopsis (Arabidopsis thaliana) cell types using confocal microscopy, conventional, and three-dimensional electron microscopy techniques. Whereas mitochondria of cells from most plant organs are always small and dispersed, shoot apical and leaf primordial meristematic cells contain small, discrete mitochondria in the cell periphery and one large, mitochondrial mass in the perinuclear region. Serial thin-section reconstructions of high-pressure-frozen shoot apical meristem cells demonstrate that during G1 through S phase, the large, central mitochondrion has a tentaculate morphology and wraps around one nuclear pole. In G2, both types of mitochondria double their volume, and the large mitochondrion extends around the nucleus to establish a second sheet-like domain at the opposite nuclear pole. During mitosis, approximately 60% of the smaller mitochondria fuse with the large mitochondrion, whose volume increases to 80% of the total mitochondrial volume, and reorganizes into a cage-like structure encompassing first the mitotic spindle and then the entire cytokinetic apparatus. During cytokinesis, the cage-like mitochondrion divides into two independent tentacular mitochondria from which new, small mitochondria arise by fission. These cell cycle-dependent changes in mitochondrial architecture explain how these meristematic cells can achieve a high rate of mtDNA recombination and ensure the even partitioning of mitochondria between daughter cells.     

Chromosome segregation in Eubacteria

It is now clear that bacterial chromosomes rapidly separate in a manner independent of cell elongation, suggesting the existence of a mitotic apparatus in bacteria. Recent studies of bacterial cells reveal filamentous structures similar to the eukaryotic cytoskeleton, proteins that mediate polar chromosome anchoring during Bacillus subtilis sporulation, and SMC interacting proteins that are involved in chromosome condensation. A picture is thereby developing of how bacterial chromosomes are organized within the cell, how they are separated following duplication, and how these processes are coordinated with the cell cycle.     

Mitotic apparatus-surface interaction and cell division

Summary

Results of recent investigations concerning the mechanisms of animal cell division are reviewed. The mitotic apparatus was aspirated from a blastomere of a sand dollar (Echinarachnius parma) egg before second cleavage, and the time interval between removal and the appearance of the furrow in the control companion blastomere was measured. When the mitotic apparatus is removed 4 min or less before the furrows appear in the controls, furrows also develop in the operated cells. These results show that 4 min before furrowing begins, the surface changes which lead to formation of the division mechanism have become irreversible. When the mitotic apparatus of a cylindrical cell is shifted by pushing in one of the poles when the furrow appears, a new furrow develops in association with the new position of the mitotic apparatus. The same mitotic apparatus could elicit as many as 13 furrows over a 24.5 min period following the appearance of the first furrow. The results show that, in the proper geometrical circumstances, the mitotic apparatus and the surface can interact over a longer period than they do in normal cells.

By artificially constricting sand dollar eggs with a glass loop, the normal distance relations between the astral centers and the polar and equatorial surfaces can be reversed. Constricted cells cleave normally. The blocking effect of ethyl urethane can be reversed by moving the equatorial surface closer to the spindle portion of the mitotic apparatus. Relocation of other parts of the surface closer to the mitotic apparatus was ineffective. These results help elucidate the geometrical relations that are essential for furrow formation between the mitotic apparatus and the surface.

In cylindrical sand dollar eggs, single asters and the widely separated asters of a broken mitotic apparatus can cause furrow-like constrictions in the adjacent cylindrical surface. This reaction can be blocked by treating cells with ethyl urethane, which reduces astral size. The nature of the shape change that the aster causes depends upon the surface region affected. These results aid in understanding the nature of the change in surface physical activity caused by the mitotic apparatus.     

Invariant mRNA and mitotic protein breakdown solves the Russian Doll problem of the cell cycle

It has been proposed that cyclical gene expression occurs at a large number of different times during the cell cycle. The existence of a large number of cycle-specific variations in mRNA and protein during the eukaryotic cell cycle raises the problem of how cell-cycle variations are regulated. This is the “infinite regression” or Russian Doll problem where postulating a cell-cycle specific control element pushes the explanation of cell-cycle variation back one step to the problem of how that control element varies during the cell cycle.PCR studies on unperturbed cells indicate Cyclin mRNA content is invariant during the cell cycle. Furthermore, calculations reveal that variations in mRNA content do not account for observed protein variations.Continuous and constant gene expression during the cell cycle, continuous protein accumulation, and protein breakdown only within the mitotic window solves the Russian Doll problem or infinite regression problem. These results, and theoretical ideas support an alternative view of the cell cycle where many of the proposed control systems do not exist.     

AN INDIRECT METHOD TO ASSAY FOR MITOTIC CENTERS IN SAND DOLLAR (DENDRASTER EXCENTRICUS) EGGS

It is possible consistently to induce sea urchin and sand dollar eggs to cleave directly from one cell into four cells. This is done by exposing the fertilized eggs to benzimidazole for 20 to 30 min beginning about early metaphase. The mitotic apparatus regresses, the cells do not cleave, and shortly after they are returned to normal sea water an early-prophase-appearing nucleus is present in each cell. Each cell then organizes a tetrapolar tetrahedral mitotic apparatus de novo, instead of transforming a bipolar mitotic apparatus into a tetrapolar figure, and cleaves one-to-four. In another type of experiment, it appears that sand dollar eggs exposed to mercaptoethanol during the first period of mitotic center duplication have only half as many centers by first cleavage metaphase as the normal controls. This is consistent with an earlier report by Mazia et al (1960). Using this same experimental technique, it was demonstrated that benzimidazole, on the contrary, does not interfere with mitotic center duplication in sand dollar eggs. A labeling experiment demonstrated that benzimidazole does not interfere markedly with the normal pattern of incorporation of C¹⁴-thymidine into the DNA of sea urchin eggs. The data reported here suggest that judicious treatment of sand dollar eggs (and probably sea urchin eggs, too) with benzimidazole can induce the eggs to cleave into as many cells as there were mitotic centers sometime earlier, for example at early metaphase of the first cleavage division. This provides a very useful tool for studies on the process of mitotic center duplication.