Kinase activation of ClC-3 accelerates cytoplasmic condensation during mitotic cell rounding

"Mitotic cell rounding" describes the rounding of mammalian cells before dividing into two daughter cells. This shape change requires coordinated cytoskeletal contraction and changes in osmotic pressure. While considerable research has been devoted to understanding mechanisms underlying cytoskeletal contraction, little is known about how osmotic gradients are involved in cell division. Here we describe cytoplasmic condensation preceding cell division, termed "premitotic condensation" (PMC), which involves cells extruding osmotically active Cl(-) via ClC-3, a voltage-gated channel/transporter. This leads to a decrease in cytoplasmic volume during mitotic cell rounding and cell division. Using a combination of time-lapse microscopy and biophysical measurements, we demonstrate that PMC involves the activation of ClC-3 by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in human glioma cells. Knockdown of endogenous ClC-3 protein expression eliminated CaMKII-dependent Cl(-) currents in dividing cells and impeded PMC. Thus, kinase-dependent changes in Cl(-) conductance contribute to an outward osmotic pressure in dividing cells, which facilitates cytoplasmic condensation preceding cell division.     

Effect of cytomegalovirus on cell cycle progression and formation of pathological mitoses in cultured human diploid fibroblasts

The effect of cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G2, or M); at the same time, at least part of infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This fact suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of cytomegalovirus.     

Chalones and the Control of Normal, Regenerative, and Neoplastic Growth of the Skin

SYNOPSIS Chalones,inhibitors of cell dmsion have been isolatedand studied from a number of mammalian tissues, most notably,the epidermis The epidermal rhalone is a glycoprotein It exhibitsconsiderable, but not complete specificity The epidermal chalone decreases mitotic activity by inhibitingcells in the G 2 phase of the cell cycle from entering mitosis,and probably also by inhibiting ceils in the G 1 phase of thecell cycle from entering mitosis To inhibit cells in G 2 fromentering mitosis the chilone requnes adrenalin, and for maximalactivity hydrocortisone It is not known if idrenalin and hydrocortisoneare required for chalone inhibition of cells in G 1 In addition to inhibiting cell division in normal epidermalcells the epidermal chalone can inhibit cell division in regeneratingepidermal cells induced to proliferate by chemical damage Thephase of the cell cycle in which the chalone inhibits legeneratingepidermal cells from entering mitosis is not known Epidermal tumors contain a decreased amount of chalone Mitosisin epidermal tumors is inhibited by treatment with epidermalchalone Tumor cells are inhibitedfrom entering mitosis fromeither the G 1 or G 2 phases of the cell cycle Chalones are said to inhibit mitosis by a negative feedbackmechanism However, experiments which presumably result in adecrease in chalone concentration do not result in an increasein mitotic activity It is suggested that if chalones are physiological controllers of cell division they do not act by a simplenegative feedback mechanism but require the action of a substanceto decrease their concentration     

Optical volume and mass measurements show that mammalian cells swell during mitosis

The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells.     

Centriole inheritance

Early cell biologists perceived centrosomes to be permanent cellular structures. Centrosomes were observed to reproduce once each cycle and to orchestrate assembly a transient mitotic apparatus that segregated chromosomes and a centrosome to each daughter at the completion of cell division. Centrosomes are composed of a pair of centrioles buried in a complex pericentriolar matrix. The bulk of microtubules in cells lie with one end buried in the pericentriolar matrix and the other extending outward into the cytoplasm. Centrioles recruit and organize pericentriolar material. As a result, centrioles dominate microtubule organization and spindle assembly in cells born with centrosomes. Centrioles duplicate in concert with chromosomes during the cell cycle. At the onset of mitosis, sibling centrosomes separate and establish a bipolar spindle that partitions a set of chromosomes and a centrosome to each daughter cell at the completion of mitosis and cell division. Centriole inheritance has historically been ascribed to a template mechanism in which the parental centriole contributed to, if not directed, assembly of a single new centriole once each cell cycle. It is now clear that neither centrioles nor centrosomes are essential to cell proliferation. This review examines the recent literature on inheritance of centrioles in animal cells.Key words: centrosome, centriol, spindle, mitosis, microtubule, cell cycle, checkpoints     

Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.     

Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis

Mitotic chromosome condensation is normally dependent on the previous completion of replication. Caffeine spectacularly deranges cell cycle controls after DNA polymerase inhibition or DNA damage; it induces the condensation, in cells that have not completed replication, of fragmented nuclear structures, analogous to the S-phase prematurely condensed chromosomes seen when replicating cells are fused with mitotic cells. Caffeine has been reported to induce S-phase condensation in cells where replication is arrested, by accelerating cell cycle progression as well as by uncoupling it from replication; for, in BHK or CHO hamster cells arrested in early S-phase and given caffeine, condensed chromosomes appear well before the normal time at which mitosis occurs in cells released from arrest. However, we have found that this apparent acceleration depends on the technique of synchrony and cell line employed. In other cells, and in synchronized hamster cells where the cycle has not been subjected to prolonged continual arrest, condensation in replication-arrested cells given caffeine occurs at the same time as normal mitosis in parallel populations where replication is allowed to proceed. This caffeine-induced condensation is therefore "premature" with respect to the chromatin structure of the S-phase nucleus, but not with respect to the timing of the normal cycle. Caffeine in replication-arrested cells thus overcomes the restriction on the formation of mitotic condensing factors that is normally imposed during DNA replication, but does not accelerate the timing of condensation unless cycle controls have previously been disturbed by synchronization procedures.     

Effect of the Cytomegalovirus on Cell Cycle Progression and Pathological Mitoses in Cultured Human Diploid Fibroblasts

The effect of the cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G ₂, or M); at the same time, at least part of the infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained ³H-thymidine label above chromosomes. This suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of the cytomegalovirus.     

Cell cycle specific expression and nucleolar localization of human J-domain containing co-chaperon Mrj

J-domain containing co-chaperone Mrj (mammalian relative to DnaJ) has been implicated in diverse cellular functions including placental development and inhibition of Huntingtin mediated cytotoxicity. It has also been shown to interact with keratin intermediate filaments. Since keratins undergo extensive reorganization during cell division, its interactor Mrj might also play an important role in the regulation of cell cycle. In support of this hypothesis, we report the up-regulation of Mrj protein in M-phase of HeLa cells implicating its role in mitosis related activities. The protein is dispersed throughout the cell during late mitosis and is localized in nucleolus during interphase, confirming that the activity of Mrj is regulated by its cell cycle specific expression together with its differential subcellular localization.     

Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts

We have examined the effects of elevating the intracellular levels of p34cdc2 kinase by microinjection into living mammalian cells. These studies reveal rapid and dramatic changes in cell shape with cells becoming round and losing the bulk of their cell-substratum contact. Such effects were induced at all times in the cell cycle except at S phase and were fully reversible at S phase or mitosis. Similar results were obtained with the homogeneous catalytic subunit of p34cdc2 kinase or p34cdc2 kinase associated with cyclin B. These alterations were accompanied by a marked reduction in interphase microtubules without the spindle formation, actin microfilament redistribution, and premature chromatin condensation. Although these changes closely mimic the events occurring during early phases of mitosis, p34cdc2 kinase-injected cells were not induced to pass further into division. These data provide detailed evidence that p34cdc2 kinase plays a major prerequisite role in the rearrangement of cellular structures associated with mammalian cell mitosis.     

Kinetics of Cell Volume Changes of Murine Lymphoma Cells Subjected to Different Agents In Vitro

Cell volume distributions obtained with an electronic particle analyzer were used to study the changes in volume of individual cells in the absence of cell division. Cultures of murine lymphoma (strain L5178-Y) cells in suspension were used in these studies. During a division delay following ionizing radiation, individual cells increased exponentially in volume with equal rate constants; these rate constants were indistinguishable from that describing the increase in cell number of an unirradiated population. When an originally log phase population of cells was prevented from increasing in number by inhibitors of DNA synthesis, individual cells increased exponentially in volume for about one generation time with the same rate constant as observed after exposure to ionizing radiation; thereafter, only the cells defining the upper half of the volume distribution continued to increase in volume, and they apparently did so with a first order rate constant proportional to their amount of DNA exceeding that present in one diploid complement of chromosomes in G(1). Cells arrested in mitosis with colchicine increased in volume for approximately 4 hr after which they remained constant in volume for almost one generation time; eventually these cells again increased in size. Inhibitors of protein and RNA synthesis inhibited the cell volume growth of irradiated cells.     

Microinjection of antibodies to centromere protein CENP-A arrests cells in interphase but does not prevent mitosis

Centromere protein CENP-A is a histone H3-like protein associated specifically with the centromere and represents one of the human autoantigens identified by sera taken from patients with the CREST variant of progressive systemic sclerosis. Injection of whole human autoimmune serum to the centromere into interphase cells disrupts some mitotic events. It has been assumed that this effect is due to CENP-E and CENP-C autoantigens, because of the effects of injecting monospecific sera to those proteins into culture cells. Here we have used an antibody raised against an N-terminal peptide of the human autoantigen CENP-A to determine its function in mitosis and during cell cycle progression. Affinity-purified anti-CENP-A antibodies injected into the nucleus during the early replication stages of the cell cycle caused cells to arrest in interphase before mitosis. These cells showed highly condensed small nuclei, a granular cytoplasm and loss of their division capability. On the other hand, microinjection of nocodazole-blocked HeLa cells in mitosis resulted in the typical punctate staining pattern of CENP-A for centromeres during different stages of mitosis and apparently normal cell division. This was corroborated by time-lapse imaging microscopy analysis of mid-interphase-injected cells, revealing that they undergo mitosis and divide properly. However, a significant delay throughout the progression of mitotic stages was observed. These results suggest that CENP-A is involved predominantly in an essential interphase event at the centromere before mitosis. This may include chromatin assembly at the kinetochore coordinate with late replication of satellite DNA to form an active centromere. Received: 3 August 1998 / Accepted: 18 September 1998     

A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited

The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed 'A quantitative model for cdc2 control of S phase and mitosis in fission yeast'. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications.     

Teilungsverhalten der Chromatophoren in bezug auf die Mitose während des Lebenszyklus vonDiatoma hiemale var.mesodon

The vegetative life cycle ofDiatoma hiemale var.mesodon (Ehr.)Grun. living in a spring has been studied under natural conditions. In the beginning the cells have a constant number of 8 chromatophores which are divided into 16 during cell growth. Chloroplast division is finished before nuclear division starts. The young daughter cells have again 8 chromatophores. In the course of cell division a plastic remodelling of the chromatophores and a simplifying of their shape occurs. Besides single cells also populations have been studied to follow the temporal progress of chromatophore division, mitosis and cell growth. The results are evaluated by indices and demonstrated by a diagram. The maximum of chromatophore divisions preceds the maximum of mitoses by several hours, while the cell growth is in correlation with the chromatophore division. Minima of the other parameters were found before mitosis is starting and after it is finished. Our results are discussed with regard to the semiautonomy of the plastids. From the morphological point of view this concept is supported by the mode of division and by the anticipation of the chromatophore division. The number of chromatophores at the beginning (8) and at the end (16) of the life cycle is constant. The life cycle is classified into stages of cell growth, chromatophore division, stagnation, mitosis and differentiation of the daughter cells.     

Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.     

Cell cycle regulation of VCIP135 deubiquitinase activity and function in p97/p47-mediated Golgi reassembly

In mammalian cells, the inheritance of the Golgi apparatus into the daughter cells during each cycle of cell division is mediated by a disassembly and reassembly process, and this process is precisely controlled by phosphorylation and ubiquitination. VCIP135 (valosin-containing protein p97/p47 complex–interacting protein, p135), a deubiquitinating enzyme required for p97/p47-mediated postmitotic Golgi membrane fusion, is phosphorylated at multiple sites during mitosis. However, whether phosphorylation directly regulates VCIP135 deubiquitinase activity and Golgi membrane fusion in the cell cycle remains unknown. We show that, in early mitosis, phosphorylation of VCIP135 by Cdk1 at a single residue, S130, is sufficient to inactivate the enzyme and inhibit p97/p47-mediated Golgi membrane fusion. At the end of mitosis, VCIP135 S130 is dephosphorylated, which is accompanied by the recovery of its deubiquitinase activity and Golgi reassembly. Our results demonstrate that phosphorylation and ubiquitination are coordinated via VCIP135 to control Golgi membrane dynamics in the cell cycle.     

Cell cycle and apoptosis: Common pathways to life and death

Programmed cell death, or apoptosis, is a highly regulated process used to eliminate unwanted or damaged cells from multicellular organisms. The morphology of cells undergoing apoptosis is similar to cells undergoing both normal mitosis and an aberrant form of mitosis called mitotic catastrophe. During each of these processes, cells release substrate attachments, lose cell volume, condense their chromatin, and disassemble the nuclear lamina. The morphological similarities among cells undergoing these processes suggest that the underlying biochemical changes also may be related. The susceptibility of cells to apoptosis frequently depends on the differentiation state of the cell. Additionally, cell cycle checkpoints appear to link the cell cycle to apoptosis. Deregulation of the cell cycle components has been shown to induce mitotic catastrophe and also may be involved in triggering apoptosis. Some apoptotic cells express abnormal levels of cell cycle proteins and often contain active Cdc2, the primary kinase active during mitosis. Although cell cycle components may not be involved in all forms of apoptosis, in many instances cell proliferation and cell death may share common pathways.     

A quantitative theory of liver regeneration in the rat. II: matching an improved mitotic inhibitor model to the data

A model of liver regeneration is put forward in which the rate of liver growth is controlled both by a liver-produced mitotic inhibitor and by the availability of parenchymal cells to enter the mitotic cycle. The model can be expressed as a pair of coupled differential equations, the first describing the dependance of inhibitor concentration on liver size and inhibitor decay and the second specifying the dependance of liver growth on inhibitor concentration and entry of cells into the mitotic cycle. The model is tested by comparing its solutions to the published data on mitotic indices following partial hepatectomy. For such a comparison, it is necessary to specify the cell-cycle time and the inhibitor dose-response function and half-life. If a negative exponential dose-response function, an inhibitor half-life of 11·4 h, and a cycle time of 18·25 h are postulated, the solutions match the data of Fabrikant (1968) who found that there were two waves of mitosis with a period of quiescence between them. The data of Grisham (1962), characterized by a single peak of mitosis, is matched by the theory using similar inhibitor properties but a shorter cell-cycle time (13·25 h); this causes the two peaks to overlap. In both cases, a better fit is obtained if the second cell cycle is longer than the first by 2–3 h. This suggests that cells enter a G₀ period after mitosis. A mechanism for littoral cell division, which occurs some 24 h after parenchymal cell division, is put forward in which the former cells depend on the enlargement of the latter for the stimulus to divide.