AN ELECTRON MICROSCOPIC STUDY OF THE DEVELOPMENT OF THE CLEAVAGE FURROW IN MAMMALIAN CELLS

The process of cytoplasmic cleavage has been studied in thin sections of rat erythroblasts and the cells of mouse leukemia and Walker 256 carcinoma of the rat. The development of the cleavage furrow begins in relation to the mid-body, which, earlier, appears on the equatorial plane in association with the continuous fibers of the spindle. The earliest evidence of a cleavage furrow is the presence of a vesicle or vesicles close to the mid-body. Subsequently, many smaller vesicles are seen in the equatorial plane. The cleavage furrow probably develops by the fusion of these vesicles so that a new plasma membrane is formed between the daughter cells, and extends from the telophase intercellular bridge to the cell margin. During the stage of formation of the vesicles, cisternae, believed to be part of the endoplasmic reticulum, assume an intimate relationship with the cleavage plane, and they may perhaps be involved in the formation of the vesicles.     

Centrosome separation and central spindle assembly act in redundant pathways that regulate microtubule density and trigger cleavage furrow formation

The mitotic spindle provides the spatial cue that coordinates cytokinesis with nuclear division. However, the specific property of the mitotic spindle that mediates this spatial regulation remains obscure, in part because different aspects of the mitotic spindle appear to have furrow inducing activity in different systems. We show that in C. elegans embryos, although the central spindle is usually dispensable for furrow initiation, it becomes essential for furrow formation when the extent of centrosome separation during anaphase is reduced. Measurements of microtubule density demonstrate that furrow formation occurs in the vicinity of a local minimum of microtubule density. Reduction of the extent of spindle elongation or disruption of the central spindle causes delayed formation of the cleavage furrow. These data suggest that reduced microtubule density triggers cleavage furrow initiation and demonstrate that redundant mechanisms direct efficient formation of the cleavage furrow.     

THE FINE STRUCTURE OF THE MID-BODY OF THE RAT ERYTHROBLAST

The development of the mid-body has been studied in mitotic erythroblasts of the rat bone marrow by means of thin sections examined with the electron microscope. A differentiated region on the continuous spindle fibers, consisting of a localized increase in density, is observed at the equatorial plane. The mid-body seems to develop by the aggregation of such denser lengths of spindle fiber. Its appearance precedes that of the cleavage furrow. A plate-like arrangement of fibrillary material lies transversely across the telophase intercellular bridge. Later, this material becomes amorphous and assumes the form of a dense ring closely applied to a ridge in the plasma membrane encircling the middle of the bridge. Although the mid-body forms in association with the spindle fibers, it is a structurally distinct part, and the changes which it undergoes are not shared by the rest of the bundle of continuous fibers.     

A quantitative study on anaphase movement of chromosomes in living grasshopper spermatocytes

Summary The present investigation has been undertaken to obtain data for the analysis of the chromosome movement at anaphase and the formation of a cleavage furrow. The study is based on simultaneous measurements of the spindle and cell diameters as well as of the chromosome separation in living spermatocyte divisions of the grasshoppers, Podisma sapporense and Acrydium japonicum.Evidence from the present investigation shows that the movement of chromosomes to the poles and the elongation of the spindle are separated in time; the spindle length remains unchanged through out anaphase. Spindle elongation is not associated with the separation of daughter chromosomes. The cell, and the spindle as well, elongate after the chromosomes have reached the poles. Cell elongation may follow the stretching of the spindle, and cause sufficient tension to distort the cell wall, resulting in the subsequent formation of a cleavage furrow.Contribution No. 327 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan. Aided by a grant from the Scientific Research Fund of the Ministry of Education.     

Cytokinesis in cells undergoing mitosis without genome replication

Chinese hamster ovary cells can be forced to enter mitosis without prior DNA replication by treatment with hydroxyurea and caffeine. Cells treated in this way assemble a spindle that functions normally except that it does not accomplish anaphase spindle elongation (anaphase B). The chromatin detaches from the unreplicated kinetochores, which fragment, but establish microtubule attachments and migrate to the metaphase plate. Partitioning of the kinetochore fragments ensues on the normal schedule. Typical midbodies and cleavage furrows are established and daughter cells of equal size are produced. These results imply that intact chromosomes are not necessary for correct cleavage furrow placement but that kinetochores might be. Further, it is clear that cleavage furrow placement does not depend on anaphase spindle elongation.     

The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells

Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase-anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 microM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.     

Contractile ring-independent localization of DdINCENP, a protein important for spindle stability and cytokinesis

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone, and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule-depolymerizing drugs, suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. Although not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring.     

Endomitotic Megakaryocytes that Form a Bipolar Spindle Exhibit Cleavage Furrow Ingression Followed by Furrow Regression

Megakaryocyte (MK) differentiation is marked by the development of progressive polyploidy, due to repeated incomplete cell cycles in which mitosis is aborted during anaphase, a process termed endomitosis. We have postulated that anaphase in endomitotic MKs diverges from diploid mitosis at a point distal to the assembly of the midzone, possibly involving impaired cleavage furrow progression. To define the extent of furrow initiation and ingression in endomitosis, we performed time-lapse imaging of MKs expressing yellow fluorescent protein (YFP)-tubulin and monitored shape change as they progressed through anaphase. We found that in early endomitotic cells that have a bipolar spindle, cleavage furrows form that can undergo significant ingression, but furrows regress to produce polyploid cells. Compared to cells that divide, cells that exhibit furrow regression have a slower rate of furrow ingression and do not furrow as deeply. More highly polyploid MKs undergoing additional endomitotic cycles also show measurable furrowing that is followed by regression, but the magnitude of the shape change is less than seen in the early MKs. This suggests that in the earliest endomitotic cycles when there is formation of a bipolar spindle, the failure of cytokinesis occurs late, following assembly and initial constriction of the actin/myosin ring, whereas in endomitotic MKs that are already polyploid there is secondary inhibition of furrow progression. This behavior of furrow ingression followed by regression may explain why midbody remnants are occasionally observed in polyploid MKs. This finding has important implications for the potential mechanisms for cytokinesis failure in endomitosis.     

Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery

The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.     

Loss of Spatial Control of the Mitotic Spindle Apparatus in a Chlamydomonas Reinhardtii Mutant Strain Lacking Basal Bodies

The bld2-1 mutation in the green alga Chlamydomonas reinhardtii is the only known mutation that results in the loss of centrioles/basal bodies and the loss of coordination between spindle position and cleavage furrow position during cell division. Based on several different assays, bld2-1 cells lack basal bodies in >99% of cells. The stereotypical cytoskeletal morphology and precise positioning of the cleavage furrow observed in wild-type cells is disrupted in bld2-1 cells. The positions of the mitotic spindle and of the cleavage furrow are not correlated with respect to each other or with a specific cellular landmark during cell division in bld2-1 cells. Actin has a variable distribution during mitosis in bld2-1 cells, but this aberrant distribution is not correlated with the spindle positioning defect. In both wild-type and bld2-1 cells, the position of the cleavage furrow is coincident with a specialized set of microtubules found in green algae known as the rootlet microtubules. We propose that the rootlet microtubules perform the functions of astral microtubules and that functional centrioles are necessary for the organization of the cytoskeletal superstructure critical for correct spindle and cleavage furrow placement in Chlamydomonas.     

Asters play only a dispensable role in the induction of the cleavage furrow in the blastomeres of early Xenopus embryos

Kuroda et al. (2001) of our laboratory have previously revealed that exposure of early Xenopus embryos to 150 mm urethane results in complete suppression of formation of the asters and the cleavage furrow, as well as significant reduction of the size of the spindle in the blastomeres, allowing only 1 or 2 cycles of mitosis but not cytokinesis. In the course of closer examination of the effect of urethane on the cleavage of blastomeres of early Xenopus embryos, we unexpectedly discovered that exposure of early Xenopus embryos to 75 mm urethane did not prevent cell division at all, though asters were not detected in the blastomeres. Instead, they contained a spindle that appeared rather normal. They also formed the diastema, a thin yolk-free structure, which is considered to play an essential role in the induction of the cleavage furrow. Essentially the same results were obtained in the exposure of embryos to vinblastine, a well-known microtubule inhibitor: exposure of embryos to 20 micro g/mL vinblastine resulted in complete suppression of cleavage of the blastomeres, where formation of both the spindle and asters were perfectly suppressed. By contrast, exposure of embryos to 5 microg/mL vinblastine did not prevent cleavage in the blastomeres though asters were not detected, whereas the rather normal spindle was formed. Thus, there was a close correlation between the formation of the normal spindle, not asters, and that of the cell division furrow and the diastema in the blastomeres of early Xenopus embryos. We suggest that while the spindle plays an essential role, asters are likely to play only a dispensable role in the induction of the cleavage furrow in even very large cells like the blastomeres of early Xenopus embryos.     

Microtubules are the only structural constituent of the spindle apparatus required for induction of cell cleavage

Structural constituents of the spindle apparatus essential for cleavage induction remain undefined. Findings from various cell types using different approaches suggest the importance of all structural constituents, including asters, the central spindle, and chromosomes. In this study, we systematically dissected the role of each constituent in cleavage induction in grasshopper spermatocytes and narrowed the essential one down to bundled microtubules. Using micromanipulation, we produced "cells" containing only asters, a truncated central spindle lacking both asters and chromosomes, or microtubules alone. We show that furrow induction occurs under all circumstances, so long as sufficient microtubules are present. Microtubules, as the only spindle structural constituent, undergo dramatic, stage-specific reorganizations, radiating toward cell cortex in "metaphase," disassembling in "anaphase," and bundling into arrays in "telophase." Furrow induction usually occurs at multisites around microtubule bundles, but only those induced by sustained bundles ingress. We suggest that microtubules, regardless of source, are the only structural constituent of the spindle apparatus essential for cleavage furrow induction.     

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.     

Presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo

The present study examines the indispensability of a nucleus or nucleus-deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)-damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV-damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV-damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV-damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus-deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos.     

Polo-like kinase controls vertebrate spindle elongation and cytokinesis

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.     

Inhibition of cytokinesis and altered contractile ring morphology induced by cytochalasins in synchronized PtK2 cells

PtK2 cells and antigen affinity-purified antibodies to actin and tubulin were used to study the effects on mitosis of cytochalasin B (CB) and dihydrocytochalasin B (H₂CB). PtK2 cells were synchronized in S phase by a double thymidine block and CB or H₂CB was added at various concentrations at the time of release from the block. CB- and H₂CB-treated populations, and control populations not treated with either drug, progressed synchronously through G2 and into mitosis with similar time courses. By both phase contrast and immunofluorescence microscopy, CB- and H₂CB-treated cells appeared normal in terms of chromosome condensation, spindle formation and spindle dynamics throughout prophase, metaphase and early anaphase. At late anaphase, contractile ring staining with actin antibody was not normal. High actin antigenicity remained localized in the region of the contractile ring; however, it appeared atypically as a punctate line of fluorescence across the midzone. Although some degree of furrowing was often seen to occur, at suitable concentrations of CB or H₂CB only binucleate G1 cells formed. Scanning electron microscopy (SEM) of normal and CB- and H₂CB-treated cells verified that cleavage furrowing did not proceed normally in treated cells. Large numbers of microvilli and surface blebs occurred in the normally smooth furrow region in these treated populations. These results suggest that intact microfilament function is not necessary for progression from S phase into mitosis, for spindle formation or for chromosome movement. They indicate that CB and H₂CB lead to formation of binucleated cells by causing aberrant cleavage furrowing and inhibition of contractile ring microfilaments.     

ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO : Direct Microtubule Counts

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK₁) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.     

Localization of Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.     

Vesicles and actin are targeted to the cleavage furrow via furrow microtubules and the central spindle

During cytokinesis, cleavage furrow invagination requires an actomyosin-based contractile ring and addition of new membrane. Little is known about how this actin and membrane traffic to the cleavage furrow. We address this through live analysis of fluorescently tagged vesicles in postcellularized Drosophila melanogaster embryos. We find that during cytokinesis, F-actin and membrane are targeted as a unit to invaginating furrows through formation of F-actin-associated vesicles. F-actin puncta strongly colocalize with endosomal, but not Golgi-derived, vesicles. These vesicles are recruited to the cleavage furrow along the central spindle and a distinct population of microtubules (MTs) in contact with the leading furrow edge (furrow MTs). We find that Rho-specific guanine nucleotide exchange factor mutants, pebble (pbl), severely disrupt this F-actin-associated vesicle transport. These transport defects are a consequence of the pbl mutants' inability to properly form furrow MTs and the central spindle. Transport of F-actin-associated vesicles on furrow MTs and the central spindle is thus an important mechanism by which actin and membrane are delivered to the cleavage furrow.     

MITOSIS IN THE OCTAFLAGELLATE PRASINOPHYTE,PYRAMIMONAS AMYLIFERA (CHLOROPHYTA)1

Cell division is described in the octaflagellate prasinophyte Pyramimonas amylifera Conrad and is compared in related genera. Basal bodies replicate at preprophase and move toward the poles. Cells remain motile throughout division. The nuclear envelope disperses and chromosomes begin to condense at prophase. Pairs of multilayered kinetochores are evident on the chromosomes of the metaphase plate. Spindle microtubules extending from the region of the basal bodies and rhizoplasts attach to the kinetochores or extend from pole to pole. Numerous vesicles and ribosomes have entered the nuclear region and the incipient cleavage furrow invaginates. The chromosomes move toward the poles at anaphase leaving a broad interzonal spindle between the two chromosomal plates. The nuclear envelope reforms first around the chromatin on the side adjacent to the spindle poles and later on the interzonal side. The cleavage furrow progresses into the interzonal spindle at telophase. By late telophase the nucleoli have reformed and the chromosomes have decondensed. The interzonal spindle has not been observed late in telophase. As the cleavage furrow nears completion the cells begin to twist and contort, ultimately separating the two cells.