The mechanism and control of cytokinesis.

Cytokinesis is under active investigation in each of the dominant experimental model systems. During 1996 and 1997, several developments necessitated the reassessment of the prevailing model for cytokinesis. In addition, the inventory of proteins required for cytokinesis has grown considerably. However, a molecular understanding of cytokinesis still remains elusive.

CDK1 inactivation regulates anaphase spindle dynamics and cytokinesis in vivo.

Through association with CDK1, cyclin B accumulation and destruction govern the G2/M/G1 transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BDelta90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.

CYK-4: A Rho family gtpase activating protein (GAP) required for central spindle formation and cytokinesis.

During cytokinesis of animal cells, the mitotic spindle plays at least two roles. Initially, the spindle positions the contractile ring. Subsequently, the central spindle, which is composed of microtubule bundles that form during anaphase, promotes a late step in cytokinesis. How the central spindle assembles and functions in cytokinesis is poorly understood. The cyk-4 gene has been identified by genetic analysis in Caenorhabditis elegans. Embryos from cyk-4(t1689ts) mutant hermaphrodites initiate, but fail to complete, cytokinesis. These embryos also fail to assemble the central spindle. We show that the cyk-4 gene encodes a GTPase activating protein (GAP) for Rho family GTPases. CYK-4 activates GTP hydrolysis by RhoA, Rac1, and Cdc42 in vitro. RNA-mediated interference of RhoA, Rac1, and Cdc42 indicates that only RhoA is essential for cytokinesis and, thus, RhoA is the likely target of CYK-4 GAP activity for cytokinesis. CYK-4 and a CYK-4:GFP fusion protein localize to the central spindle and persist at cell division remnants. CYK-4 localization is dependent on the kinesin-like protein ZEN-4/CeMKLP1 and vice versa. These data suggest that CYK-4 and ZEN-4/CeMKLP1 cooperate in central spindle assembly. Central spindle localization of CYK-4 could accelerate GTP hydrolysis by RhoA, thereby allowing contractile ring disassembly and completion of cytokinesis.

Cell cycle. The only way out of mitosis.

The ubiquitination and destruction of cyclins provides the way out of mitosis. A multiprotein complex polyubiquitinates cyclin B and promotes the separation of sister chromatids.

Mitosis: don't get mad, get even.

The 'mitotic spindle checkpoint' ensures that, before a cell exits from mitosis, all of its chromosomes are aligned on the spindle to form the metaphase plate. Mad2 is an essential component of this checkpoint system and it binds specifically to unattached kinetochores.

Chromosome segregation: Samurai separation of Siamese sisters.

How do cells ensure that sister chromatids are precisely partitioned in mitosis? New studies on budding yeast have revealed that sister chromatid separation at anaphase requires endoproteolytic cleavage of a protein that maintains the association between sister chromatids.

Cell polarity. The importance of being polar.

Cell polarization is often accompanied by cytoskeletal rearrangements. Two signalling proteins, a GTPase and a kinase, are required for both actin and microtubule rearrangements. Are these two systems coupled?

Depletion of syntaxins in the early Caenorhabditis elegans embryo reveals a role for membrane fusion events in cytokinesis.

BACKGROUND: During cytokinesis, the plasma membrane of the parent cell is resolved into the two plasma membranes of the daughter cells. Membrane fusion events mediated by the machinery that participates in intracellular vesicle trafficking might contribute to this process. Two classes of molecules that are required for membrane fusion are the t-SNAREs and the v-SNAREs. The t-SNAREs (syntaxins) comprise a multi-gene family that has been suggested to mediate, at least in part, selective membrane fusion events in the cell. RESULTS: We have analyzed the genome of Caenorhabditis elegans and identified eight syntaxin genes. RNA-mediated interference (RNAi) was used to produce embryos deficient in individual syntaxins and these embryos were phenotypically characterized. Embryos deficient in one syntaxin, Syn-4, became multinucleate because of defects in karyomere fusion and cytokinesis. Syn-4 localized both to ingressing cleavage furrows and to punctate structures surrounding nuclei as they reformed during interphase. CONCLUSIONS: Our analyses indicate that both cytokinesis and reformation of the nuclear envelope are dependent on SNARE-mediated membrane fusion.

Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis.

BACKGROUND: In animal cells, cytokinesis begins shortly after the sister chromatids move to the spindle poles. The inner centromere protein (Incenp)has been implicated in both chromosome segregation and cytokinesis, but it is not known exactly how it mediates these two distinct processes. RESULTS: We identified two Caenorhabditis elegans proteins, ICP-1 and ICP-2, with significant homology in their carboxyl termini to the corresponding region of vertebrate Incenp. Embryos depleted of ICP-1 by RNA-mediated interference had defects in both chromosome segregation and cytokinesis. Depletion of the Aurora-like kinase AIR-2 resulted in a similar phenotype. The carboxy-terminal region of Incenp is also homologous to that in Sli15p, a budding yeast protein that functions with the yeast Aurora kinase Ipl1p. ICP-1 bound C. elegans AIR-2 in vitro, and the corresponding mammalian orthologs Incenp and AIRK2 could be co-immunoprecipitated from cell extracts. A significant fraction of embryos depleted of ICP-1 and AIR-2 completed one cell division over the course of several cell cycles. ICP-1 promoted the stable localization of ZEN-4 (also known as CeMKLP1), a kinesin-like protein required for central spindle assembly. CONCLUSIONS: ICP-1 and AIR-2 are part of a complex that is essential for chromosome segregation and for efficient completion of cytokinesis. We propose that this complex acts by promoting dissolution of sister chromatid cohesion and the assembly of the central spindle.

Cytoplasmic flows localize injected oskar RNA in Drosophila oocytes.

BACKGROUND: The oskar (osk) gene encodes a determinant of posterior identity in Drosophila, and the localization of osk RNA to the pole plasm at the posterior pole of the oocyte is essential for development of the embryo. The mechanisms by which osk RNA is localized are unknown. RESULTS: To study the mechanisms underlying localization of osk RNA, we have injected fluorescently labelled RNA into oocytes at stages 9, 10 and 11. Injected osk RNA localizes to the pole plasm, reproducing localization of the endogenous RNA. In oocytes at stages 10 and 11, the long-range movement of injected osk RNA is promoted by a vigorous, microtubule-dependent cytoplasmic flow, or ooplasmic streaming. Treatment with colchicine, a microtubule-destabilizing drug, inhibits ooplasmic streaming and prevents localization of the RNA from an injection site distal to the posterior pole. If the RNA is injected close to the posterior pole, however, it localizes even in the presence of colchicine. Similarly, in small oocytes, such as stage 9 oocytes, localization of injected osk RNA is insensitive to colchicine. CONCLUSIONS: These results reveal that microtubule-dependent cytoplasmic flows could contribute to the long-range transport of osk RNA, whereas microtubule-independent processes could mediate short-range transport. These results also highlight the role of the osk RNA anchor in the localization process.

A requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos.

BACKGROUND: During cytokinesis in animal cells, an equatorial actomyosin-based contractile ring divides the cell into two daughter cells. The position of the contractile ring is specified by a signal that emanates from the mitotic spindle. This signal has not been identified and it is not understood how the components of the contractile ring assemble. It is also unclear how the ring constricts or how new plasma membrane inserts specifically behind the leading edge of the constricting furrow. The Rho family of small GTPases regulate polarized changes in cell growth and cell shape by affecting the formation of actin structures beneath the plasma membrane, but their role in cytokinesis is unclear. RESULTS: We have studied the function of two Rho family members during the early cell divisions of Xenopus embryos by injecting modified forms of Rho and Cdc42. Both inhibition and constitutive activation of either GTPase blocked cytokinesis. Furrow specification occurred normally, but ingression of the furrow was inhibited. Newly inserted cleavage membranes appeared aberrantly on the outer surface of the embryo. Microinjected Rho localized to the cortex and regulated the levels of cortical F-actin. CONCLUSIONS: These results show that Rho regulates the assembly of actin filaments in the cortex during cytokinesis, that local activation of Rho is important for proper constriction of the contractile furrow, and that Cdc42 plays a role in furrow ingression. Moreover, our observations reveal that furrow ingression and membrane insertion are not strictly linked. Neither Rho nor Cdc42 appear to be required for establishment of the cell-division plane.

Separase is required for chromosome segregation during meiosis I in Caenorhabditis elegans.

BACKGROUND: Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms. During animal cell mitosis, cohesin is lost in two steps. A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase. In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit. Homolog segregation in S. cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms. In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic "prophase pathway." RESULTS: Inactivation of separase in C. elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents their timely disjunction. It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis. Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the "eggshell." CONCLUSIONS: Separase is essential for homologous chromosome disjunction during meiosis I. Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells. Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes.

Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.

Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.

Animal cell cytokinesis.

Cytokinesis creates two daughter cells endowed with a complete set of chromosomes and cytoplasmic organelles. This conceptually simple event is mediated by a complex and dynamic interplay between the microtubules of the mitotic spindle, the actomyosin cytoskeleton, and membrane fusion events. For many decades the study of cytokinesis was driven by morphological studies on specimens amenable to physical manipulation. The studies led to great insights into the cellular structures that orchestrate cell division, but the underlying molecular machinery was largely unknown. Molecular and genetic approaches have now allowed the initial steps in the development of a molecular understanding of this fundamental event in the life of a cell. This review provides an overview of the literature on cytokinesis with a particular emphasis on the molecular pathways involved in the division of animal cells.

Cyclin is degraded by the ubiquitin pathway.

Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.

Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor.

We have used frog egg extracts that assemble mitotic spindles to identify the event that triggers sister chromatid separation. Adding a nondegradable form of cyclin B prevents maturation-promoting factor (MPF) inactivation but does not block sister chromatid separation, showing that MPF inactivation is not needed to initiate anaphase. In contrast, adding an N-terminal fragment of cyclin, which acts as a specific competitor for cyclin degradation, produces a dose-dependent delay in MPF inactivation and sister chromatid separation. Methylated ubiquitin, which inhibits ubiquitin-mediated proteolysis, also delays sister chromatid separation, suggesting that ubiquitin-mediated proteolysis is necessary to initiate anaphase. The N-terminal cyclin fragment inhibits chromosome separation even in extracts that contain only nondegradable forms of cyclin, suggesting that proteins other than the known cyclins must be degraded to dissolve the linkage between sister chromatids.

Cyclin activation of p34cdc2.

The gradual accumulation of cyclin in the frog egg induces an abrupt and concerted activation of p34cdc2 that initiates mitosis. Activation is delayed even after the accumulation of cyclin to a critical threshold concentration. We have reproduced these unusual kinetic properties of p34cdc2 activation in vitro using bacterially expressed cyclin proteins and extracts derived from Xenopus eggs. Abrupt activation follows a lag period, the length of which is independent of the concentration of cyclin. The threshold concentration of cyclin and the length of the lag period are regulated by INH, an inhibitor of MPF activation in oocytes recently identified as a type 2A protein phosphatase. Binding to cyclin induces both tyrosine and threonine phosphorylation of the previously unphosphorylated p34cdc2, rendering it inactivated. The concerted transition into mitosis involves both a reduction in the rate of p34cdc2 phosphorylation on tyrosine and an increase in its rate of dephosphorylation.

Cyclin is a component of maturation-promoting factor from Xenopus.

Highly purified maturation-promoting factor (MPF) from Xenopus eggs contains both cyclin B1 and cyclin B2 as shown by Western blotting and immunoprecipitation using Xenopus anti-B-type cyclin antibodies. Immunoprecipitates with these antibodies display the histone H1 kinase activity characteristic of MPF, for which exogenously added B1 and B2 cyclins are both substrates. Protein kinase activity against cyclin oscillates in maturing oocytes and activated eggs with the same kinetics as p34cdc2 kinase activity. These data indicate that B-type cyclin is the other component of MPF besides p34cdc2.