Dannel McCollum

Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN

Proper completion of mitosis requires careful coordination of numerous cellular events. It is crucial, for example, that cells do not initiate spindle disassembly and cytokinesis until chromosomes have been properly segregated. Cells have developed numerous safeguards or checkpoints to delay exit from mitosis and initiation of the next cell cycle in response to defects in late mitosis. In this review, we discuss recent work on two homologous signaling pathways in budding and fission yeast, termed the mitotic exit network (MEN) and septation initiation network (SIN), respectively, that are essential for coordinating completion of mitosis and cytokinesis with other mitotic events.

Cytokinesis in Eukaryotes

SUMMARY Cytokinesis is the final event of the cell division cycle, and its completion results in irreversible partition of a mother cell into two daughter cells. Cytokinesis was one of the first cell cycle events observed by simple cell biological techniques; however, molecular characterization of cytokinesis has been slowed by its particular resistance to in vitro biochemical approaches. In recent years, the use of genetic model organisms has greatly advanced our molecular understanding of cytokinesis. While the outcome of cytokinesis is conserved in all dividing organisms, the mechanism of division varies across the major eukaryotic kingdoms. Yeasts and animals, for instance, use a contractile ring that ingresses to the cell middle in order to divide, while plant cells build new cell wall outward to the cortex. As would be expected, there is considerable conservation of molecules involved in cytokinesis between yeast and animal cells, while at first glance, plant cells seem quite different. However, in recent years, it has become clear that some aspects of division are conserved between plant, yeast, and animal cells. In this review we discuss the major recent advances in defining cytokinesis, focusing on deciding where to divide, building the division apparatus, and dividing. In addition, we discuss the complex problem of coordinating the division cycle with the nuclear cycle, which has recently become an area of intense research. In conclusion, we discuss how certain cells have utilized cytokinesis to direct development.

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression

BACKGROUND In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete. RESULTS We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine. CONCLUSIONS S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast.

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p–Cdc14p downstream of Spg1p–Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2–cyclin activity promotes Sid1p–Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.

Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor

The Hippo pathway kinase LATS2 promotes contact inhibition of growth. How LATS2 is activated in response to changes in cell density is unknown. It is found that tight junction protein AMOTL2 is a novel activator of LATS2, raising the possibility that tight junction assembly promotes LATS2-dependent inhibition of cell proliferation.

Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast

A great deal is now known about how cells regulate entry into mitosis, but only recently have the mechanisms controlling exit from mitosis and cytokinesis begun to be revealed. In the budding yeast Saccharomyces cerevisiae, Mob1p interacts with the Dbf2p kinase and cells containing mutations in these genes arrest in late anaphase [1] [2]. Proteins related to Mob1p are present in both plants and animals, but information about Mob1p function has been obtained only from budding yeast. Here, we describe the identification and characterization of Mob1p from Schizosaccharomyces pombe. Mob1p associates with the Sid2p kinase and like Sid2p, Mob1p is required for the initiation of cytokinesis, but not for mitotic exit. Mob1p localizes to the spindle pole body (SPB) and to the cell-division site during cell division, suggesting that it might be involved in transducing the signal to initiate cell division from the SPB to the division site. Mob1p is required for Sid2p localization, and Mob1p localization requires the function of the cdc7, cdc11, cdc14, spg1, sid1, sid2, and sid4 genes, suggesting that together with Sid2p, Mob1p functions at the end of the signaling cascade required to regulate the onset of cytokinesis at the end of mitosis.

The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe

Fission yeast mutants defective in actomyosin ring formation and function exhibit a prolonged G2 delay following cytokinesis failure. This G2 delay depends on the SIN, a signaling network essential for cytokinesis, and the non-essential Cdc14p family phosphatase, Clp1p/Flp1p and has been proposed to signify a cytokinesis checkpoint mechanism. However, the physiological relevance of this proposed Clp1p/Flp1p-dependent checkpoint is unclear because all previous studies were carried out using mutations in essential actomyosin ring components under fully restrictive conditions and thus these cells would have died regardless of the presence of the checkpoint. Here we show that delays in cytokinesis caused by minor perturbations to different components of the cytokinetic machinery, which normally cause only mild defects, become lethal when Clp1p/Flp1p is inactivated. In addition, we show that Clp1p/Flp1p does not function simply to inhibit further rounds of nuclear division, but also allows damaged actomyosin rings to be maintained to facilitate completion of cell division. Ectopic activation of the SIN significantly bypasses the requirement of Clp1p/Flp1p for G2 delay as well as for completion of cytokinesis. We conclude that the Clp1p/Flp1p-dependent cytokinesis checkpoint provides a previously unrecognized cell survival advantage when the cell division apparatus is mildly perturbed.

Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression.

The molecular mechanisms that temporally and spatially coordinate cell morphogenesis with the cell cycle remain poorly understood. Here we describe the characterization of fission yeast Mob2p, a novel protein required for regulating cell polarity and cell cycle control. Deletion of mob2 is lethal and causes cells to become spherical, with depolarized actin and microtubule cytoskeletons. A decrease in Mob2p protein level results in a defect in the activation of bipolar growth. This phenotype is identical to that of mutants defective in the orb6 protein kinase gene, and we find that Mob2p physically interacts with Orb6p. In addition, overexpression of Mob2p, like that of Orb6p, results in a delay in the onset of mitosis. Mob2p localizes to the cell periphery and cytoplasm throughout the cell cycle and to the division site during late anaphase and telophase. Mob2p is unable to localize to the cell middle in mutants defective in actomyosin ring and septum formation. Our results suggest that Mob2p, along with Orb6p, is required for coordinating polarized cell growth during interphase with the onset of mitosis.

Dma1 prevents mitotic exit and cytokinesis by inhibiting the septation initiation network (SIN).

In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) triggers cytokinesis after mitosis. We investigated the relationship between Dma1p, a spindle checkpoint protein and cytokinesis inhibitor, and the SIN. Deletion of dma1 inactivates the spindle checkpoint and allows precocious SIN activation, while overexpressing Dma1p reduces SIN signaling. Dma1p seems to function by inhibiting the SIN activator, Plo1p kinase, since dma1 overexpression and deletion phenotypes suggest that Dma1p antagonizes Plo1p localization. Furthermore, failure to maintain high cyclin-dependent kinase (CDK) activity during spindle checkpoint activation in dma1 deletion cells requires Plo1p. Dma1p itself localizes to spindle pole bodies through interaction with Sid4p. Our observations suggest that Dma1p functions to prevent mitotic exit and cytokinesis during spindle checkpoint arrest by inhibiting SIN signaling.

Angiomotins link F-actin architecture to Hippo pathway signaling

Angiomotin proteins, together with LATS kinase, regulate the Hippo pathway transcriptional coactivator YAP in response to changes in the F-actin cytoskeleton. Competition between F-actin and YAP for binding to angiomotins makes YAP regulation responsive to F-actin levels. Phosphorylation by LATS can switch angiomotins from F-actin to YAP binding.