Abbreviations used in this paper: MBP, maltose-binding protein; MS, mass spectrometry; PCH, S. pombe cdc15 homology; TAP, tandem affi nity purifi cation; TEV, tobacco etch virus; YE, yeast extract.

Summary Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.

Summary Cdc14-family phosphatases play a conserved role in promoting mitotic exit and cytokinesis by dephosphorylating substrates of cyclin dependent kinase (Cdk). Cdc14-family phosphatases have been best studied in yeast (for review see [1] [2]), where budding yeast Cdc14 and its fission yeast homolog Clp1 are regulated in part by their localization, with both proteins thought to be sequestered in the nucleolus in interphase. Cdc14/Clp1 are released from the nucleolus in mitosis, and in late mitosis a conserved signaling pathway termed the MEN/SIN acts through an unknown mechanism to keep Cdc14 and Clp1 respectively out of the nucleolus [3-6]. Here we show that the most downstream SIN component, the Ndr-family kinase Sid2, acts to maintain Clp1 in the cytoplasm in late mitosis by phosphorylating Clp1 directly and thereby creating binding sites for the 14-3-3 protein Rad24. Mutation of the Sid2 phosphorylation sites on Clp1 disrupts the interaction between Clp1 and Rad24, and causes premature return of Clp1 to the nucleolus during cytokinesis. Loss of Clp1 from the cytoplasm in telophase renders cells sensitive to perturbation of the actomyosin ring, but does not affect other functions of Clp1. Because all components of this pathway are conserved, this might be a broadly conserved mechanism for regulation of Cdc14-family phosphatases.

Summary In eukaryotes, cytokinesis generally involves an acto-myosin ring, the contraction of which promotes daughter cells segregation. Assembly of the contractile ring is tightly controlled in space and time (see[1–4] for reviews). In the fission yeast, contractile ring components are first organized by the Anillin-like protein Mid1 [5, 6] and Cdr2 kinase [7–9] into medial cortical nodes. These nodes then coalesce laterally into a functional compact contractile ring [10–13]. Although Mid1 is present at the medial cortex throughout G2 phase [14], recruitment of contractile ring components to nodes starts only at mitotic onset [12] indicating that this event is cell cycle regulated. Polo kinases are key temporal coordinators of mitosis and cytokinesis [1] and the Polo-like kinase Plo1 [15] has long been implicated in Mid1 regulation [16]: Plo1 activates Mid1 nuclear export at mitotic onset [16], coupling division plane specification to nuclear position [7]. Here, we provide evidence that Plo1 also triggers the recruitment of contractile ring components into medial cortical nodes. Plo1 binds at least two independent sites on Mid1, including a consensus site phosphorylated by Cdc2. Plo1 phosphorylates several residues within the first 100 amino acids of Mid1, which directly interact with the IQGAP Rng2 [17], and influences the timing of Myosin II recruitment. Plo1 thereby facilitates contractile ring assembly at mitotic onset.

The fission yeast F-BAR proteins Cdc15 and Imp2 and their combined SH3-domain partners appear to act as “molecular glue” to stabilize the interaction between the plasma membrane and a complex network of proteins at the division site that mediates cell division.

Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.

SummaryEukaryotes remodel the nucleus during mitosis using a variety of mechanisms that differ in the timing and the extent of nuclear envelope (NE) breakdown. Here, we probe the principles enabling this functional diversity by exploiting the natural divergence in NE management strategies between the related fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus [1, 2, 3]. We show that inactivation of Ned1, the phosphatidic acid phosphatase of the lipin family, by CDK phosphorylation is both necessary and sufficient to promote NE expansion required for “closed” mitosis in S. pombe. In contrast, Ned1 is not regulated during division in S. japonicus, thus limiting membrane availability and necessitating NE breakage. Interspecies gene swaps result in phenotypically normal divisions with the S. japonicus lipin acquiring an S. pombe-like mitotic phosphorylation pattern. Our results provide experimental evidence for the mitotic regulation of phosphatidic acid flux and suggest that the regulatory networks governing lipin activity diverged in evolution to give rise to strikingly dissimilar mitotic programs.

Phosphorylation by the DYRK kinase Pom1 is one of two major signals for proper division site placement in Schizosaccharomyces pombe. Pom1 phosphorylation of F-BAR protein Cdc15 inhibits its membrane and protein binding, thus inhibiting scaffolding of the cytokinetic ring and preventing mislocalized division.