SIN-like pathway kinases regulate the end of mitosis in the methylotrophic yeast Ogataea polymorpha

The Mitotic exit network (MEN) is a conserved signalling pathway essential for termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Amongst O. polymorpha protein kinases that have some similarity to ScCdc15, only two had no other obvious homologues in S. cerevisiae and these were named OpHCD1 and OpHCD2 for homologue candidate of ScCdc15. A search in other yeast species revealed that OpHcd2 has an armadillo type fold in the C-terminal region as found in SpCdc7 kinases of the fission yeast Schizosaccharomyces pombe, which are homologues of ScCdc15; while OpHcd1 is homologous to SpSid1 kinase, a component of the Septation Initiation Network (SIN) of S. pombe not present in the MEN. Since the deletion of either OpHCD1 or OpHCD2 resulted in lethality under standard growth conditions, conditional mutants were constructed by introducing an ATP analog sensitive mutation. For OpHCD2, we constructed and used new genetic tools for O. polymorpha that combined the Tet promoter and the improved auxin-degron systems. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. These results suggest a SIN-like signalling pathway regulates termination of mitosis in O. polymorpha and that the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

MOB: Pivotal Conserved Proteins in Cytokinesis, Cell Architecture and Tissue Homeostasis

The MOB family proteins are constituted by highly conserved eukaryote kinase signal adaptors that are often essential both for cell and organism survival. Historically, MOB family proteins have been described as kinase activators participating in Hippo and Mitotic Exit Network/ Septation Initiation Network (MEN/SIN) signaling pathways that have central roles in regulating cytokinesis, cell polarity, cell proliferation and cell fate to control organ growth and regeneration. In metazoans, MOB proteins act as central signal adaptors of the core kinase module MST1/2, LATS1/2, and NDR1/2 kinases that phosphorylate the YAP/TAZ transcriptional co-activators, effectors of the Hippo signaling pathway. More recently, MOBs have been shown to also have non-kinase partners and to be involved in cilia biology, indicating that its activity and regulation is more diverse than expected. In this review, we explore the possible ancestral role of MEN/SIN pathways on the built-in nature of a more complex and functionally expanded Hippo pathway, by focusing on the most conserved components of these pathways, the MOB proteins. We discuss the current knowledge of MOBs-regulated signaling, with emphasis on its evolutionary history and role in morphogenesis, cytokinesis, and cell polarity from unicellular to multicellular organisms.

A novel checkpoint pathway controls actomyosin ring constriction trigger in fission yeast

In fission yeast, the septation initiation network (SIN) ensures temporal coordination between actomyosin ring (CAR) constriction with membrane ingression and septum synthesis. However, questions remain about CAR regulation under stress conditions. We show that Rgf1p (Rho1p GEF), participates in a delay of cytokinesis under cell wall stress (blankophor, BP). BP did not interfere with CAR assembly or the rate of CAR constriction, but did delay the onset of constriction in the wild type cells but not in the rgf1Δ cells. This delay was also abolished in the absence of Pmk1p, the MAPK of the cell integrity pathway (CIP), leading to premature abscission and a multi-septated phenotype. Moreover, cytokinesis delay correlates with maintained SIN signaling and depends on the SIN to be achieved. Thus, we propose that the CIP participates in a checkpoint, capable of triggering a CAR constriction delay through the SIN pathway to ensure that cytokinesis terminates successfully.

Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein Sid4 at the spindle pole body (SPB), and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Pololike kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

The kinase domain of CK1 enzymes contains the localization cue essential for compartmentalized signaling at the spindle pole

CK1 protein kinases contribute to multiple biological processes, but how they are tailored to function in compartmentalized signaling events is largely unknown. Hhp1 and Hhp2 (Hhp1/2) are the soluble CK1 family members in Schizosaccharomyces pombe. One of their functions is to inhibit the septation initiation network (SIN) during a mitotic checkpoint arrest. The SIN is assembled by Sid4 at spindle pole bodies (SPBs), and though Hhp1/2 colocalize there, it is not known how they are targeted there or whether their SPB localization is required for SIN inhibition. Here, we establish that Hhp1/2 localize throughout the cell cycle to SPBs, as well as to the nucleus, cell tips, and division site. We find that their catalytic domains but not their enzymatic function are used for SPB targeting and that this targeting strategy is conserved in human CK1δ/ε localization to centrosomes. Further, we pinpoint amino acids in the Hhp1 catalytic domain required for SPB interaction; mutation of these residues disrupts Hhp1 association with the core SPB protein Ppc89, and the inhibition of cytokinesis in the setting of spindle stress. Taken together, these data have enabled us to define a molecular mechanism used by CK1 enzymes to target a specific cellular locale for compartmentalized signaling.

Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein, Sid4, at the SPB, and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Polo-like kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive auto-ubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.