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.

Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.

In the fission yeast Schizosaccharomyces pombe, pheromone signaling engages a signaling pathway composed of a G protein-coupled receptor, Ras, and a mitogen-activated protein kinase (MAPK) cascade that triggers sexual differentiation and gamete fusion. Cell-cell fusion requires local cell wall digestion, which relies on an initially dynamic actin fusion focus that becomes stabilized upon local enrichment of the signaling cascade on the structure. We constructed a live-reporter of active Ras1 (Ras1-guanosine triphosphate [GTP]) that shows Ras activity at polarity sites peaking on the fusion structure before fusion. Remarkably, constitutive Ras1 activation promoted fusion focus stabilization and fusion attempts irrespective of cell pairing, leading to cell lysis. Ras1 activity was restricted by the guanosine triphosphatase-activating protein Gap1, which was itself recruited to sites of Ras1-GTP and was essential to block untimely fusion attempts. We propose that negative feedback control of Ras activity restrains the MAPK signal and couples fusion with cell-cell engagement.

In many organisms, positive and negative signals cooperate to position the division site for cytokinesis. In the rod-shaped fission yeast Schizosaccharomyces pombe, symmetric division is achieved through anillin/Mid1-dependent positive cues released from the central nucleus and negative signals from the DYRK-family polarity kinase Pom1 at cell tips. Here we establish that Pom1's kinase activity prevents septation at cell tips even if Mid1 is absent or mislocalized. We also find that Pom1 phosphorylation of F-BAR protein Cdc15, a major scaffold of the division apparatus, disrupts Cdc15's ability to bind membranes and paxillin, Pxl1, thereby inhibiting Cdc15's function in cytokinesis. A Cdc15 mutant carrying phosphomimetic versions of Pom1 sites or deletion of Cdc15 binding partners suppresses division at cell tips in cells lacking both Mid1 and Pom1 signals. Thus, inhibition of Cdc15-scaffolded septum formation at cell poles is a key Pom1 mechanism that ensures medial division.

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8.

Myosin-1s are monomeric actin-based motors that function at membranes. Myo1 is the single myosin-1 isoform in Schizosaccharomyces pombe that works redundantly with Wsp1-Vrp1 to activate the Arp2/3 complex for endocytosis. Here, we identified Ank1 as an uncharacterized cytoplasmic Myo1 binding partner. We found that in ank1Δ cells, Myo1 dramatically redistributed from endocytic patches to decorate the entire plasma membrane and endocytosis was defective. Biochemical analysis and structural predictions suggested that the Ank1 ankyrin repeats bind the Myo1 lever arm and the Ank1 acidic tail binds the Myo1 TH1 domain to prevent TH1-dependent Myo1 membrane binding. Indeed, Ank1 over-expression precluded Myo1 membrane localization and recombinant Ank1 blocked purified Myo1 liposome binding in vitro. Based on biochemical and cell biology analyses, we propose budding yeast Ank1 and human OSTF1 are functional Ank1 orthologs and that cytoplasmic sequestration by small ankyrin repeat proteins is a conserved mechanism regulating myosin-1s in endocytosis.

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans. PAR-1 is also required for A-P axis determination in Drosophila. Here we show that the Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome and is regulated by prenylation and by phosphorylation by protein kinase A. We show that protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome.

Ubiquitination and deubiquitination of proteins are reciprocal events involved in many cellular processes, including the cell cycle. During mitosis, the metaphase to anaphase transition is regulated by the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C). Although the E3 ubiquitin ligase function of the APC/C has been well characterized, it is not clear whether deubiquitinating enzymes (DUBs) play a role in reversing APC/C substrate ubiquitination. Here we performed a genetic screen to determine what DUB, if any, antagonizes the function of the APC/C in the fission yeast Schizosaccharomyces pombe. We found that deletion of ubp8, encoding the Spt-Ada-Gcn5-Acetyl transferase (SAGA) complex associated DUB, suppressed temperature-sensitive phenotypes of APC/C mutants cut9-665, lid1-6, cut4-533, and slp1-362. Our analysis revealed that Ubp8 antagonizes APC/C function in a mechanism independent of the spindle assembly checkpoint and proteasome activity. Notably, suppression of APC/C mutants was linked to loss of Ubp8 catalytic activity and required histone H2B ubiquitination. On the basis of these data, we conclude that Ubp8 antagonizes APC/C function indirectly by modulating H2B ubiquitination status.

Cell-cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure-the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion.

Many eukaryotic cells divide by assembling and constricting an actin- and myosin-based contractile ring (CR) that is physically linked to the plasma membrane (PM). In this study, we report that Schizosaccharomyces pombe cells lacking efr3, which encodes a conserved PM scaffold for the phosphatidylinositol-4 kinase Stt4, build CRs that can slide away from the cell middle during anaphase in a myosin V-dependent manner. The Efr3-dependent CR-anchoring mechanism is distinct from previously reported pathways dependent on the Fes/CIP4 homology Bin-Amphiphysin-Rvs167 (F-BAR) protein Cdc15 and paxillin Pxl1. In efr3Δ, the concentrations of several membrane-binding proteins were reduced in the CR and/or on the PM. Our results suggest that proper PM lipid composition is important to stabilize the central position of the CR and resist myosin V-based forces to promote the fidelity of cell division.

Cell fusion is universal in eukaryotes for fertilization and development, but what signals this process is unknown. Here, we show in Schizosaccharomyces pombe that fusion does not require a dedicated signal but is triggered by spatial focalization of the same pheromone-GPCR (G-protein-coupled receptor)-MAPK signaling cascade that drives earlier mating events. Autocrine cells expressing the receptor for their own pheromone trigger fusion attempts independently of cell-cell contact by concentrating pheromone release at the fusion focus, a dynamic actin aster underlying the secretion of cell wall hydrolases. Pheromone receptor and MAPK cascade are similarly enriched at the fusion focus, concomitant with fusion commitment in wild-type mating pairs. This focalization promotes cell fusion by immobilizing the fusion focus, thus driving local cell wall dissolution. We propose that fusion commitment is imposed by a local increase in MAPK concentration at the fusion focus, driven by a positive feedback between fusion focus formation and focalization of pheromone release and perception.

Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.

The small GTPase Cdc42 is critical for cell polarization in eukaryotic cells. In rod-shaped fission yeast Schizosaccharomyces pombe cells, active GTP-bound Cdc42 promotes polarized growth at cell poles, while inactive Cdc42-GDP localizes ubiquitously also along cell sides. Zones of Cdc42 activity are maintained by positive feedback amplification involving the formation of a complex between Cdc42-GTP, the scaffold Scd2, and the guanine nucleotide exchange factor (GEF) Scd1, which promotes the activation of more Cdc42. Here, we use the CRY2-CIB1 optogenetic system to recruit and cluster a cytosolic Cdc42 variant at the plasma membrane and show that this leads to its moderate activation also on cell sides. Surprisingly, Scd2, which binds Cdc42-GTP, is still recruited to CRY2-Cdc42 clusters at cell sides in individual deletion of the GEFs Scd1 or Gef1. We show that activated Cdc42 clusters at cell sides are able to recruit Scd1, dependent on the scaffold Scd2. However, Cdc42 activity is not amplified by positive feedback and does not lead to morphogenetic changes, due to antagonistic activity of the GTPase activating protein Rga4. Thus, the cell architecture is robust to moderate activation of Cdc42 at cell sides.

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

Schizosaccharomyces pombe cdc15 homology (PCH) family members participate in numerous biological processes, including cytokinesis, typically by bridging the plasma membrane via their F-BAR domains to the actin cytoskeleton. Two SH3 domain-containing PCH family members, Cdc15 and Imp2, play critical roles in S. pombe cytokinesis. Although both proteins localize to the contractile ring, with Cdc15 preceding Imp2, only cdc15 is an essential gene. Despite these distinct roles, the SH3 domains of Cdc15 and Imp2 cooperate in the essential process of recruiting other proteins to stabilize the contractile ring. To better understand the connectivity of this SH3 domain-based protein network at the CR and its function, we used a biochemical approach coupled to proteomics to identify additional proteins (Rgf3, Art1, Spa2, and Pos1) that are integrated into this network. Cell biological and genetic analyses of these SH3 partners implicate them in a range of activities that ensure the fidelity of cell division, including promoting cell wall metabolism and influencing cell morphogenesis.

Concentration gradients regulate many cell biological and developmental processes. In rod-shaped fission yeast cells, polar cortical gradients of the DYRK family kinase Pom1 couple cell length with mitotic commitment by inhibiting a mitotic inducer positioned at midcell. However, how Pom1 gradients are established is unknown. Here, we show that Tea4, which is normally deposited at cell tips by microtubules, is both necessary and, upon ectopic cortical localization, sufficient to recruit Pom1 to the cell cortex. Pom1 then moves laterally at the plasma membrane, which it binds through a basic region exhibiting direct lipid interaction. Pom1 autophosphorylates in this region to lower lipid affinity and promote membrane release. Tea4 triggers Pom1 plasma membrane association by promoting its dephosphorylation through the protein phosphatase 1 Dis2. We propose that local dephosphorylation induces Pom1 membrane association and nucleates a gradient shaped by the opposing actions of lateral diffusion and autophosphorylation-dependent membrane detachment.

Secretory vesicle clusters transported on actin filaments by myosin V motors for local secretion underlie various cellular processes, such as neurotransmitter release at neuronal synapses,1 hyphal steering in filamentous fungi,2,3 and local cell wall digestion preceding the fusion of yeast gametes.4 During fission yeast Schizosaccharomyces pombe gamete fusion, the actin fusion focus assembled by the formin Fus1 concentrates secretory vesicles carrying cell wall digestive enzymes.5,6,7 The position and coalescence of the vesicle focus are controlled by local signaling and actin-binding proteins to prevent inappropriate cell wall digestion that would cause lysis,6,8,9,10 but the mechanisms of focusing have been elusive. Here, we show that the regulatory N terminus of Fus1 contains an intrinsically disordered region (IDR) that mediates Fus1 condensation in vivo and forms dense assemblies that exclude ribosomes. Fus1 lacking its IDR fails to concentrate in a tight focus and causes cell lysis during attempted cell fusion. Remarkably, the replacement of Fus1 IDR with a heterologous low-complexity region that forms molecular condensates fully restores Fus1 focusing and function. By contrast, the replacement of Fus1 IDR with a domain that forms more stable oligomers restores focusing but poorly supports cell fusion, suggesting that condensation is tuned to yield a selectively permeable structure. We propose that condensation of actin structures by an IDR may be a general mechanism for actin network organization and the selective local concentration of secretory vesicles.

In non-motile fungi, sexual reproduction relies on strong morphogenetic changes in response to pheromone signaling. We report here on a systematic screen for morphological abnormalities of the mating process in fission yeast Schizosaccharomyces pombe. We derived a homothallic (self-fertile) collection of viable deletions, which, upon visual screening, revealed a plethora of phenotypes affecting all stages of the mating process, including cell polarization, cell fusion and sporulation. Cell fusion relies on the formation of the fusion focus, an aster-like F-actin structure that is marked by strong local accumulation of the myosin V Myo52, which concentrates secretion at the fusion site. A secondary screen for fusion-defective mutants identified the myosin V Myo51-associated coiled-coil proteins Rng8 and Rng9 as critical for the coalescence of the fusion focus. Indeed, rng8Δ and rng9Δ mutant cells exhibit multiple stable dots at the cell-cell contact site, instead of the single focus observed in wildtype. Rng8 and Rng9 accumulate on the fusion focus, dependent on Myo51 and tropomyosin Cdc8. A tropomyosin mutant allele, which compromises Rng8/9 localization but not actin binding, similarly leads to multiple stable dots instead of a single focus. By contrast, myo51 deletion does not strongly affect fusion focus coalescence. We propose that focusing of the actin filaments in the fusion aster primarily relies on Rng8/9-dependent cross-linking of tropomyosin-actin filaments.