Cell division intersects with cell geometry.

Single-celled organisms monitor cell geometry and use this information to control cell division. Such geometry-sensing mechanisms control both the decision to enter into cell division and the physical orientation of the chromosome segregation machinery, suggesting that signals controlling cell division may be linked to the mechanisms that ensure proper chromosome segregation.

Regulation of cell size and Wee1 kinase by elevated levels of the cell cycle regulatory protein kinase Cdr2.

Many cell cycle regulatory proteins catalyze cell cycle progression in a concentration-dependent manner. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr2 promotes mitotic entry by organizing cortical oligomeric nodes that lead to inhibition of Wee1, which itself inhibits the cyclin-dependent kinase Cdk1. cdr2Δ cells lack nodes and divide at increased size due to overactive Wee1, but it has not been known how increased Cdr2 levels might impact Wee1 and cell size. It also has not been clear if and how Cdr2 might regulate Wee1 in the absence of the related kinase Cdr1/Nim1. Using a tetracycline-inducible expression system, we found that a 6× increase in Cdr2 expression caused hyperphosphorylation of Wee1 and reduction in cell size even in the absence of Cdr1/Nim1. This overexpressed Cdr2 formed clusters that sequestered Wee1 adjacent to the nuclear envelope. Cdr2 mutants that disrupt either kinase activity or clustering ability failed to sequester Wee1 and to reduce cell size. We propose that Cdr2 acts as a dosage-dependent regulator of cell size by sequestering its substrate Wee1 in cytoplasmic clusters, away from Cdk1 in the nucleus. This mechanism has implications for other clustered kinases, which may act similarly by sequestering substrates.

Dynamic regulation of Cdr1 kinase localization and phosphorylation during osmotic stress.

Environmental conditions modulate cell cycle progression in many cell types. A key component of the eukaryotic cell cycle is the protein kinase Wee1, which inhibits the cyclin-dependent kinase Cdk1 in yeast through human cells. In the fission yeast Schizosaccharomyces pombe, the protein kinase Cdr1 is a mitotic inducer that promotes mitotic entry by phosphorylating and inhibiting Wee1. Cdr1 and Wee1 both localize to punctate structures, termed nodes, on the medial cortex, but it has been unknown whether node localization can be altered by physiological signals. Here we investigated how environmental conditions regulate Cdr1 signaling for cell division. Osmotic stress induced hyperphosphorylation of the mitotic inducer Cdr1 for several hours, and cells delayed division for the same time period. This stress-induced hyperphosphorylation required both Cdr1 autophosphorylation and the stress-activated protein kinase Sty1. During osmotic stress, Cdr1 exited cortical nodes and localized in the cytoplasm. Using a series of truncation mutants, we mapped a C-terminal domain that is necessary and sufficient for Cdr1 node localization and found that Sty1 directly phosphorylates this domain in vitro Sty1 was not required for Cdr1 exit from nodes, indicating the existence of additional regulatory signals. Both Cdr1 phosphorylation and node localization returned to basal levels when cells adapted to osmotic conditions and resumed cell cycle progression. In summary, we identified a mechanism that prevents Cdr1 colocalization with its inhibitory target Wee1 during osmotic stress. Dynamic regulation of protein localization to cortical nodes might represent a strategy to modulate entry into mitosis under differing environmental conditions.

Eisosomes provide membrane reservoirs for rapid expansion of the yeast plasma membrane.

Cell surface area rapidly increases during mechanical and hypoosmotic stresses. Such expansion of the plasma membrane requires 'membrane reservoirs' that provide surface area and buffer membrane tension, but the sources of this membrane remain poorly understood. In principle, the flattening of invaginations and buds within the plasma membrane could provide this additional surface area, as recently shown for caveolae in animal cells. Here, we used microfluidics to study the rapid expansion of the yeast plasma membrane in protoplasts, which lack the rigid cell wall. To survive hypoosmotic stress, yeast cell protoplasts required eisosomes, protein-based structures that generate long invaginations at the plasma membrane. Both budding yeast and fission yeast protoplasts lacking eisosomes were unable to expand like wild-type protoplasts during hypoosmotic stress, and subsequently lysed. By performing quantitative fluorescence microscopy on single protoplasts, we also found that eisosomes disassembled as surface area increased. During this process, invaginations generated by eisosomes at the plasma membrane became flattened, as visualized by scanning electron microscopy. We propose that eisosomes serve as tension-dependent membrane reservoirs for expansion of yeast cells in an analogous manner to caveolae in animal cells.

Quantitative phosphoproteomics reveals pathways for coordination of cell growth and division by the conserved fission yeast kinase pom1.

Complex phosphorylation-dependent signaling networks underlie the coordination of cellular growth and division. In the fission yeast Schizosaccharomyces pombe, the Dual specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK) family protein kinase Pom1 regulates cell cycle progression through the mitotic inducer Cdr2 and controls cell polarity through unknown targets. Here, we sought to determine the phosphorylation targets of Pom1 kinase activity by SILAC-based phosphoproteomics. We defined a set of high-confidence Pom1 targets that were enriched for cytoskeletal and cell growth functions. Cdr2 was the only cell cycle target of Pom1 kinase activity that we identified in cells. Mutation of Pom1-dependent phosphorylation sites in the C terminus of Cdr2 inhibited mitotic entry but did not impair Cdr2 localization. In addition, we found that Pom1 phosphorylated multiple substrates that function in polarized cell growth, including Tea4, Mod5, Pal1, the Rho GAP Rga7, and the Arf GEF Syt22. Purified Pom1 phosphorylated these cell polarity targets in vitro, confirming that they are direct substrates of Pom1 kinase activity and likely contribute to regulation of polarized growth by Pom1. Our study demonstrates that Pom1 acts in a linear pathway to control cell cycle progression while regulating a complex network of cell growth targets.

Eisosomes Regulate Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Cortical Clusters and Mitogen-activated Protein (MAP) Kinase Signaling upon Osmotic Stress.

Eisosomes are multiprotein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P2 regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast Schizosaccharomyces pombe. Activation of the MAPK Pmk1 was impaired by mutations in the phosphatidylinositol (PI) 5-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P2 clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI 5-kinase Its3 and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P2 clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.

A Pil1-Sle1-Syj1-Tax4 functional pathway links eisosomes with PI(4,5)P2 regulation.

Stable compartments of the plasma membrane promote a wide range of cellular functions. In yeast cells, cytosolic structures called eisosomes generate prominent cortical invaginations of unknown function. Through a series of genetic screens in fission yeast, we found that the eisosome proteins Pil1 and Sle1 function with the synaptojanin-like lipid phosphatase Syj1 and its ligand Tax4. This genetic pathway connects eisosome function with the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] in cells. Defects in PI(4,5)P2 regulation led to eisosome defects, and we found that the core eisosome protein Pil1 can bind to and tubulate liposomes containing PI(4,5)P2. Mutations in components of the Pil1-Sle1-Syj1-Tax4 pathway suppress the growth and morphology defects of TORC2 mutants, indicating that eisosome-dependent regulation of PI(4,5)P2 feeds into signal transduction pathways. We propose that the geometry of membrane invaginations generates spatial and temporal signals for lipid-mediated signaling events in cells.

A spatial gradient coordinates cell size and mitotic entry in fission yeast.

Many eukaryotic cell types undergo size-dependent cell cycle transitions controlled by the ubiquitous cyclin-dependent kinase Cdk1 (refs 1-4). The proteins that control Cdk1 activity are well described but their links with mechanisms monitoring cell size remain elusive. In the fission yeast Schizosaccharomyces pombe, cells enter mitosis and divide at a defined and reproducible size owing to the regulated activity of Cdk1 (refs 2, 3). Here we show that the cell polarity protein kinase Pom1, which localizes to cell ends, regulates a signalling network that contributes to the control of mitotic entry. This network is located at cortical nodes in the middle of interphase cells, and these nodes contain the Cdk1 inhibitor Wee1, the Wee1-inhibitory kinases Cdr1 (also known as Nim1) and Cdr2, and the anillin-like protein Mid1. Cdr2 establishes the hierarchical localization of other proteins in the nodes, and receives negative regulatory signals from Pom1. Pom1 forms a polar gradient extending from the cell ends towards the cell middle and acts as a dose-dependent inhibitor of mitotic entry, working through the Cdr2 pathway. As cells elongate, Pom1 levels decrease at the cell middle, leading to mitotic entry. We propose that the Pom1 polar gradient and the medial cortical nodes generate information about cell size and coordinate this with mitotic entry by regulating Cdk1 through Pom1, Cdr2, Cdr1 and Wee1.

PP2A-B56 regulates Mid1 protein levels for proper cytokinesis in fission yeast.

Protein phosphorylation regulates many steps in the cell division process including cytokinesis. In the fission yeast S. pombe, the anillin-like protein Mid1 sets the cell division plane and is regulated by phosphorylation. Multiple protein kinases act on Mid1, but no protein phosphatases have been shown to regulate Mid1. Here, we discovered that the conserved protein phosphatase PP2A-B56 is required for proper cytokinesis by promoting Mid1 protein levels. We find that par1Δ cells lacking the primary B56 subunit divide asymmetrically due to the assembly of misplaced cytokinetic rings that slide toward cell tips. These par1Δ mutants have reduced whole-cell levels of Mid1 protein, leading to reduced Mid1 at the cytokinetic ring. Restoring proper Mid1 expression suppresses par1Δ cytokinesis defects. This work identifies a new PP2A-B56 pathway regulating cytokinesis through Mid1, with implications for control of cytokinesis in other organisms.

Design principles of Cdr2 node patterns in fission yeast cells.

Pattern-forming networks have diverse roles in cell biology. Rod-shaped fission yeast cells use pattern formation to control the localization of mitotic signaling proteins and the cytokinetic ring. During interphase, the kinase Cdr2 forms membrane-bound multiprotein complexes termed nodes, which are positioned in the cell middle due in part to the node inhibitor Pom1 enriched at cell tips. Node positioning is important for timely cell cycle progression and positioning of the cytokinetic ring. Here, we combined experimental and modeling approaches to investigate pattern formation by the Pom1-Cdr2 system. We found that Cdr2 nodes accumulate near the nucleus, and Cdr2 undergoes nucleocytoplasmic shuttling when cortical anchoring is reduced. We generated particle-based simulations based on tip inhibition, nuclear positioning, and cortical anchoring. We tested model predictions by investigating Pom1-Cdr2 localization patterns after perturbing each positioning mechanism, including in both anucleate and multinucleated cells. Experiments show that tip inhibition and cortical anchoring alone are sufficient for the assembly and positioning of nodes in the absence of the nucleus, but that the nucleus and Pom1 facilitate the formation of unexpected node patterns in multinucleated cells. These findings have implications for spatial control of cytokinesis by nodes and for spatial patterning in other biological systems.

The fission yeast cell size control system integrates pathways measuring cell surface area, volume, and time.

Eukaryotic cells tightly control their size, but the relevant aspect of size is unknown in most cases. Fission yeast divide at a threshold cell surface area (SA) due, in part, to the protein kinase Cdr2. We find that fission yeast cells only divide by SA under a size threshold. Mutants that divide at a larger size shift to volume-based divisions. Diploid cells divide at a larger size than haploid cells do, but they maintain SA-based divisions, and this indicates that the size threshold for changing from surface-area-based to volume-based control is set by ploidy. Within this size control system, we found that the mitotic activator Cdc25 accumulates like a volume-based sizer molecule, whereas the mitotic cyclin Cdc13 accumulates in the nucleus as a timer. We propose an integrated model for cell size control based on multiple signaling pathways that report on distinct aspects of cell size and growth, including cell SA (Cdr2), cell volume (Cdc25), and time (Cdc13). Combined modeling and experiments show how this system can generate both sizer- and adder-like properties.

Design principles of Cdr2 node patterns in fission yeast cells.

Pattern forming networks have diverse roles in cell biology. Rod-shaped fission yeast cells use pattern formation to control the localization of mitotic signaling proteins and the cytokinetic ring. During interphase, the kinase Cdr2 forms membrane-bound multiprotein complexes termed nodes, which are positioned in the cell middle due in part to the node inhibitor Pom1 enriched at cell tips. Node positioning is important for timely cell cycle progression and positioning of the cytokinetic ring. Here, we combined experimental and modeling approaches to investigate pattern formation by the Pom1-Cdr2 system. We found that Cdr2 nodes accumulate near the nucleus, and Cdr2 undergoes nucleocytoplasmic shuttling when cortical anchoring is reduced. We generated particle-based simulations based on tip inhibition, nuclear positioning, and cortical anchoring. We tested model predictions by investigating Pom1-Cdr2 localization patterns after perturbing each positioning mechanism, including in both anucleate and multinucleated cells. Experiments show that tip inhibition and cortical anchoring alone are sufficient for the assembly and positioning of nodes in the absence of the nucleus, but that the nucleus and Pom1 facilitate the formation of unexpected node patterns in multinucleated cells. These findings have implications for spatial control of cytokinesis by nodes and for spatial patterning in other biological systems.

Arf6 anchors Cdr2 nodes at the cell cortex to control cell size at division.

Fission yeast cells prevent mitotic entry until a threshold cell surface area is reached. The protein kinase Cdr2 contributes to this size control system by forming multiprotein nodes that inhibit Wee1 at the medial cell cortex. Cdr2 node anchoring at the cell cortex is not fully understood. Through a genomic screen, we identified the conserved GTPase Arf6 as a component of Cdr2 signaling. Cells lacking Arf6 failed to divide at a threshold surface area and instead shifted to volume-based divisions at increased overall size. Arf6 stably localized to Cdr2 nodes in its GTP-bound but not GDP-bound state, and its guanine nucleotide exchange factor (GEF), Syt22, was required for both Arf6 node localization and proper size at division. In arf6Δ mutants, Cdr2 nodes detached from the membrane and exhibited increased dynamics. These defects were enhanced when arf6Δ was combined with other node mutants. Our work identifies a regulated anchor for Cdr2 nodes that is required for cells to sense surface area.

Connecting cell polarity signals to the cytokinetic machinery in yeast and metazoan cells.

Polarized growth and cytokinesis are two fundamental cellular processes that exist in virtually all cell types. Mechanisms for asymmetric distribution of materials allow for cells to grow in a polarized manner. This gives rise to a variety of cell shapes seen throughout all cell types. Following polarized growth during interphase, dividing cells assemble a cytokinetic ring containing the protein machinery to constrict and separate daughter cells. Here, we discuss how cell polarity signaling pathways act on cytokinesis, with a focus on direct regulation of the contractile actomyosin ring (CAR). Recent studies have exploited phosphoproteomics to identify new connections between cell polarity kinases and CAR proteins. Existing evidence suggests that some polarity kinases guide the local organization of CAR proteins and structures while also contributing to global organization of the division plane within a cell. We provide several examples of this regulation from budding yeast, fission yeast, and metazoan cells. In some cases, kinase-substrate connections point to conserved processes in these different organisms. We point to several examples where future work can indicate the degree of conservation and divergence in the cell division process of these different organisms.

Pak1 kinase controls cell shape through ribonucleoprotein granules.

Fission yeast cells maintain a rod shape due to conserved signaling pathways that organize the cytoskeleton for polarized growth. We discovered a mechanism linking the conserved protein kinase Pak1 with cell shape through the RNA-binding protein Sts5. Pak1 (also called Shk1 and Orb2) prevents Sts5 association with P bodies by directly phosphorylating its intrinsically disordered region (IDR). Pak1 and the cell polarity kinase Orb6 both phosphorylate the Sts5 IDR but at distinct residues. Mutations preventing phosphorylation in the Sts5 IDR cause increased P body formation and defects in cell shape and polarity. Unexpectedly, when cells encounter glucose starvation, PKA signaling triggers Pak1 recruitment to stress granules with Sts5. Through retargeting experiments, we reveal that Pak1 localizes to stress granules to promote rapid dissolution of Sts5 upon glucose addition. Our work reveals a new role for Pak1 in regulating cell shape through ribonucleoprotein granules during normal and stressed growth conditions.

Sequestration of the exocytic SNARE Psy1 into multiprotein nodes reinforces polarized morphogenesis in fission yeast.

Polarized morphogenesis is achieved by targeting or inhibiting growth in distinct regions. Rod-shaped fission yeast cells grow exclusively at their ends by restricting exocytosis and secretion to these sites. This growth pattern implies the existence of mechanisms that prevent exocytosis and growth along nongrowing cell sides. We previously identified a set of 50-100 megadalton-sized node structures along the sides of fission yeast cells that contained the interacting proteins Skb1 and Slf1. Here, we show that Skb1-Slf1 nodes contain the syntaxin-like soluble N-ethylmaleimide-sensitive factor attachment protein receptor Psy1, which mediates exocytosis in fission yeast. Psy1 localizes in a diffuse pattern at cell tips, where it likely promotes exocytosis and growth, but is sequestered in Skb1-Slf1 nodes at cell sides where growth does not occur. Mutations that prevent node assembly or inhibit Psy1 localization to nodes lead to aberrant exocytosis at cell sides and increased cell width. Genetic results indicate that this Psy1 node mechanism acts in parallel to actin cables and Cdc42 regulation. Our work suggests that sequestration of syntaxin-like Psy1 at nongrowing regions of the cell cortex reinforces cell morphology by restricting exocytosis to proper sites of polarized growth.

The yeast actin cytoskeleton: from cellular function to biochemical mechanism.

All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. These events are driven by the coordinated activities of a set of 20 to 30 highly conserved actin-associated proteins, in addition to many cell-specific actin-associated proteins and numerous upstream signaling molecules. The combined activities of these factors control with exquisite precision the spatial and temporal assembly of actin structures and ensure dynamic turnover of actin structures such that cells can rapidly alter their cytoskeletons in response to internal and external cues. One of the most exciting principles to emerge from the last decade of research on actin is that the assembly of architecturally diverse actin structures is governed by highly conserved machinery and mechanisms. With this realization, it has become apparent that pioneering efforts in budding yeast have contributed substantially to defining the universal mechanisms regulating actin dynamics in eukaryotes. In this review, we first describe the filamentous actin structures found in Saccharomyces cerevisiae (patches, cables, and rings) and their physiological functions, and then we discuss in detail the specific roles of actin-associated proteins and their biochemical mechanisms of action.

The phosphatase inhibitor Sds23 promotes symmetric spindle positioning in fission yeast.

A hallmark of cell division in eukaryotic cells is the formation and elongation of a microtubule (MT)-based mitotic spindle. Proper positioning of the spindle is critical to ensure equal segregation of the genetic material to the resulting daughter cells. Both the timing of spindle elongation and constriction of the actomyosin contractile ring must be precisely coordinated to prevent missegregation or damage to the genetic material during cellular division. Here, we show that Sds23, an inhibitor of protein phosphatases, contributes to proper positioning of elongating spindles in fission yeast cells. We found that sds23∆ mutant cells exhibit asymmetric spindles that initially elongate asymmetrically toward one end of the dividing cell. Spindle asymmetry in sds23∆ cells results from a defect that is distinct from previously identified mechanisms, including MT protrusions and enlarged vacuoles. Combined with our previous work, this study demonstrates that Sds23, an inhibitor of PP2A-family protein phosphatases, promotes proper positioning of both the bipolar spindle and cytokinetic ring during fission yeast cell division. These two steps ensure the overall symmetry and fidelity of the cell division process.

Fission yeast Pak1 phosphorylates anillin-like Mid1 for spatial control of cytokinesis.

Protein kinases direct polarized growth by regulating the cytoskeleton in time and space and could play similar roles in cell division. We found that the Cdc42-activated polarity kinase Pak1 colocalizes with the assembling contractile actomyosin ring (CAR) and remains at the division site during septation. Mutations in pak1 led to defects in CAR assembly and genetic interactions with cytokinesis mutants. Through a phosphoproteomic screen, we identified novel Pak1 substrates that function in polarized growth and cytokinesis. For cytokinesis, we found that Pak1 regulates the localization of its substrates Mid1 and Cdc15 to the CAR. Mechanistically, Pak1 phosphorylates the Mid1 N-terminus to promote its association with cortical nodes that act as CAR precursors. Defects in Pak1-Mid1 signaling lead to misplaced and defective division planes, but these phenotypes can be rescued by synthetic tethering of Mid1 to cortical nodes. Our work defines a new signaling mechanism driven by a cell polarity kinase that promotes CAR assembly in the correct time and place.

Cell Biology: Marine Yeasts Deepen the Sea of Diversity.

Fewer than 10% of fungal species have been discovered, and the diversity and ecological roles of marine species are particularly enigmatic. A new study shows that exploration of this untapped fungal biodiversity may expand our understanding of basic cellular functions such as growth, polarization, and division.