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.

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.

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.

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.

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.

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.

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.

A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1.

To enter into mitosis, cells must shut off the cell cycle inhibitor Wee1. SAD family protein kinases regulate Wee1 signaling in yeast and humans. In Schizosaccharomyces pombe, two SAD kinases (Cdr1/Nim1 and Cdr2) act as upstream inhibitors of Wee1. Previous studies found that S. pombe Cdr1/Nim1 directly phosphorylates and inhibits Wee1 in vitro, but different results were obtained for budding yeast and human SAD kinases. Without a full understanding of Cdr1 action on Wee1, it has been difficult to assess the in vivo relevance and conservation of this mechanism. Here, we show that both Cdr1 and Cdr2 promote Wee1 phosphorylation in cells, but only Cdr1 inhibits Wee1 kinase activity. Inhibition occurs when Cdr1 phosphorylates a cluster of serine residues linking α-helices G and H of the Wee1 kinase domain. This region is highly divergent among different Wee1 proteins, consistent with distinct regulatory mechanisms. A wee(4A) mutant that impairs phosphorylation by Cdr1 delays mitotic entry and causes elongated cells. By disrupting and retargeting Cdr1 localization, we show that Cdr1 inhibition of Wee1 occurs in cells at cortical nodes formed by Cdr2. On the basis of our results, we propose a two-step model for inhibition of Wee1 by Cdr1 and Cdr2 at nodes.

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.

The phosphatase inhibitor Sds23 regulates cell division symmetry in fission yeast.

Animal and fungal cells divide through the assembly, anchoring, and constriction of a contractile actomyosin ring (CAR) during cytokinesis. The timing and position of the CAR must be tightly controlled to prevent defects in cell division, but many of the underlying signaling events remain unknown. The conserved heterotrimeric protein phosphatase PP2A controls the timing of events in mitosis, and upstream pathways including Greatwall-Ensa regulate PP2A activity. A role for PP2A in CAR regulation has been less clear, although loss of PP2A in yeast causes defects in cytokinesis. Here, we report that Sds23, an inhibitor of PP2A family protein phosphatases, promotes the symmetric division of fission yeast cells through spatial control of cytokinesis. We found that sds23∆ cells divide asymmetrically due to misplaced CAR assembly, followed by sliding of the CAR away from its assembly site. These mutant cells exhibit delayed recruitment of putative CAR anchoring proteins including the glucan synthase Bgs1. Our observations likely reflect a broader role for regulation of PP2A in cell polarity and cytokinesis because sds23∆ phenotypes were exacerbated when combined with mutations in the fission yeast Ensa homologue, Igo1. These results identify the PP2A regulatory network as a critical component in the signaling pathways coordinating cytokinesis.

Stable Pom1 clusters form a glucose-modulated concentration gradient that regulates mitotic entry.

Control of cell size requires molecular size sensors that are coupled to the cell cycle. Rod-shaped fission yeast cells divide at a threshold size partly due to Cdr2 kinase, which forms nodes at the medial cell cortex where it inhibits the Cdk1-inhibitor Wee1. Pom1 kinase phosphorylates and inhibits Cdr2, and forms cortical concentration gradients from cell poles. Pom1 inhibits Cdr2 signaling to Wee1 specifically in small cells, but the time and place of their regulatory interactions were unclear. We show that Pom1 forms stable oligomeric clusters that dynamically sample the cell cortex. Binding frequency is patterned into a concentration gradient by the polarity landmarks Tea1 and Tea4. Pom1 clusters colocalize with Cdr2 nodes, forming a glucose-modulated inhibitory threshold against node activation. Our work reveals how Pom1-Cdr2-Wee1 operates in multiprotein clusters at the cortex to promote mitotic entry at a cell size that can be modified by nutrient availability.

Eisosomes.

Moseley discusses the molecular and mechanical functions of eisosomes - invaginations from the yeast plasma membrane.

Cell size-dependent regulation of Wee1 localization by Cdr2 cortical nodes.

Cell size control requires mechanisms that link cell growth with Cdk1 activity. In fission yeast, the protein kinase Cdr2 forms cortical nodes that include the Cdk1 inhibitor Wee1 along with the Wee1-inhibitory kinase Cdr1. We investigated how nodes inhibit Wee1 during cell growth. Biochemical fractionation revealed that Cdr2 nodes were megadalton structures enriched for activated Cdr2, which increases in level during interphase growth. In live-cell total internal reflection fluorescence microscopy videos, Cdr2 and Cdr1 remained constant at nodes over time, but Wee1 localized to nodes in short bursts. Recruitment of Wee1 to nodes required Cdr2 kinase activity and the noncatalytic N terminus of Wee1. Bursts of Wee1 localization to nodes increased 20-fold as cells doubled in size throughout G2. Size-dependent signaling was caused in part by the Cdr2 inhibitor Pom1, which suppressed Wee1 node bursts in small cells. Thus, increasing Cdr2 activity during cell growth promotes Wee1 localization to nodes, where inhibitory phosphorylation of Wee1 by Cdr1 and Cdr2 kinases promotes mitotic entry.

Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division.

Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in a highly polarized manner, and genetic screens have identified many protein kinases, including the CaMKK-like Ssp1 and the MARK/PAR-1 family kinase Kin1, that are required for polarized growth and cell shape, but their functional mechanisms and connections have been unknown [1-5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4-7]. We performed a large-scale phosphoproteomic screen and found that Kin1 phosphorylates itself and Pal1 to promote growth at cell tips, and these proteins are interdependent for localization to growing cell tips. Additional Kin1 substrates for cell polarity and cytokinesis (Tea4, Mod5, Cdc15, and Cyk3) were also phosphorylated by a second kinase, the DYRK family member Pom1 [8]. Kin1 and Pom1 were enriched at opposite ends of growing cells, and they phosphorylated largely non-overlapping sites on shared substrates. Combined inhibition of both Kin1and Pom1 led to synthetic defects in their shared substrates Cdc15 and Cyk3, confirming a non-redundant functional connection through shared substrates. These findings uncover a new Ssp1-Kin1 signaling pathway, and define its functional and mechanistic connection with Pom1 signaling for cell polarity and cytokinesis. These kinases are conserved in many eukaryotes including humans, suggesting that similar connections and mechanisms might operate in a broad range of cells.

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.

Transient activation of fission yeast AMPK is required for cell proliferation during osmotic stress.

The heterotrimeric kinase AMPK acts as an energy sensor to coordinate cell metabolism with environmental status in species from yeast through humans. Low intracellular ATP leads to AMPK activation through phosphorylation of the activation loop within the catalytic subunit. Other environmental stresses also activate AMPK, but it is unclear whether cellular energy status affects AMPK activation under these conditions. Fission yeast AMPK catalytic subunit Ssp2 is phosphorylated at Thr-189 by the upstream kinase Ssp1 in low-glucose conditions, similar to other systems. Here we find that hyperosmotic stress induces strong phosphorylation of Ssp2-T189 by Ssp1. Ssp2-pT189 during osmotic stress is transient and leads to transient regulation of AMPK targets, unlike sustained activation by low glucose. Cells lacking this activation mechanism fail to proliferate after hyperosmotic stress. Activation during osmotic stress requires energy sensing by AMPK heterotrimer, and osmotic stress leads to decreased intracellular ATP levels. We observed mitochondrial fission during osmotic stress, but blocking fission did not affect AMPK activation. Stress-activated kinases Sty1 and Pmk1 did not promote AMPK activation but contributed to subsequent inactivation. Our results show that osmotic stress induces transient energy stress, and AMPK activation allows cells to manage this energy stress for proliferation in new osmotic states.