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Wee1 localization to nodes requires Cdr2 kinase activity and the Wee1 N terminus. (A) Schematic of Wee1 and Cdr2 functional domains. Values represent amino acid positions. (B) The Wee1 N terminus interacts with WT Cdr2 but not with kinase-dead Cdr2(E177A) in the yeast two-hybrid assay. Transformants were selected on a double-dropout (DDO) plate, and interactions were tested on a quadruple-dropout plate containing aureobasidin, X-gal, and 3-AT (QDO/A/X/3AT) plate. Positive interactions are indicated by growth of blue colonies on selective plates. (C) Localization of the indicated Wee1 constructs overexpressed from the P81nmt1 promoter. Middle–focal plane widefield images with inverted contrast are shown, and insets show enlarged views of dashed boxes. Bars, 5 µm. (D) Length of dividing septated cells of the indicated genotypes (means ± SD; n > 50 cells). ****, P < 0.0001; n.s., P > 0.05.
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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...
Citations
... Nodes are assembled and organized by the conserved protein kinase Cdr2, which binds the membrane through a KA1 domain (Martin and Berthelot-Grosjean, 2009;Moseley et al., 2009;Rincon et al., 2014). This domain also has clustering activity for node formation, and Cdr2 nodes then recruit downstream components such as anillin-like Mid1, cell cycle kinases Cdr1 and Wee1, and other proteins (Almonacid et al., 2009;Rincon et al., 2014;Guzmán-Vendrell et al., 2015;Allard et al., 2018). Two inhibitory signals from cell tips help to position Cdr2 nodes in the cell middle. ...
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.
... Nodes are assembled and organized by the conserved protein kinase Cdr2, which binds the membrane through a KA1 domain (Martin and Berthelot-Grosjean, 2009;Moseley et al., 2009;Rincon et al., 2014). This domain also has clustering activity for node formation, and Cdr2 nodes then recruit downstream components such as anillin-like Mid1, cell cycle kinases Cdr1 and Wee1, and other proteins (Almonacid et al., 2009;Rincon et al., 2014;Guzmán-Vendrell et al., 2015;Allard et al., 2018). Two inhibitory signals from cell tips help to position Cdr2 nodes in the cell middle. ...
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.
... Conversely, mutations in cdc25+ increase cell size, while cdc25+ overexpression reduces cell size (8). Wee1 activity in cells is regulated in a size-dependent manner by the protein kinases Cdr1 and Cdr2 (9), which are conserved SAD family kinases. Cdr1 (also called Nim1) directly phosphorylates Wee1 to inhibit its kinase activity (10)(11)(12)(13). ...
... Cdr2 also promotes Wee1 inhibition in cells but does not appear to inhibit Wee1 kinase activity directly (15). Rather, Cdr2 forms oligomeric "nodes" at the plasma membrane and recruits both Cdr1 and Wee1 to these structures (9,(16)(17)(18). Consistent with this mechanism, loss-of-function mutations in cdr2+ increase cell size (15,19,20). ...
... A second open question addressed in our work is whether Cdr2 regulates Wee1 and cell size independently of Cdr1/ Nim1. Recent work has led to the model that Cdr2 nodes act as scaffolds to promote inhibitory phosphorylation of Wee1 by Cdr1 (9,13). In this model, the function of Cdr2 on Wee1 and cell size requires Cdr1. ...
Many cell cycle regulatory proteins catalyze cell cycle progression in a concentration-dependent manner. In the fission yeast S. 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 6X 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.
... Interphase cells grow linearly at their tips (Fantes and Nurse, 1977;Moreno et al., 1989) until they reach a threshold size of twice the initial cell length (Gu and Oliferenko, 2019;Neumann and Nurse, 2007). During this period, the interphase node components also double in number (Allard et al., 2018;Pan et al., 2014). Interphase nodes are thought to sense the cell surface area to control cell size at the time of division (Facchetti et al., 2019a;Pan et al., 2014). ...
... Using the same scaled fire LUT plugin in ImageJ (Willet et al., 2019) measured ≤15 nodes marked with Mid1 in wild-type cells that had a yellow, orange, or white intensity but did not count dim purple or red intensity nodes. Allard et al., 2018 used the superior (140 nm) resolution of Airyscan microscopy and an automated particle tracking plugin to count ~50 nodes tagged with Cdr2-mEGFP during interphase. However, the algorithm used in the plugin was designed to find only local intensity maxima and did not count the dim nodes with low intensities. ...
... All of the nodes considered here were within minutes of condensing into contractile rings. Third, following Allard et al., 2018 we used Airyscan microscopy to image cytokinesis nodes. The higher resolution of Airyscan allowed us to detect and measure the fluorescence of dim individual cytokinesis nodes better than by conventional confocal microscopy. ...
Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here, we used the ~140 nm resolution of Airyscan super-resolution microscopy to measure the fluorescence intensity of small, single cytokinesis nodes marked with Blt1-mEGFP in live fission yeast cells early in mitosis. The ratio of the total Blt1-mEGFP fluorescence in the broad band of cytokinesis nodes to the average fluorescence of a single node gives about 190 single cytokinesis nodes in wild-type fission yeast cells early in mitosis. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although large diameter rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The Pom1 kinase restricts cytokinesis nodes from the ends of cells, but the surface density of Pom1 on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, when the concentrations of either kinase Pom1 or kinase Cdr2 were varied with the nmt1 promoter, the numbers of cytokinesis nodes increased above a baseline of about ~190 with the total cellular concentration of either kinase.
... In the study of cell size dynamics, a core issue is to understand the size homeostasis strategies in various cell types, especially in fission yeast [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. There are three popular phenomenological models of cell size control leading to size homeostasis [52]: (i) the timer strategy which implies a constant time between successive divisions regardless of initial size; (ii) the sizer strategy which implies cell division upon attainment of a critical size, and (iii) the adder strategy which implies a constant size addition between consecutive generations. ...
Unlike many single-celled organisms, the growth of fission yeast cells within a cell cycle is not exponential. It is rather characterized by three distinct phases (elongation, septation, and reshaping), each with a different growth rate. Experiments also showed that the distribution of cell size in a lineage can be bimodal, unlike the unimodal distributions measured for the bacterium Escherichia coli . Here we construct a detailed stochastic model of cell size dynamics in fission yeast. The theory leads to analytic expressions for the cell size and the birth size distributions, and explains the origin of bimodality seen in experiments. In particular, our theory shows that the left peak in the bimodal distribution is associated with cells in the elongation phase, while the right peak is due to cells in the septation and reshaping phases. We show that the size control strategy, the variability in the added size during a cell cycle, and the fraction of time spent in each of the three cell growth phases have a strong bearing on the shape of the cell size distribution. Furthermore, we infer all the parameters of our model by matching the theoretical cell size and birth size distributions to those from experimental single-cell time-course data for seven different growth conditions. Our method provides a much more accurate means of determining the size control strategy (timer, adder or sizer) than the standard method based on the slope of the best linear fit between the birth and division sizes. We also show that the variability in added size and the strength of size control in fission yeast depend weakly on the temperature but strongly on the culture medium. More importantly, we find that stronger size homeostasis and larger added size variability are required for fission yeast to adapt to unfavorable environmental conditions.
... The SAD kinase Cdr2 is a key component of the signaling network that prevents mitotic entry until cells reach a critical and reproducible size (Martin and Berthelot-Grosjean, 2009;Moseley et al., 2009;Allard et al., 2018). The pivotal role of Cdr2 in cell cycle regulation prompted us to investigate whether overexpression of cdr2 + rescued the defect of G2/M transition in ksg1-208 cells. ...
... The integration of cell growth and cell cycle progression ensures the reproducible cell division size. The key component of this integration in fission yeast is Cdr2, which organizes cortical nodes in the cell middle to sense cell size and promote mitotic entry (Breeding et al., 1998;Kanoh and Russell, 1998;Martin and Berthelot-Grosjean, 2009;Moseley et al., 2009;Rincon et al., 2017;Allard et al., 2018;Facchetti et al., 2019). The activity of Cdr2 increases as cells grow in the G2 phase while the total level keeps constant throughout the cell cycle (Deng et al., 2014). ...
... In eukaryotes, the cell cycle G2/M phase transition is controlled by mitotic cyclin-dependent kinase complex (Cdc2-cyclin B), which is inactivated by Wee1 family protein kinases and activated by the opposing phosphatase Cdc25 Nurse, 1986, 1987;Karlsson-Rosenthal and Millar, 2006;Glover, 2012). Previous studies have shown that Cdr2 recruits Wee1 to cortical nodes through interacting with the N-terminal of Wee1 and negatively regulates the latter together with another SAD family kinase Cdr1 to promote mitotic entry (Allard et al., 2018). However, a recent study showed that both Cdr1 and Cdr2 promote the phosphorylation of Wee1 in vivo, but only Cdr1 inhibits the kinase activity of Wee1 (Opalko et al., 2019). ...
Aberration in the control of cell cycle contributes to the development and progression of many diseases including cancers. Ksg1 is a Schizosaccharomyces pombe fission yeast homolog of mammalian phosphoinositide-dependent protein kinase 1 (PDK1) which is regarded as a signaling hub for human tumorigenesis. A previous study reported that Ksg1 plays an important role in cell cycle progression, however, the underlying mechanism remains elusive. Our genomic library screen for novel elements involved in Ksg1 function identified two serine/threonine kinases, namely SAD family kinase Cdr2 and another PDK1 homolog Ppk21, as multicopy suppressors of the thermosensitive phenotype of ksg1-208 mutant. We found that overexpression of Ppk21 or Cdr2 recovered the defective cell cycle transition of ksg1-208 mutant. In addition, ksg1-208 Δ ppk21 cells showed more marked defects in cell cycle transition than each single mutant. Moreover, overexpression of Ppk21 failed to recover the thermosensitive phenotype of the ksg1-208 mutant when Cdr2 was lacking. Notably, the ksg1-208 mutation resulted in abnormal subcellular localization and decreased abundance of Cdr2, and Ppk21 deletion exacerbated the decreased abundance of Cdr2 in the ksg1-208 mutant. Intriguingly, expression of a mitotic inducer Cdc25 was significantly decreased in ksg1-208 , Δ ppk21 , or Δ cdr2 cells, and overexpression of Ppk21 or Cdr2 partially recovered the decreased protein level of Cdc25 in the ksg1-208 mutant. Altogether, our findings indicated that Cdr2 is a novel downstream effector of PDK1 homologs Ksg1 and Ppk21, both of which cooperatively participate in regulating cell cycle progression, and Cdc25 is involved in this process in fission yeast.
... Multiple mechanisms contribute to cell size-dependent Cdk1 activation in S. pombe. The protein kinase Wee1 phosphorylates and inhibits Cdk1 in small cells (Russell and Nurse, 1987a;Gould and Nurse, 1989), while a regulatory network progressively inhibits Wee1 as cell size increases (Lucena et al., 2017;Allard et al., 2018;Opalko et al., 2019). The concentrations of mitotic inducers including Cdc13 (mitotic cyclin) and Cdc25 (protein phosphatase that counteracts Wee1) increase as cells grow (Moreno et al., 1990;Keifenheim et al., 2017;Patterson et al., 2019). ...
... Cdr2 forms oligomeric "nodes," which are stable structures tethered to the plasma membrane and positioned in the cell middle (Morrell et al., 2004). Cdr2 recruits both Cdr1 and Wee1 to these sites, and Cdr2 kinase activity correlates with the dwell time of Wee1 at individual nodes Martin and Berthelot-Grosjean, 2009;Allard et al., 2018). Since Cdr2 is progressively activated as cells grow, Wee1 spends more time at nodes as cells grow larger, resulting in its cell size-dependent phosphorylation and inhibition (Deng et al., 2014;Allard et al., 2018). ...
... Cdr2 recruits both Cdr1 and Wee1 to these sites, and Cdr2 kinase activity correlates with the dwell time of Wee1 at individual nodes Martin and Berthelot-Grosjean, 2009;Allard et al., 2018). Since Cdr2 is progressively activated as cells grow, Wee1 spends more time at nodes as cells grow larger, resulting in its cell size-dependent phosphorylation and inhibition (Deng et al., 2014;Allard et al., 2018). Importantly, this mechanism acts at the plasma membrane, consistent with its role connecting cell surface area with the G2/M transition. ...
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.
... Tome-1 has previously been identified as a potential interactor of the E-cadherin complex (Guo et al., 2014), suggesting a putative link between E-cadherin adhesions and Wee1 degradation. Intriguingly, in budding yeast Wee1 regulation couples the timing of mitotic entry to cell growth, inducing division only once cells have achieved their appropriate size (Allard et al., 2017). This suggests an evolutionary conserved role for Wee1 in sensing physical cues and coordinating cell divisions with cell/tissue size. ...
Epithelial cell divisions must be tightly coordinated with cell loss to preserve epithelial integrity. However, it is not well understood how the rate of epithelial cell division adapts to changes in cell number, for instance during homeostatic turnover or upon wounding of epithelia. Here, we show epithelial cells sense local cell density through mechanosensitive E-cadherin adhesions to control G2/M cell cycle progression. We demonstrate that tensile forces on E-cadherin adhesions are reduced as local cell density increases, which prompts the accumulation of the G2 checkpoint kinase Wee1. This elevated abundance of Wee1 results in inhibitory phoshorylation of Cdk1, and thereby establishes a pool of cells that is temporarily halted in G2-phase. Importantly, these cells are readily triggered to divide upon epithelial wounding, due to the consequent increase in intercellular forces and resulting degradation of Wee1. Our data thus demonstrate that epithelial cell division is controlled by a mechanical G2 checkpoint, which is regulated by cell density-dependent intercellular forces sensed and transduced by E-cadherin adhesions.
... Fission yeast has been an attractive model organism for several decades in studies of eukaryotic cellular growth [1][2][3][4][5][6][7]. Revealing the rules of cellular growth is crucial in understanding how size control mechanisms maintain size homeostasis in steady-state cell cultures, and this point has also been extensively studied in fission yeast [5,[7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. ...
Fission yeast is commonly used as a model organism in eukaryotic cell growth studies. To describe the cells' length growth patterns during the mitotic cycle, different models have been proposed previously as linear, exponential, bilinear and biexponential ones. The task of discriminating among these patterns is still challenging. Here, we have analyzed 298 individual cells altogether , namely from three different steady-state cultures (wild-type, wee1-50 mutant and pom1Δ mutant). We have concluded that in 190 cases (63.8%) the bilinear model was more adequate than either the linear or the exponential ones. These 190 cells were further examined by separately analyzing the linear segments of the best fitted bilinear models. Linear and exponential functions have been fitted to these growth segments to determine whether the previously fitted bilinear functions were really correct. The majority of these growth segments were found to be linear; nonetheless, a significant number of exponential ones were also detected. However, exponential ones occurred mainly in cases of rather short segments (<40 min), where there were not enough data for an accurate model fitting. By contrast, in long enough growth segments (≥40 min), linear patterns highly dominated over exponential ones, verifying that overall growth is probably bilinear.
... The molecular output of Cdr2 pathway signaling is inhibitory phosphorylation of Wee1, which can be monitored by SDS-PAGE mobility shifts (Allard et al., 2018(Allard et al., , 2019Opalko et al., 2019). The slower migrating, hyperphosphorylated form of Wee1 was lost in arf6∆ and syt22∆, similar to cdr2∆ (Fig 1H). ...
... Further, Arf6 and Cdr2 were highly colocalized at nodes (Fig 2B), identifying Arf6 as a new node component. The Arf6-mNG signal at individual nodes was stable by time lapse microscopy (Fig 2C), similar to Cdr2 and Cdr1 but distinct from Wee1 (Pan et al., 2014;Allard et al., 2018). To test the timing of Arf6 node localization, we used Sad1 and Rlc1 to track mitosis and cytokinesis respectively. ...
... Gels were transferred to nitrocellulose using Trans-blot Turbo Transfer System (Bio-rad). Wee1 was probed using anti-Wee1 antibody (Allard et al., 2018). Cdc2 was used as a loading control using anti-cdc2 antibody (Santa Cruz Biotechnologies; SC-53217). ...
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 GEF (guanine nucleotide exchange factor) 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.
