Biology subjects

Das, M. E.

Publications and source records attributed to Das, M. E..

13 recordsLinked to original sources

bioRxiv2025

Rax1/2 promote memory of growth by early Ras1 activation to maintain polarity across generations

As polarized cells divide, daughter cells must re-establish polarity at the correct site to maintain their shape. Fission yeast cells are bipolar, however growth initiates first at the old end of the cell that was inherited from the mother. This dominance of the old end is dependent on that end having grown in the previous generation, suggesting cells must be able to recognize when an end has a previous history of growth. We find that the proteins Rax1/2 act as markers for memory of growth by localizing to growing cell ends and remaining through division. Rax1/2 then promote activation of Ras1-GTPase at dividing cell ends by recruitment of its GEF Efc25. Localized Ras1 activity at the old ends ensures timely activation of the Rho GTPase Cdc42 to initiate polarized growth, providing an advantage to the old end. Through this work, we demonstrate the role of a Ras-GTPase in positioning the site of polarization and maintaining cell shape across multiple generations.

cell biology↗

bioRxiv2025

Differential gene expression drives cell-cycle-dependent transition from monopolar to bipolar growth in fission yeast.

Cell polarity is important for maintaining cell structure and function. In S. pombe, after division, cells are monopolar and grow from the old end that existed from the previous generation. Cells transition to bipolar growth once the cell reaches a certain size, when the new end generated after division initiates growth. Bipolarity occurs as a consequence of cell growth, where the two cell ends have sufficient growth-promoting factors. However, the transition to bipolarity does not occur in G1-arrested cells even with enhanced cell growth, suggesting a role for the cell-cycle phase in this process. To identify how the cell cycle impacts monopolar to bipolar transition, we performed high-throughput mRNA sequencing to detect differentially expressed genes in G1/S-arrested and G2-phase cells of the cell cycle mutant cdc10-129. Gene expression analysis using the DESeq2 R package identified 65 unique genes upregulated in G1/S phase and 35 in G2 phase cells. Enrichment analysis shows that G1/S phase cells upregulated the MAPK pheromone-response pathway, protein folding, rRNA processing, and heat-shock protein binding. G2 phase cells showed upregulation of plasma membrane maintenance and cell wall organization. Protein-protein interaction networks identified the cdc15-hob3-rho1-bgs1 hub in G2 phase cells. These genes are known to promote bipolar growth and regulate cell wall biogenesis. In G1/S phase cells spk1-byr2-ste11 hub was identified, which is required for pheromone-response and nutritional stress-dependent G1-arrest. We find that Spk1 also prevents precocious bipolar growth. Thus, we hypothesize that the pheromone response pathway, in addition to promoting G1-G0 transition, also prevents bipolar growth. In nutrient-rich conditions, this pathway is downregulated, G1-arrest is alleviated, the cell cycle progresses, and bipolarity occurs. Our findings suggest that in nutrition-rich conditions, stress response pathways are downregulated enabling transition from monopolar to bipolar growth, along with cell cycle progression.

cell biology↗

bioRxiv2025

Scd1 diffuses end to end along the cytoplasm to facilitate Cdc42 activation and bipolar growth

The conserved GTPase Cdc42 is a major regulator of polarized growth in most eukaryotes. In Schizosaccharomyces pombe, Cdc42 activity displays anticorrelated oscillatory dynamics between the growing ends enabling bipolarity. Cdc42 at each end is activated only when the opposite end loses activity. This suggests that a regulator of Cdc42 likely travels end-to-end to activate Cdc42. The oscillatory dynamics between the growing ends have also been observed in Cdc42 activator Scd1, its scaffold Scd2. It is unclear how these proteins move between the ends to facilitate bipolarity. We find that Scd1 does not travel between the cell ends via actin-mediated delivery. Instead, we show that Scd1 is mostly cytoplasmic and diffuses between the cell ends. The rate of diffusion is not entirely proportional to increasing the mass of Scd1 and cells lacking the inhibitor Pak1 kinase show decreased diffusion. Moreover, we show that Scd1 diffuses at a much faster rate compared to its scaffold Scd2. These findings suggest that Scd1 diffusion is not random and is regulated by Pak1 kinase. We find that decreasing the rate of diffusion disrupts Cdc42 oscillatory dynamics and results in monopolarity. Our results show that end-to-end Scd1 diffusion drives Cdc42 oscillatory dynamics and regulates cell polarity. SIGNIFICANCE STATEMENTO_LICdc42 activation shows oscillatory dynamics between the sites of growth C_LIO_LIThe Cdc42 GEF Scd1 diffuses from site of activation to the opposite end to facilitate these oscillatory dynamics C_LIO_LIThis diffusion is not random and likely depends on intrinsic properties of the Scd1 protein. C_LI

cell biology↗

bioRxiv2024

Endocytic Patch Dynamics are Differentially Regulated at Distinct Cell Sites in Fission Yeast

Endocytosis promotes polarity and growth in eukaryotes. In Schizosaccharomyces pombe fission yeast, endocytosis occurs at the polarized cell ends and division site and at the non-polarized cell sides. Our characterization of endocytic actin patches show that they are differentially regulated. The patches at the cell ends and division site internalize successfully while those at the sides are weak and erratic. The major regulator of cell polarity, Cdc42, and its target Pak1 kinase only localize to the cell ends and division site. We find that these proteins regulate assembly and internalization of patches at these sites but not at the cell sides. Moreover, Cdc42 specifically activated by the GEF Gef1 promotes proper patch dynamics. Endocytosis requires phosphorylation of the Type I Myosin Myo1 by the Pak1 kinase. Myo1 localizes to the cell ends, division site, and the cell sides. We find that unlike Cdc42 and Pak1, Myo1 also promotes patch assembly at the cell sides. Our data indicate that while Myo1 can globally promote branched actin assembly, successful endocytic patch dynamics and internalization at polarized sites require Cdc42 and Pak1 kinase. SUMMARY STATEMENTEndocytic patch dynamics are differentially regulated at distinct sites such as the cell ends, division site and the cell sides by Cdc42 and its downstream targets Pak1 kinase and the Type 1 myosin.

cell biology↗

bioRxiv2024

The Septin Cytoskeleton excludes the Cdc42 GAP Rga4 from growing sites to promote bipolarity in fission yeast

The polarized growth promoter Cdc42 is inactivated at the growing cell ends during mitosis and reactivated after division. In fission yeast, the Cdc42 inactivator/GAP Rga4 localizes to the cell ends during mitosis and is restricted to the sides in interphase. We show that this cell-cycle-dependent Rga4 localization is septin-dependent. Septins form linear filaments along the sides during interphase and recede to form a medial ring during mitosis. In septin mutants, Rga4 is mobile, homogeneously distributed, and increasingly localizes to the cell ends and cytoplasm, similar to mitotic cells. Accordingly, septin mutants are monopolar with disrupted Cdc42 activation dynamics. This regulation appears to be indirect, via septin-dependent lipid organization on the cortex. We find that the septin cytoskeleton prevents excessive phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and its upstream kinase Its3 at the plasma membrane. Limiting PI(4,5)P2 levels enhances Rga4 puncta at the cortex. Our data describe an unusual form of polarity regulation where the septin cytoskeleton restricts GAP localization via membrane lipid organization. SummaryCell-cycle-dependent Rga4 localization pattern is controlled by the septin cytoskeleton to promote bipolar growth. In interphase, the septin cytoskeleton at the cell sides restricts Rga4 mobility and localization away from the cell ends to promote proper Cdc42 activation dynamics.

cell biology↗

bioRxiv2023

The Arp2/3 complex promotes periodic removal of Pak1-mediated negative feedback to facilitate anticorrelated Cdc42 oscillations.

The conserved GTPase Cdc42 is a major regulator of polarized growth in most eukaryotes. Cdc42 periodically cycles between active and inactive states at sites of polarized growth. These periodic cycles are caused by positive feedback and time-delayed negative feedback loops. In the bipolar yeast S. pombe, both growing ends must regulate Cdc42 activity. At each cell end, Cdc42 activity recruits the Pak1 kinase which prevents further Cdc42 activation thus establishing negative feedback. It is unclear how Cdc42 activation returns to the end after Pak1-dependent negative feedback. Using genetic and chemical perturbations, we find that disrupting branched actin-mediated endocytosis disables Cdc42 reactivation at the cell ends. With our experimental data and mathematical models, we show that endocytosis-dependent Pak1 removal from the cell ends allows the Cdc42 activator Scd1 to return to that end to enable reactivation of Cdc42. Moreover, we show that Pak1 elicits its own removal via activation of endocytosis. In agreement with these observations, our model and experimental data show that in each oscillatory cycle, Cdc42 activation increases followed by an increase in Pak1 recruitment at that end. These findings provide a deeper insight into the self-organization of Cdc42 regulation and reveal previously unknown feedback with endocytosis in the establishment of cell polarity.

cell biology↗

bioRxiv2022

Cdc42 prevents precocious Rho1 activation during cytokinesis in a Pak1-dependent manner

Cytokinesis consists of a series of coordinated multi-step events that partition a dividing cell. Accurate regulation of cytokinesis is essential for proliferation and genome integrity. In fission yeast, these coordinated events ensure that the actomyosin ring and septum start ingressing only after chromosome segregation. How cytokinetic events are coordinated remains unclear. The GTPase Cdc42 is required for the delivery of certain cell wall-building enzymes while the GTPase Rho1 is required for activation of these enzymes. Here we show that Cdc42 prevents early Rho1 activation during cytokinesis. Using an active Rho-probe, we show that even though the Rho1 activators Rgf1 and Rgf3 localize to the division site in early anaphase, Rho1 is not activated until late anaphase, just before the onset of ring constriction. We find that loss of Cdc42 activation enables precocious Rho1 activation in early anaphase. Furthermore, this inhibition of Rho1 activation is dependent on the downstream Cdc42 effector Pak1 kinase. Disrupting pak1 function results in early Rho1 activation accompanied by precocious septum deposition and ring constriction. We provide functional and genetic evidence which indicates that Pak1 regulates Rho1 activation likely via the regulation of its GEF Rgf1. Our work proposes a mechanism of Rho1 regulation by active Cdc42 to coordinate timely septum formation and cytokinesis fidelity.

cell biology↗

bioRxiv2021

Cdc42 GTPase activating proteins Rga4 and Rga6 coordinate septum synthesis and membrane trafficking at the division plane during cytokinesis

Fission yeast cytokinesis is driven by simultaneous septum synthesis, membrane furrowing and actomyosin ring constriction. The septum consists of a primary septum flanked by secondary septa. First, delivery of the glucan synthase Bgs1 and membrane vesicles initiate primary septum synthesis and furrowing. Next, Bgs4 is delivered for secondary septum formation. It is unclear how septum synthesis is coordinated with membrane furrowing. Cdc42 promotes delivery of Bgs1 but not Bgs4. We find that after primary septum initiation, Cdc42 inactivators Rga4 and Rga6 localize to the division site. In rga4{Delta}rga6{Delta} mutants Cdc42 activity is enhanced during late cytokinesis and cells take longer to separate. Electron micrographs of the division site in these mutants exhibit malformed septum with irregular membrane structures. These mutants have a larger division plane with enhanced Bgs1 delivery but fail to enhance accumulation of Bgs4 and several exocytic proteins. Additionally, these mutants show endocytic defects at the division site. This suggests that Cdc42 regulates only specific membrane trafficking events. Our data indicate that while active Cdc42 promotes primary septum synthesis, as cytokinesis progresses Rga4 and Rga6 localize to the division site to decrease Cdc42 activity. This couples specific membrane trafficking events with septum formation to allow proper septum morphology. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/472679v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@ef042aorg.highwire.dtl.DTLVardef@1fa81adorg.highwire.dtl.DTLVardef@157a4a5org.highwire.dtl.DTLVardef@fa516d_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisThe GTPase Cdc42 regulates cytokinesis in cell-walled fission yeast. Active Cdc42 promotes the initiation of septum (new cell wall) synthesis to physically divide daughter cells. Here we show that Cdc42 activity must be decreased at the later stages of cytokinesis to enable proper septum formation. Mutants lacking Cdc42 inactivators, Rga4 and Rga6, lead to membrane trafficking defects and malformed septa consequently delaying cell separation.

cell biology↗

bioRxiv2020

Cdc42 reactivation at growth sites is regulated by cell-cycle-dependent removal of its GAP Rga4 in fission yeast

In fission yeast, polarized cell growth stops during division and resumes after cell separation. We uncoupled these sequential events by delaying cytokinesis with a temporary Latrunculin A treatment. Mitotic cells recovering from treatment initiate end growth without cell separation, displaying a polar elongation sans separation (PrESS) phenotype. PrESS cell ends reactivate Cdc42, a major regulator of polarized growth, before cell separation, but at a fixed time after anaphase B. A candidate screen implicates Rga4, a negative regulator of Cdc42, in this process. We show that Rga4 appears punctate at the cell sides during G2, but is diffuse during mitosis, extending to the ends. While the Morphogenesis Orb6 (MOR) pathway is known to promote cell separation and growth by activating protein synthesis, we find that for polarized growth, removal of Rga4 from the ends is also necessary. Therefore, we propose that growth resumes after division once the MOR pathway is activated and the ends lose Rga4 in a cell-cycle-dependent manner.

cell biology↗

bioRxiv2020

Cdc42 GTPase Activating Proteins (GAPs) Maintain Generational Inheritance of Cell Polarity and Cell Shape in Fission Yeast

The highly conserved small GTPase Cdc42 regulates polarized cell growth and morphogenesis from yeast to humans. We previously reported that Cdc42 activation exhibits oscillatory dynamics in Schizosaccharomyces pombe cells. Mathematical modeling suggests that this dynamic behavior enables a variety of symmetric and asymmetric Cdc42 distributions to coexist in cell populations. For individual wild type cells, however, growth follows a stereotypical pattern where Cdc42 distribution is initially asymmetrical in young daughter cells and becomes more symmetrical as cell volume increases, enabling bipolar growth activation. To explore whether different states of Cdc42 activation are possible in a biological context, we examined S. pombe rga4{Delta} mutant cells, lacking the Cdc42 GTPase activating protein (GAP) Rga4. We found that monopolar rga4{Delta} mother cells divide asymmetrically leading to the emergence of both symmetric and asymmetric Cdc42 distributions in rga4{Delta} daughter cells. Using genetic screening approaches to identify mutants that alter the rga4{Delta} phenotype, we tested the predictions of different computational models that reproduce the unequal fate of daughter cells. We found experimentally that the unequal distribution of active Cdc42 GTPase in daughter cells is consistent with an unequal inheritance of another Cdc42 GAP, Rga6, in the two daughter cells. Our findings highlight the crucial role of Cdc42 GAP protein localization in determining the morphological fate of cell progeny and ensuring consistent Cdc42 activation and growth patterns across generations.

cell biology↗

bioRxiv2019

Cdc42 promotes Bgs1 recruitment for septum synthesis and glucanase localization for cell separation during cytokinesis in fission yeast.

Cytokinesis in fission yeast involves actomyosin ring constriction concurrent to septum synthesis followed by septum digestion resulting in cell separation. A recent report indicates that endocytosis is required for septum synthesis and cell separation. The conserved GTPase Cdc42 is required for membrane trafficking and promotes endocytosis. Cdc42 is activated by Guanine nucleotide exchange factors (GEFs). Cdc42 GEFs have been shown to promote timely initiation of septum synthesis and proper septum morphology. Here we show that Cdc42 promotes the recruitment of the major primary septum synthesizing enzyme Bgs1 and consequent ring constriction. Cdc42 is also required for proper localization of the septum digesting glucanases at the division site. Thus, Cdc42 is required to promote multiple steps during cytokinesis.

cell biology↗

bioRxiv2019

F-BAR Cdc15 Promotes Gef1-mediated Cdc42 Activation During Cytokinesis and Cell Polarization in S. pombe

Cdc42, a Rho-family GTPase, is a master regulator of cell polarity. Recently it has been shown that Cdc42 also facilitates proper cytokinesis in the fission yeast, Schizosaccharomyces pombe. Cdc42 is activated by two partially redundant GEFs Gef1 and Scd1. Although both the GEFs activate Cdc42, their deletion mutants display distinct phenotypes, indicating that they are differentially regulated, by an unknown mechanism. During cytokinesis, Gef1 localizes to the division site and activates Cdc42 to initiate ring constriction and septum ingression. Here we report that the F-BAR domain containing Cdc15 promotes Gef1 localization to its functional sites. We show that cdc15 promotes Gef1 association with the cytokinetic nodes to activate Cdc42 during ring assembly. Moreover, cdc15 phospho-mutants phenocopy polarity phenotypes of gef1 mutants. In a hypermorphic cdc15 mutant, Gef1 localizes precociously to the division site, and is readily detected at the cortical patches and the cell cortex. Correspondingly, the hypermorphic cdc15 mutant shows increased bipolarity during interphase and precocious Cdc42 activation at the division site during cytokinesis. Finally, loss of gef1 in hypermorphic cdc15 mutants abrogates the increased bipolarity and precocious Cdc42 activation phenotype. We did not see any change in the localization of the other GEF Scd1 in a Cdc15-dependent manner. Taken together our data indicates that Cdc15 promotes Cdc42 activation specifically via Gef1 localization to the division site to facilitate proper cytokinesis and to the cell cortex to promote bipolarity.

cell biology↗

bioRxiv2018

A novel interplay between GEFs orchestrates Cdc42 activation in cell polarity and cytokinesis

Cdc42, a conserved regulator of cell polarity, is activated by two GEFs, Gef1 and Scd1, in fission yeast. While gef1 and scd1 mutants exhibit distinct phenotypes, how they do so is unclear given that they activate the same GTPase. Using the GEF localization pattern during cytokinesis as a paradigm, we report a novel interplay between Gef1 and Scd1 that spatially modulates Cdc42. We find that Gef1 promotes Scd1 localization to the division site during cytokinesis and to the new end during polarized growth through the recruitment of the scaffold Scd2 via a Cdc42 feedforward pathway. Gef1-mediated Scd1 recruitment at the new end enables the transition from monopolar to bipolar growth. Reciprocally, Scd1 restricts Gef1 localization to prevent ectopic Cdc42 activation during cytokinesis to promote cell separation and during interphase to maintain cell shape. Our findings reveal an elegant regulatory pattern in which Gef1 establishes new sites of Scd1-mediated Cdc42 activity, while Scd1 restricts Gef1 to functional sites. We propose that crosstalk between GEFs is a conserved mechanism that orchestrates Cdc42 activation during complex cellular processes.\n\nSummary StatementCdc42 GEFs Gef1 and Scd1 crosstalk to fine-tune Cdc42 activity. This crosstalk promotes bipolar growth and maintains cell shape in fission yeast.

cell biology↗