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1. F-BAR Cdc15 Promotes Cdc42 Activation During Cytokinesis and Cell Polarization in Schizosaccharomyces pombe

   https://doi.org/10.1534/genetics.119.302649  

   Hercyk, Brian S; Das, Maitreyi E (December 2019, Genetics)

   Abstract 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 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 protein Cdc15 promotes Gef1 localization to its functional sites. We show that cdc15 promotes Gef1 association with cortical puncta at the incipient division site to activate Cdc42 during ring assembly. Moreover, cdc15 phospho-mutants phenocopy the 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. Our data indicate that Cdc15 facilitates Cdc42 activation at the division site during cytokinesis at the cell cortex to promote bipolarity and this is mediated by promoting Gef1 localization to these sites. 

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2. A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis

   https://doi.org/10.1242/jcs.236018  

   Hercyk, Brian S.; Rich-Robinson, Julie; Mitoubsi, Ahmad S.; Harrell, Marcus A.; Das, Maitreyi E. (January 2019, Journal of Cell Science)

   Cdc42, a conserved regulator of cell polarity, is activated by two GEFs, Gef1 and Scd1, in fission yeast. Why the cell needs two GEFs is unclear, given that they are partially redundant and 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 through the recruitment of the scaffold Scd2 via a Cdc42 feedforward pathway. Similarly, in interphase Gef1 promotes Scd1 recruitment at the new end to enable the transition from monopolar to bipolar growth. Reciprocally, Scd1 restricts Gef1 localization to prevent ectopic Cdc42 activation during cytokinesis to promote cell separation, and to maintain cell shape during interphase. Our findings reveal an elegant regulatory pattern in which Gef1 primes Cdc42 activation at new sites to initiate Scd1-dependent polarized growth, while Scd1 restricts Gef1 to sites of polarization. We propose that crosstalk between GEFs is a conserved mechanism that orchestrates Cdc42 activation during complex cellular processes. 

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3. Coordinating septum formation and the actomyosin ring during cytokinesis in Schizosaccharomyces pombe

   https://doi.org/10.1111/mmi.14387  

   Hercyk, Brian S.; Onwubiko, Udo N.; Das, Maitreyi E. (December 2019, Molecular Microbiology)

   Summary During cytokinesis, animal and fungal cells form a membrane furrow via actomyosin ring constriction. Our understanding of actomyosin ring‐driven cytokinesis stems extensively from the fission yeast model system. However, unlike animal cells, actomyosin ring constriction occurs simultaneously with septum formation in fungi. While the formation of an actomyosin ring is essential for cytokinesis in fission yeast, proper furrow formation also requires septum deposition. The molecular mechanisms of spatiotemporal coordination of septum deposition with actomyosin ring constriction are poorly understood. Although the role of the actomyosin ring as a mechanical structure driving furrow formation is better understood, its role as a spatiotemporal landmark for septum deposition is not widely discussed. Here we review and discuss the recent advances describing how the actomyosin ring spatiotemporally regulates membrane traffic to promote septum‐driven cytokinesis in fission yeast. Finally, we explore emerging questions in cytokinesis, and discuss the role of extracellular matrix during cytokinesis in other organisms. 

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4. A Cdc42 GEF, Gef1, through endocytosis organizes F-BAR Cdc15 along the actomyosin ring and promotes concentric furrowing

   https://doi.org/10.1242/jcs.223776  

   Onwubiko, Udo N.; Mlynarczyk, Paul J.; Wei, Bin; Habiyaremye, Julius; Clack, Amanda; Abel, Steven M.; Das, Maitreyi E. (January 2019, Journal of Cell Science)

   During cytokinesis, fission yeast coordinates actomyosin ring constriction with septum ingression, resulting in concentric furrow formation by a poorly defined mechanism. We report that cells lacking the Cdc42 activator Gef1, combined with an activated allele of the formin, Cdc12, display non-concentric furrowing. Non-concentrically furrowing cells display uneven distribution of the scaffold Cdc15 along the ring. This suggests that after ring assembly, uniform Cdc15 distribution along the ring enables proper furrow formation. We find that after assembly Cdc15 is recruited to the ring in an Arp2/3 complex-dependent manner and is decreased in the activated cdc12 mutant. Cdc15 at cortical endocytic patches show increased levels and extended lifetimes in gef1 and activated cdc12 mutants. We hypothesize endocytosis helps recruit Cdc15 to assembled rings; uneven Cdc15 distribution at the ring occurs when endocytic patches contain increased Cdc15 levels and patch-association rate is slow. Based on this, we developed a mathematical model that captures experimentally observed Cdc15 distributions along the ring. We propose that, at the ring, Gef1 and endocytic events promote uniform Cdc15 organization to enable proper septum ingression and concentric furrow formation. 

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5. Spatiotemporal Coordination of Rac1 and Cdc42 at the Whole Cell Level during Cell Ruffling

   https://doi.org/10.3390/cells12121638  

   Hladyshau, Siarhei; Stoop, Jorik P.; Kamada, Kosei; Nie, Shuyi; Tsygankov, Denis (June 2023, Cells)

   Rho-GTPases are central regulators within a complex signaling network that controls cytoskeletal organization and cell movement. The network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, along with their numerous effectors that provide mutual regulation through feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling, using a simulation model that couples GTPase signaling with cell morphodynamics and captures the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of time-lapsed recordings of cell dynamics and GTPase activity. Our data-driven modeling approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data. 

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