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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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Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1
The Cdc42 guanosine triphosphatase (GTPase) plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site is selected in the G1 phase. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet how Rsr1 regulates cell polarization is not fully understood. Here, we show that Rsr1-GDP interacts with the scaffold protein Bem1 in early G1, likely hindering the role of Bem1 in Cdc42 polarization and polarized secretion. Consistent with these in vivo observations, mathematical modeling predicts that Bem1 is unable to promote Cdc42 polarization in early G1 in the presence of Rsr1-GDP. We find that a part of the Bem1 Phox homology domain, which overlaps with a region interacting with the exocyst component Exo70, is necessary for the association of Bem1 with Rsr1-GDP. Overexpression of the GDP-locked Rsr1 interferes with Bem1-dependent Exo70 polarization. We thus propose that Rsr1 functions in spatial and temporal regulation of polarity establishment by associating with distinct polarity factors in its GTP- and GDP-bound states.
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- Award ID(s):
- 1813071
- PAR ID:
- 10148626
- Date Published:
- Journal Name:
- Molecular Biology of the Cell
- Volume:
- 30
- Issue:
- 20
- ISSN:
- 1059-1524
- Page Range / eLocation ID:
- 2543 to 2557
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Morphogenesis in fungi and animals is directed by polarization of small GTPases Cdc42 and Rac. In the budding yeastSaccharomyces cerevisiaecompetition between polarity patches results in one polarized patch and the growth of a single bud. Here, we describe cell polarity in the yeastAureobasidium pullulans, which establishes multiple coexisting polarity sites yielding multiple buds during a single cell division cycle. Polarity machinery components oscillate in their abundance in these coexisting sites but do so independently of one another, pointing to a lack of global coupling between sites. Previous theoretical work has demonstrated that negative feedback in a polarity circuit could promote coexistence of multiple polarity sites, and time-delayed negative feedback is known to cause oscillations. We show that both these features of negative feedback depend on a protein we identified as Pak1, and that Pak1 requires Rac1 but not Cdc42 for its localization. This work shows how conserved signaling networks can be modulated for distinct morphogenic programs even within the constraints of fungal budding.more » « less
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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.more » « less
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Cells polarize for growth, motion, or mating through regulation of membrane-bound small GTPases between active GTP-bound and inactive GDP-bound forms. Activators (GEFs, GTP exchange factors) and inhibitors (GAPs, GTPase activating proteins) provide positive and negative feedbacks. We show that a reaction–diffusion model on a curved surface accounts for key features of polarization of model organism fission yeast. The model implements Cdc42 membrane diffusion using measured values for diffusion coefficients and dissociation rates and assumes a limiting GEF pool (proteins Gef1 and Scd1), as in prior models for budding yeast. The model includes two types of GAPs, one representing tip-localized GAPs, such as Rga3; and one representing side-localized GAPs, such as Rga4 and Rga6, that we assume switch between fast and slow diffusing states. After adjustment of unknown rate constants, the model reproduces active Cdc42 zones at cell tips and the pattern of GEF and GAP localization at cell tips and sides. The model reproduces observed tip-to-tip oscillations with periods of the order of several minutes, as well as asymmetric to symmetric oscillations transitions (corresponding to NETO “new end take off”), assuming the limiting GEF amount increases with cell size.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1091/mbc.E19-02-0106
