Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor

Okada, Satoshi; Lee, Mid Eum; Bi, Erfei; Park, Hay-Oak

doi:10.1016/bs.mie.2017.01.011

Cited by 17 publications

(24 citation statements)

References 29 publications

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“…Average relative recovery of PBD is shown below each lane. The WT control lanes have also been used for validation of the PBD-RFP biosensor ( Okada et al , 2017 ). (B) Cdc42 D76A interacts with Rsr1 equally well as WT Cdc42.…”

Section: Resultsmentioning

confidence: 99%

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The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization

Kang

Miller

Guegueniat

et al. 2018

MBoC

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The Cdc42 GTPase plays a central role in polarity development in many species. In budding yeast, Cdc42 is essential for polarized growth at the proper site and also for spontaneous cell polarization in the absence of spatial cues. Cdc42 polarization is critical for multiple events in the G1 phase prior to bud emergence, including bud-site assembly, polarization of the actin cytoskeleton, and septin filament assembly to form a ring at the new bud site. Yet the mechanism by which Cdc42 polarizes is not fully understood. Here we report that biphasic Cdc42 polarization in the G1 phase is coupled to stepwise assembly of the septin ring for bud emergence. We show that the Rsr1 GTPase shares a partially redundant role with Gic1 and Gic2, two related Cdc42 effectors, in the first phase of Cdc42 polarization in haploid cells. We propose that the first phase of Cdc42 polarization is mediated by positive feedback loops that function in parallel—one involving Rsr1 via local activation of Cdc42 in response to spatial cues and another involving Gic1 or Gic2 via reduction of diffusion of active Cdc42.

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Section: Resultsmentioning

confidence: 99%

“…To quantify Cdc42 polarization, the fluorescence intensity of PBD-RFP clusters was measured by a threshold method using an ImageJ macro ( Okada et al , 2013 ; Okada et al , 2017 ). Briefly, mean projections were generated from five best z-sections at each time point, and then a threshold method was used after background subtraction.…”

Section: Methodsmentioning

confidence: 99%

The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization

Kang

Miller

Guegueniat

et al. 2018

MBoC

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“…Gic2p-PBD-tdTomato clustering was quantified as described in Okada et al (2017) with the following modifications. Raw fluorescence and DIC images were imported in ImageJ.…”

Section: Image Analysismentioning

confidence: 99%

Regulation of intrinsic polarity establishment by a differentiation-type MAPK pathway in S. cerevisiae

Prabhakar

Chow

Siegel

et al. 2020

Journal of Cell Science

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All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in the yeast Saccharomyces cerevisiae, which is controlled by the Rho GTPase Cdc42p. The prevailing view of bud emergence does not account for regulation by extrinsic cues. Here, we show that the filamentous growth mitogen activated protein kinase (fMAPK) pathway regulates bud emergence under nutrient-limiting conditions. The fMAPK pathway regulated the expression of polarity targets including the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and active during the period of the cell cycle leading up to bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

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“…Because there was some difficulty visualizing bud scars within a bud scar from the static images, we took a complementary approach to determine the role of Rga1 at the old division sites. We monitored Cdc42 polarization in rax1∆ cells by time-lapse imaging using the p21-binding domain of Gic2 fused to tdTomato (PBD-RFP), a biosensor for active Cdc42, which specifically interacts with Cdc42-GTP ( Ozbudak et al ., 2005 ; Tong et al ., 2007 ; Okada et al., 2017 ). The transcriptional repressor Whi5 fused to GFP was used as a cell-cycle marker, since its nuclear exit divides the G1 phase into two temporal steps, T 1 and T 2 ( Di Talia et al ., 2007 ).…”

Section: Resultsmentioning

confidence: 99%

Fine-tuning the orientation of the polarity axis by Rga1, a Cdc42 GTPase-activating protein

Miller¹,

Lee³

et al. 2017

MBoC

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MBoC | ARTICLE Fine-tuning the orientation of the polarity axis by Rga1, a Cdc42 GTPase-activating protein ABSTRACT In yeast and animal cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate cell polarization. In budding yeast, selection of a bud site directs polarity establishment and subsequently determines the plane of cell division. Rga1, a Cdc42 GTPase-activating protein, prevents budding within the division site by inhibiting Cdc42 repolarization. A protein complex including Nba1 and Nis1 is involved in preventing rebudding at old division sites, yet how these proteins and Rga1 might function in negative polarity signaling has been elusive. Here we show that Rga1 transiently localizes to the immediately preceding and older division sites by interacting with Nba1 and Nis1. The LIM domains of Rga1 are necessary for its interaction with Nba1, and loss of this interaction results in premature delocalization of Rga1 from the immediately preceding division site and, consequently, abnormal bud-site selection in daughter cells. However, such defects are minor in mother cells of these mutants, likely because the G1 phase is shorter and a new bud site is established prior to delocalization of Rga1. Indeed, our biphasic mathematical model of Cdc42 polarization predicts that premature delocalization of Rga1 leads to more frequent Cdc42 repolarization within the division site when the first temporal step in G1 is assumed to last longer. Spatial distribution of a Cdc42 GAP in coordination with G1 progression may thus be critical for fine-tuning the orientation of the polarity axis in yeast.

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Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor

Cited by 17 publications

References 29 publications

The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization

The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization

Regulation of intrinsic polarity establishment by a differentiation-type MAPK pathway in S. cerevisiae

Fine-tuning the orientation of the polarity axis by Rga1, a Cdc42 GTPase-activating protein

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