Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast

Lee, Mid Eum; Lo, Wing-Cheong; Miller, Kristi E.; Chou, Ching-Shan; Park, Hay-Oak

doi:10.1242/jcs.166538

Cited by 25 publications

(62 citation statements)

References 56 publications

(85 reference statements)

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“…The highest Cdc42-GTP peak appeared at multiple positions near the septin ring in daughter cells until the Cdc42-GTP polar cap became stable at the boundary of T 1 and T 2 ( i.e ., right before the new septin clouds appear at the new bud site) (Fig. 4; See details in Lee et al, 2015). Thus, the Cdc42 biosensor represents a powerful method for uncovering novel Cdc42 activation dynamics and mechanisms underlying cell polarization.…”

Section: Application Of the Cdc42 Biosensormentioning

confidence: 97%

“…2–5)(Ozbudak et al 2005; Tong, Gao, Howell, Bose, Lew & Bi 2007; Lo et al 2013; Okada et al 2013; Kang et al. 2014; Lee et al 2015). …”

Section: Generation Of a Biosensor Of Active Cdc42mentioning

confidence: 99%

“…Alternatively, poly-lysine-coated glass-bottom dishes can be used. Mounting cells in the dish often improves the spatial resolution of a ring structure at the mother-bud neck (such as the septin ring) by positioning yeast cells vertically (i.e., the mother-bud axis is parallel to the direction of capturing Z-stacks)(Lee et al 2015).

Apply 10 µL of ConA solution into a dish and spread it with the side of a pipet tip, and air dry for about 20 min in a box to prevent dust.

Rinse the dish twice with water.

Place 100 µL of cell suspension and incubate for 20 min.

Wash three times with synthetic complete medium to remove cells that are not stuck to the glass bottom.

Place a thin layer of 0.6% agarose containing media (cooled down after melting).

For long-term imaging, place 500µL of liquid medium over the solidified agarose layer.

…”

Section: Detection and Quantification Of Cdc42 Activation In Vivomentioning

confidence: 99%

“…The Cdc42 effector Gic2 contains a conserved CRIB motif, also known as PBD (p21-binding domain), which binds specifically to Cdc42-GTP, and localizes to the sites of polarized growth in a Cdc42-dependent manner (Jaquenoud & Peter 2000). A fragment of Gic2 1–208 containing the PBD has been tagged with fluorescent molecules and used for monitoring Cdc42-GTP in live cells (Ozbudak, Becskei & van Oudenaarden 2005; Tong, Gao, Howell, Bose, Lew & Bi 2007; Lo, Lee, Narayan, Chou & Park 2013; Okada, Leda, Hanna, Savage, Bi & Goryachev 2013; Kang, Lee & Park 2014; Lee, Lo, Miller, Chou & Park 2015). By using this affinity-based biosensor, we can monitor dynamics of Cdc42 activation in specific processes in vivo .…”

Section: Introductionmentioning

confidence: 99%

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Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor

Okada

Lee

et al. 2017

Methods in Enzymology

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Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding (CRIB) motif from one of the Cdc42 effector proteins. Because it binds specifically to the GTP-bound Cdc42, this biosensor can be used to monitor Cdc42 activation in vivo. This or similar biosensors can be widely used for studying GTPase signaling in other cell types because of the conserved CRIB motif present among GTPase targets.

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Section: Application Of the Cdc42 Biosensormentioning

confidence: 97%

“…2–5)(Ozbudak et al 2005; Tong, Gao, Howell, Bose, Lew & Bi 2007; Lo et al 2013; Okada et al 2013; Kang et al. 2014; Lee et al 2015). …”

Section: Generation Of a Biosensor Of Active Cdc42mentioning

confidence: 99%

Apply 10 µL of ConA solution into a dish and spread it with the side of a pipet tip, and air dry for about 20 min in a box to prevent dust.

Rinse the dish twice with water.

Place 100 µL of cell suspension and incubate for 20 min.

Wash three times with synthetic complete medium to remove cells that are not stuck to the glass bottom.

Place a thin layer of 0.6% agarose containing media (cooled down after melting).

For long-term imaging, place 500µL of liquid medium over the solidified agarose layer.

…”

Section: Detection and Quantification Of Cdc42 Activation In Vivomentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor

Okada

Lee

et al. 2017

Methods in Enzymology

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“…A very similar model of this class with Bem1-mediated nonlinear feedback [GEF] ~ RT 2 has been proposed by Park, Chou, and colleagues (Lo et al. , 2013; Lee et al. , 2015).…”

Section: Six Mechanistic Metamodels Of Cdc42 Polarizationmentioning

confidence: 99%

Many roads to symmetry breaking: molecular mechanisms and theoretical models of yeast cell polarity

Goryachev

Leda

2017

MBoC

116

111

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Mathematical modeling has been instrumental in identifying common principles of cell polarity across diverse systems. These principles include positive feedback loops that are required to destabilize a spatially uniform state of the cell. The conserved small G-protein Cdc42 is a master regulator of eukaryotic cellular polarization. Here we discuss recent developments in studies of Cdc42 polarization in budding and fission yeasts and demonstrate that models describing symmetry-breaking polarization can be classified into six minimal classes based on the structure of positive feedback loops that activate and localize Cdc42. Owing to their generic system-independent nature, these model classes are also likely to be relevant for the G-protein–based symmetry-breaking systems of higher eukaryotes. We review experimental evidence pro et contra different theoretically plausible models and conclude that several parallel and non–mutually exclusive mechanisms are likely involved in cellular polarization of yeasts. This potential redundancy needs to be taken into consideration when interpreting the results of recent cell-rewiring studies.

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Spontaneous cell polarization: Feedback control of Cdc42 GTPase breaks cellular symmetry

Martin

2015

BioEssays

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Spontaneous polarization without spatial cues, or symmetry breaking, is a fundamental problem of spatial organization in biological systems. This question has been extensively studied using yeast models, which revealed the central role of the small GTPase switch Cdc42. Active Cdc42‐GTP forms a coherent patch at the cell cortex, thought to result from amplification of a small initial stochastic inhomogeneity through positive feedback mechanisms, which induces cell polarization. Here, I review and discuss the mechanisms of Cdc42 activity self‐amplification and dynamic turnover. A robust Cdc42 patch is formed through the combined effects of Cdc42 activity promoting its own activation and active Cdc42‐GTP displaying reduced membrane detachment and lateral diffusion compared to inactive Cdc42‐GDP. I argue the role of the actin cytoskeleton in symmetry breaking is not primarily to transport Cdc42 to the active site. Finally, negative feedback and competition mechanisms serve to control the number of polarization sites.

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Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast

Cited by 25 publications

References 56 publications

Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor

Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor

Many roads to symmetry breaking: molecular mechanisms and theoretical models of yeast cell polarity

Spontaneous cell polarization: Feedback control of Cdc42 GTPase breaks cellular symmetry

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