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Cell wall stress depolarizes cell growth via hyperactivation of RHO1.

Delley PA, Hall MN - J. Cell Biol. (1999)

Bottom Line: Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch.The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1.Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
Cells sense and physiologically respond to environmental stress via signaling pathways. Saccharomyces cerevisiae cells respond to cell wall stress by transiently depolarizing the actin cytoskeleton. We report that cell wall stress also induces a transient depolarized distribution of the cell wall biosynthetic enzyme glucan synthase FKS1 and its regulatory subunit RHO1, possibly as a mechanism to repair general cell wall damage. The redistribution of FKS1 is dependent on the actin cytoskeleton. Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch. WSC1 behaves like a signal transducer or a stress-specific actin landmark that both controls and responds to the actin cytoskeleton, similar to the bidirectional signaling between integrin receptors and the actin cytoskeleton in mammalian cells. The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1. Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch.

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Hyperactivated RHO1 or PKC1 is sufficient to induce actin depolarization. Wild-type (JK9-3da) cells carrying an empty vector (A–D), pGAL-RHO1* (E–H), pGAL-PKC1* (I–L), pGAL-BCK1* (M–P), or pGAL-MKK1* (Q–T) were grown at 30°C in minimal medium containing raffinose. Galactose was added to a final concentration of 2% and strains were further incubated for 2.5 h (empty vector, pGAL-RHO1* or pGAL-PKC1*) or 5 h (pGAL-BCK1* or pGAL-MKK1*). Cells containing pGAL-BCK1* or pGAL-MKK1* gave the same result when incubated in galactose for 2.5 or 5 h. Samples were fixed, stained with TRITC-phalloidin, and observed by fluorescence (A, C, E, G, I, K, M, O, Q, and S) and Nomarski (B, D, F, H, J, L, N, P, R, and T) microscopy to visualize the actin cytoskeleton and whole cells, respectively.
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Figure 3: Hyperactivated RHO1 or PKC1 is sufficient to induce actin depolarization. Wild-type (JK9-3da) cells carrying an empty vector (A–D), pGAL-RHO1* (E–H), pGAL-PKC1* (I–L), pGAL-BCK1* (M–P), or pGAL-MKK1* (Q–T) were grown at 30°C in minimal medium containing raffinose. Galactose was added to a final concentration of 2% and strains were further incubated for 2.5 h (empty vector, pGAL-RHO1* or pGAL-PKC1*) or 5 h (pGAL-BCK1* or pGAL-MKK1*). Cells containing pGAL-BCK1* or pGAL-MKK1* gave the same result when incubated in galactose for 2.5 or 5 h. Samples were fixed, stained with TRITC-phalloidin, and observed by fluorescence (A, C, E, G, I, K, M, O, Q, and S) and Nomarski (B, D, F, H, J, L, N, P, R, and T) microscopy to visualize the actin cytoskeleton and whole cells, respectively.

Mentions: The previous observation that cell wall defects increase ROM2 exchange activity toward RHO1 (Bickle et al. 1998) and the above finding that a wsc1 or rom2 mutation reduces stress-induced depolarization suggest that the depolarization process requires RHO1 hyperactivation. To examine this further, we investigated the effect of overexpressing constitutively activated RHO1 (RHO1*) on the actin cytoskeleton and FKS1 localization. As RHO1* is toxic, we constructed a plasmid-borne RHO1* allele under control of the strong and galactose-inducible GAL1 promoter (pGAL-RHO1*). This construct was not toxic in cells grown on a carbon source other than galactose (data not shown). Cells containing pGAL-RHO1* were grown to logarithmic phase in medium containing raffinose as a carbon source, and galactose was added to a final concentration of 2%. Aliquots of cells were removed at different time points after addition of galactose and processed for visualization of the actin cytoskeleton. In raffinose-containing medium, cells displayed a normal vegetative distribution of actin patches. After 1.5 h in galactose, the number of patches in the mother cell increased (data not shown). After 2.5 h in galactose, the actin cytoskeleton was depolarized in essentially all cells (Fig. 3). FKS1 was also depolarized, with similar kinetics and severity as observed for the actin cytoskeleton (data not shown). Expression of wild-type RHO1 from the GAL1 promoter (pGAL-RHO1) had no depolarizing effect (data not shown). Thus, RHO1 hyperactivation is sufficient to induce actin and FKS1 depolarization. This finding, combined with the previous findings that cell wall stress hyperactivates ROM2 exchange activity toward RHO1 (Bickle et al. 1998) and that a rom2 mutation blocks depolarization, suggests that depolarization results from RHO1 hyperactivation.


Cell wall stress depolarizes cell growth via hyperactivation of RHO1.

Delley PA, Hall MN - J. Cell Biol. (1999)

Hyperactivated RHO1 or PKC1 is sufficient to induce actin depolarization. Wild-type (JK9-3da) cells carrying an empty vector (A–D), pGAL-RHO1* (E–H), pGAL-PKC1* (I–L), pGAL-BCK1* (M–P), or pGAL-MKK1* (Q–T) were grown at 30°C in minimal medium containing raffinose. Galactose was added to a final concentration of 2% and strains were further incubated for 2.5 h (empty vector, pGAL-RHO1* or pGAL-PKC1*) or 5 h (pGAL-BCK1* or pGAL-MKK1*). Cells containing pGAL-BCK1* or pGAL-MKK1* gave the same result when incubated in galactose for 2.5 or 5 h. Samples were fixed, stained with TRITC-phalloidin, and observed by fluorescence (A, C, E, G, I, K, M, O, Q, and S) and Nomarski (B, D, F, H, J, L, N, P, R, and T) microscopy to visualize the actin cytoskeleton and whole cells, respectively.
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Figure 3: Hyperactivated RHO1 or PKC1 is sufficient to induce actin depolarization. Wild-type (JK9-3da) cells carrying an empty vector (A–D), pGAL-RHO1* (E–H), pGAL-PKC1* (I–L), pGAL-BCK1* (M–P), or pGAL-MKK1* (Q–T) were grown at 30°C in minimal medium containing raffinose. Galactose was added to a final concentration of 2% and strains were further incubated for 2.5 h (empty vector, pGAL-RHO1* or pGAL-PKC1*) or 5 h (pGAL-BCK1* or pGAL-MKK1*). Cells containing pGAL-BCK1* or pGAL-MKK1* gave the same result when incubated in galactose for 2.5 or 5 h. Samples were fixed, stained with TRITC-phalloidin, and observed by fluorescence (A, C, E, G, I, K, M, O, Q, and S) and Nomarski (B, D, F, H, J, L, N, P, R, and T) microscopy to visualize the actin cytoskeleton and whole cells, respectively.
Mentions: The previous observation that cell wall defects increase ROM2 exchange activity toward RHO1 (Bickle et al. 1998) and the above finding that a wsc1 or rom2 mutation reduces stress-induced depolarization suggest that the depolarization process requires RHO1 hyperactivation. To examine this further, we investigated the effect of overexpressing constitutively activated RHO1 (RHO1*) on the actin cytoskeleton and FKS1 localization. As RHO1* is toxic, we constructed a plasmid-borne RHO1* allele under control of the strong and galactose-inducible GAL1 promoter (pGAL-RHO1*). This construct was not toxic in cells grown on a carbon source other than galactose (data not shown). Cells containing pGAL-RHO1* were grown to logarithmic phase in medium containing raffinose as a carbon source, and galactose was added to a final concentration of 2%. Aliquots of cells were removed at different time points after addition of galactose and processed for visualization of the actin cytoskeleton. In raffinose-containing medium, cells displayed a normal vegetative distribution of actin patches. After 1.5 h in galactose, the number of patches in the mother cell increased (data not shown). After 2.5 h in galactose, the actin cytoskeleton was depolarized in essentially all cells (Fig. 3). FKS1 was also depolarized, with similar kinetics and severity as observed for the actin cytoskeleton (data not shown). Expression of wild-type RHO1 from the GAL1 promoter (pGAL-RHO1) had no depolarizing effect (data not shown). Thus, RHO1 hyperactivation is sufficient to induce actin and FKS1 depolarization. This finding, combined with the previous findings that cell wall stress hyperactivates ROM2 exchange activity toward RHO1 (Bickle et al. 1998) and that a rom2 mutation blocks depolarization, suggests that depolarization results from RHO1 hyperactivation.

Bottom Line: Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch.The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1.Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
Cells sense and physiologically respond to environmental stress via signaling pathways. Saccharomyces cerevisiae cells respond to cell wall stress by transiently depolarizing the actin cytoskeleton. We report that cell wall stress also induces a transient depolarized distribution of the cell wall biosynthetic enzyme glucan synthase FKS1 and its regulatory subunit RHO1, possibly as a mechanism to repair general cell wall damage. The redistribution of FKS1 is dependent on the actin cytoskeleton. Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch. WSC1 behaves like a signal transducer or a stress-specific actin landmark that both controls and responds to the actin cytoskeleton, similar to the bidirectional signaling between integrin receptors and the actin cytoskeleton in mammalian cells. The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1. Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch.

Show MeSH
Related in: MedlinePlus