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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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Stress-induced depolarization of WSC1 distribution is independent of a depolarized actin cytoskeleton, yet WSC1 localization is controlled by the actin cytoskeleton. Wild-type (I–X, JK9-3da) or rom2 (A–H, AS138-1b) cells expressing WSC1-HA (pWSC1-HA) were grown in minimal medium at 24°C, shifted to 37°C for 35 min or maintained at 24°C, fixed, and processed for detection of WSC1-HA by immunofluorescence (see Materials and Methods). In cells grown continuously at 24°C, LAT-A was added 35 min before fixation (Q–T). In cells shifted to 37°C, LAT-A was added at the time of shift (U–X). Fluorescence (A, C, E, G, I, K, M, O, Q, S, U, and W) and Nomarski (B, D, F, H, J, L, N, P, R, T, V, and X) images are shown.
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Figure 5: Stress-induced depolarization of WSC1 distribution is independent of a depolarized actin cytoskeleton, yet WSC1 localization is controlled by the actin cytoskeleton. Wild-type (I–X, JK9-3da) or rom2 (A–H, AS138-1b) cells expressing WSC1-HA (pWSC1-HA) were grown in minimal medium at 24°C, shifted to 37°C for 35 min or maintained at 24°C, fixed, and processed for detection of WSC1-HA by immunofluorescence (see Materials and Methods). In cells grown continuously at 24°C, LAT-A was added 35 min before fixation (Q–T). In cells shifted to 37°C, LAT-A was added at the time of shift (U–X). Fluorescence (A, C, E, G, I, K, M, O, Q, S, U, and W) and Nomarski (B, D, F, H, J, L, N, P, R, T, V, and X) images are shown.

Mentions: To determine if cell wall stress affects localization of the plasma membrane protein WSC1, we constructed a COOH terminally, 3×HA-tagged, functional WSC1 (pWSC1-HA) (see Materials and Methods). Visualization of WSC1 by indirect immunofluorescence revealed a polarized distribution similar to that of FKS1 and other proteins found at a growth (bud) site (Fig. 5). Surprisingly, WSC1 had previously been reported to be evenly distributed throughout the cell periphery (Verna et al. 1997). This discrepancy may be because of the earlier study using overexpressed WSC1, whereas we examined WSC1 expressed from its own promoter on a centromeric plasmid. To examine WSC1 localization in response to stress, growing wild-type cells containing pWSC1-HA were shifted from 24 to 37°C, incubated at the higher temperature for 35 and 120 min, and processed for visualization of WSC1 (WSC1-HA). After 35 min, WSC1 was dispersed throughout the cell periphery (Fig. 5). After 120 min at 37°C, WSC1 was repolarized (data not shown). Thus, like the actin cytoskeleton and FKS1, WSC1 displayed a transient depolarization upon heat shock. Interestingly, WSC1 was also found in internal compartments at both low and high temperatures.


Cell wall stress depolarizes cell growth via hyperactivation of RHO1.

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

Stress-induced depolarization of WSC1 distribution is independent of a depolarized actin cytoskeleton, yet WSC1 localization is controlled by the actin cytoskeleton. Wild-type (I–X, JK9-3da) or rom2 (A–H, AS138-1b) cells expressing WSC1-HA (pWSC1-HA) were grown in minimal medium at 24°C, shifted to 37°C for 35 min or maintained at 24°C, fixed, and processed for detection of WSC1-HA by immunofluorescence (see Materials and Methods). In cells grown continuously at 24°C, LAT-A was added 35 min before fixation (Q–T). In cells shifted to 37°C, LAT-A was added at the time of shift (U–X). Fluorescence (A, C, E, G, I, K, M, O, Q, S, U, and W) and Nomarski (B, D, F, H, J, L, N, P, R, T, V, and X) images are shown.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164985&req=5

Figure 5: Stress-induced depolarization of WSC1 distribution is independent of a depolarized actin cytoskeleton, yet WSC1 localization is controlled by the actin cytoskeleton. Wild-type (I–X, JK9-3da) or rom2 (A–H, AS138-1b) cells expressing WSC1-HA (pWSC1-HA) were grown in minimal medium at 24°C, shifted to 37°C for 35 min or maintained at 24°C, fixed, and processed for detection of WSC1-HA by immunofluorescence (see Materials and Methods). In cells grown continuously at 24°C, LAT-A was added 35 min before fixation (Q–T). In cells shifted to 37°C, LAT-A was added at the time of shift (U–X). Fluorescence (A, C, E, G, I, K, M, O, Q, S, U, and W) and Nomarski (B, D, F, H, J, L, N, P, R, T, V, and X) images are shown.
Mentions: To determine if cell wall stress affects localization of the plasma membrane protein WSC1, we constructed a COOH terminally, 3×HA-tagged, functional WSC1 (pWSC1-HA) (see Materials and Methods). Visualization of WSC1 by indirect immunofluorescence revealed a polarized distribution similar to that of FKS1 and other proteins found at a growth (bud) site (Fig. 5). Surprisingly, WSC1 had previously been reported to be evenly distributed throughout the cell periphery (Verna et al. 1997). This discrepancy may be because of the earlier study using overexpressed WSC1, whereas we examined WSC1 expressed from its own promoter on a centromeric plasmid. To examine WSC1 localization in response to stress, growing wild-type cells containing pWSC1-HA were shifted from 24 to 37°C, incubated at the higher temperature for 35 and 120 min, and processed for visualization of WSC1 (WSC1-HA). After 35 min, WSC1 was dispersed throughout the cell periphery (Fig. 5). After 120 min at 37°C, WSC1 was repolarized (data not shown). Thus, like the actin cytoskeleton and FKS1, WSC1 displayed a transient depolarization upon heat shock. Interestingly, WSC1 was also found in internal compartments at both low and high temperatures.

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