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Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth.

Orlando K, Sun X, Zhang J, Lu T, Yokomizo L, Wang P, Guo W - Mol. Biol. Cell (2011)

Bottom Line: We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell.Defects in one of these pathways result in Cdc42p being rerouted through the other.A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19096 College of Life Sciences, Nankai University, Tianjin, 300071, China.

ABSTRACT
Cdc42p plays a central role in asymmetric cell growth in yeast by controlling actin organization and vesicular trafficking. However, how Cdc42p is maintained specifically at the daughter cell plasma membrane during asymmetric cell growth is unclear. We have analyzed Cdc42p localization in yeast mutants defective in various stages of membrane trafficking by fluorescence microscopy and biochemical fractionation. We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell. Defects in one of these pathways result in Cdc42p being rerouted through the other. In particular, the pathway involving trafficking through endosomes may couple Cdc42p endocytosis from, and subsequent redelivery to, the plasma membrane to maintain Cdc42p polarization at the daughter cell. Although the endo-exocytotic coupling is necessary for Cdc42p polarization, it is not sufficient to prevent the lateral diffusion of Cdc42p along the cell cortex. A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking. Collectively, membrane trafficking and septins function synergistically to maintain the dynamic polarization of Cdc42p during asymmetric growth in yeast.

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Cdc42p polarization in endocytosis mutants. (A) The wild-type, rvs161Δ, and end4-1 cells were grown to log phase, incubated at 37°C for 90 min, fixed, and immunostained for Cdc42p. Cdc42p is polarized in the endocytosis mutants. Scale bar = 10 μm. (B) Quantification of Cdc42p polarization in wild-type, rvs161Δ, and end4-1 cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. (C) FRAP curves of GFP-Cdc42p in wild-type and rvs161Δ cells. The normalized fluorescence intensity is plotted over time using SigmaPlot. (D) Recovery half-times for GFP-Cdc42p in wild-type and rvs161Δ cells. Bottom and top of the box are the lower and upper quartiles, respectively. The band near the middle of the box is the median. Whiskers represent standard errors. n = 25, p ≤ 0.01. (E) The sec5-24 and sec5-24 rvs161Δ cells were grown to early log phase, shifted to 35°C for 90 min, fixed, and immunostained for Cdc42p. Although mostly depolarized in the sec5-24 mutant, Cdc42p is well polarized in the sec5-24 rvs161Δ double mutant cells. (F) Quantification of Cdc42p polarization in the sec5-24 and sec5-24 rvs161Δ double mutant cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. *, p ≤ 0.01. (G) The sec5-24 rvs161Δ cells were transformed with GFP-Cdc42, grown to early log phase, and shifted to 35°C for 90 min. GFP-Cdc42p was then observed using fluorescence microscopy. GFP-Cdc42p is enriched at daughter cell plasma membrane.
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Figure 5: Cdc42p polarization in endocytosis mutants. (A) The wild-type, rvs161Δ, and end4-1 cells were grown to log phase, incubated at 37°C for 90 min, fixed, and immunostained for Cdc42p. Cdc42p is polarized in the endocytosis mutants. Scale bar = 10 μm. (B) Quantification of Cdc42p polarization in wild-type, rvs161Δ, and end4-1 cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. (C) FRAP curves of GFP-Cdc42p in wild-type and rvs161Δ cells. The normalized fluorescence intensity is plotted over time using SigmaPlot. (D) Recovery half-times for GFP-Cdc42p in wild-type and rvs161Δ cells. Bottom and top of the box are the lower and upper quartiles, respectively. The band near the middle of the box is the median. Whiskers represent standard errors. n = 25, p ≤ 0.01. (E) The sec5-24 and sec5-24 rvs161Δ cells were grown to early log phase, shifted to 35°C for 90 min, fixed, and immunostained for Cdc42p. Although mostly depolarized in the sec5-24 mutant, Cdc42p is well polarized in the sec5-24 rvs161Δ double mutant cells. (F) Quantification of Cdc42p polarization in the sec5-24 and sec5-24 rvs161Δ double mutant cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. *, p ≤ 0.01. (G) The sec5-24 rvs161Δ cells were transformed with GFP-Cdc42, grown to early log phase, and shifted to 35°C for 90 min. GFP-Cdc42p was then observed using fluorescence microscopy. GFP-Cdc42p is enriched at daughter cell plasma membrane.

Mentions: The association of Cdc42p with the invertase vesicles indicates that endosomal compartments are involved in Cdc42p trafficking. As such, it is likely that the Cdc42p on the plasma membrane is endocytosed as cargo into these endosomal compartments before being recycled back to the bud through another round of exocytosis. Indeed, previous studies using the F-actin disrupting drug latrunculin and Arp2/3 mutants have implicated endocytosis in Cdc42p polarization (Irazoqui et al., 2005; Marco et al., 2007). Also, for protein cargoes such as the v-SNARE protein Snc1p and the cell wall stress sensor Wsc1p, endocytosis was shown to be important for counteracting the lateral diffusion along the plasma membrane to maintain their polarized localization (Valdez-Taubas and Pelham, 2003; Piao et al., 2007). Here we directly examined the localization of Cdc42p in endocytosis mutants. Rvs161p is a yeast amphiphysin homolog implicated in the internalization step of endocytosis (Lombardi and Riezman, 2001). End4p (a.k.a. Sla2p) is a transmembrane protein that links actin to clathrin coats for endocytosis (Raths et al., 1993; Henry et al., 2002; Kaksonen et al., 2003). Surprisingly, we found that neither end4-1 nor rvs161Δ had an effect on Cdc42p polarization (Figure 5A, results quantified in Figure 5B). This is different from the observation on the transmembrane cargo Snc1p, in which endocytosis is sufficient to counteract its lateral dispersal (Valdez-Taubas and Pelham, 2003). The difference between Cdc42p and Snc1p is likely caused by their different diffusion rates, which is explicated later in the Discussion.


Exo-endocytic trafficking and the septin-based diffusion barrier are required for the maintenance of Cdc42p polarization during budding yeast asymmetric growth.

Orlando K, Sun X, Zhang J, Lu T, Yokomizo L, Wang P, Guo W - Mol. Biol. Cell (2011)

Cdc42p polarization in endocytosis mutants. (A) The wild-type, rvs161Δ, and end4-1 cells were grown to log phase, incubated at 37°C for 90 min, fixed, and immunostained for Cdc42p. Cdc42p is polarized in the endocytosis mutants. Scale bar = 10 μm. (B) Quantification of Cdc42p polarization in wild-type, rvs161Δ, and end4-1 cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. (C) FRAP curves of GFP-Cdc42p in wild-type and rvs161Δ cells. The normalized fluorescence intensity is plotted over time using SigmaPlot. (D) Recovery half-times for GFP-Cdc42p in wild-type and rvs161Δ cells. Bottom and top of the box are the lower and upper quartiles, respectively. The band near the middle of the box is the median. Whiskers represent standard errors. n = 25, p ≤ 0.01. (E) The sec5-24 and sec5-24 rvs161Δ cells were grown to early log phase, shifted to 35°C for 90 min, fixed, and immunostained for Cdc42p. Although mostly depolarized in the sec5-24 mutant, Cdc42p is well polarized in the sec5-24 rvs161Δ double mutant cells. (F) Quantification of Cdc42p polarization in the sec5-24 and sec5-24 rvs161Δ double mutant cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. *, p ≤ 0.01. (G) The sec5-24 rvs161Δ cells were transformed with GFP-Cdc42, grown to early log phase, and shifted to 35°C for 90 min. GFP-Cdc42p was then observed using fluorescence microscopy. GFP-Cdc42p is enriched at daughter cell plasma membrane.
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Figure 5: Cdc42p polarization in endocytosis mutants. (A) The wild-type, rvs161Δ, and end4-1 cells were grown to log phase, incubated at 37°C for 90 min, fixed, and immunostained for Cdc42p. Cdc42p is polarized in the endocytosis mutants. Scale bar = 10 μm. (B) Quantification of Cdc42p polarization in wild-type, rvs161Δ, and end4-1 cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. (C) FRAP curves of GFP-Cdc42p in wild-type and rvs161Δ cells. The normalized fluorescence intensity is plotted over time using SigmaPlot. (D) Recovery half-times for GFP-Cdc42p in wild-type and rvs161Δ cells. Bottom and top of the box are the lower and upper quartiles, respectively. The band near the middle of the box is the median. Whiskers represent standard errors. n = 25, p ≤ 0.01. (E) The sec5-24 and sec5-24 rvs161Δ cells were grown to early log phase, shifted to 35°C for 90 min, fixed, and immunostained for Cdc42p. Although mostly depolarized in the sec5-24 mutant, Cdc42p is well polarized in the sec5-24 rvs161Δ double mutant cells. (F) Quantification of Cdc42p polarization in the sec5-24 and sec5-24 rvs161Δ double mutant cells. Fifty cells were counted for each group (n = 3). Error bars represent standard error. *, p ≤ 0.01. (G) The sec5-24 rvs161Δ cells were transformed with GFP-Cdc42, grown to early log phase, and shifted to 35°C for 90 min. GFP-Cdc42p was then observed using fluorescence microscopy. GFP-Cdc42p is enriched at daughter cell plasma membrane.
Mentions: The association of Cdc42p with the invertase vesicles indicates that endosomal compartments are involved in Cdc42p trafficking. As such, it is likely that the Cdc42p on the plasma membrane is endocytosed as cargo into these endosomal compartments before being recycled back to the bud through another round of exocytosis. Indeed, previous studies using the F-actin disrupting drug latrunculin and Arp2/3 mutants have implicated endocytosis in Cdc42p polarization (Irazoqui et al., 2005; Marco et al., 2007). Also, for protein cargoes such as the v-SNARE protein Snc1p and the cell wall stress sensor Wsc1p, endocytosis was shown to be important for counteracting the lateral diffusion along the plasma membrane to maintain their polarized localization (Valdez-Taubas and Pelham, 2003; Piao et al., 2007). Here we directly examined the localization of Cdc42p in endocytosis mutants. Rvs161p is a yeast amphiphysin homolog implicated in the internalization step of endocytosis (Lombardi and Riezman, 2001). End4p (a.k.a. Sla2p) is a transmembrane protein that links actin to clathrin coats for endocytosis (Raths et al., 1993; Henry et al., 2002; Kaksonen et al., 2003). Surprisingly, we found that neither end4-1 nor rvs161Δ had an effect on Cdc42p polarization (Figure 5A, results quantified in Figure 5B). This is different from the observation on the transmembrane cargo Snc1p, in which endocytosis is sufficient to counteract its lateral dispersal (Valdez-Taubas and Pelham, 2003). The difference between Cdc42p and Snc1p is likely caused by their different diffusion rates, which is explicated later in the Discussion.

Bottom Line: We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell.Defects in one of these pathways result in Cdc42p being rerouted through the other.A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19096 College of Life Sciences, Nankai University, Tianjin, 300071, China.

ABSTRACT
Cdc42p plays a central role in asymmetric cell growth in yeast by controlling actin organization and vesicular trafficking. However, how Cdc42p is maintained specifically at the daughter cell plasma membrane during asymmetric cell growth is unclear. We have analyzed Cdc42p localization in yeast mutants defective in various stages of membrane trafficking by fluorescence microscopy and biochemical fractionation. We found that two separate exocytic pathways mediate Cdc42p delivery to the daughter cell. Defects in one of these pathways result in Cdc42p being rerouted through the other. In particular, the pathway involving trafficking through endosomes may couple Cdc42p endocytosis from, and subsequent redelivery to, the plasma membrane to maintain Cdc42p polarization at the daughter cell. Although the endo-exocytotic coupling is necessary for Cdc42p polarization, it is not sufficient to prevent the lateral diffusion of Cdc42p along the cell cortex. A barrier function conferred by septins is required to counteract the dispersal of Cdc42p and maintain its localization in the daughter cell but has no effect on the initial polarization of Cdc42p at the presumptive budding site before symmetry breaking. Collectively, membrane trafficking and septins function synergistically to maintain the dynamic polarization of Cdc42p during asymmetric growth in yeast.

Show MeSH
Related in: MedlinePlus