Limits...
Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ - J. Cell Biol. (2001)

Bottom Line: The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells.Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis.Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Show MeSH

Related in: MedlinePlus

Localization of Exo70-GFP and Sec8-GFP in CDC42 and cdc42-6 strains. COOH terminally tagged GFP constructs were transformed into wild-type and cdc42-6 cells and examined by fluorescence microscopy. The results for wild-type (A and B) and cdc42-6 cells following a 1-h shift at 33°C (C and D) demonstrate that the polarization of the exocyst complex was unaffected in cdc42-6 cells. DIC and GFP fluorescence images were captured immediately following temperature shift. Similar were results were obtained by addition of formaldehyde directly to the media at the end of the temperature shift. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198861&req=5

fig9: Localization of Exo70-GFP and Sec8-GFP in CDC42 and cdc42-6 strains. COOH terminally tagged GFP constructs were transformed into wild-type and cdc42-6 cells and examined by fluorescence microscopy. The results for wild-type (A and B) and cdc42-6 cells following a 1-h shift at 33°C (C and D) demonstrate that the polarization of the exocyst complex was unaffected in cdc42-6 cells. DIC and GFP fluorescence images were captured immediately following temperature shift. Similar were results were obtained by addition of formaldehyde directly to the media at the end of the temperature shift. Bar, 5 μm.

Mentions: One plausible explanation for the secretory defect of cdc42-6 cells would be a failure to localize the exocyst complex to the sites of exocytosis. However, using functional Sec8-GFP and Exo70-GFP constructs, we found that in living cells these exocyst components are correctly localized in cdc42-6 cells at the restrictive temperature. Sec3-GFP constructs were also tested and showed similar results for wild-type and mutant strains at all temperatures, but are not shown here because a low signal associated with the particular constructs resulted in low percentage of cells (20–30%) with polarized staining. In a wild-type population of cells grown at either permissive or restrictive temperature, ∼70% of small-budded cells showed polarized Sec8-GFP, and 51% showed polarized Exo70-GFP (Fig. 9 A and B). In cdc42-6 cells grown for 1 h at the restrictive temperature, 65% of small-budded cells showed polarized Sec8-GFP and 56% showed polarized Exo70-GFP (Fig. 9, C and D). Therefore, we conclude that the cdc42-6 mutant is not defective for the localization of the exocyst complex.


Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ - J. Cell Biol. (2001)

Localization of Exo70-GFP and Sec8-GFP in CDC42 and cdc42-6 strains. COOH terminally tagged GFP constructs were transformed into wild-type and cdc42-6 cells and examined by fluorescence microscopy. The results for wild-type (A and B) and cdc42-6 cells following a 1-h shift at 33°C (C and D) demonstrate that the polarization of the exocyst complex was unaffected in cdc42-6 cells. DIC and GFP fluorescence images were captured immediately following temperature shift. Similar were results were obtained by addition of formaldehyde directly to the media at the end of the temperature shift. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198861&req=5

fig9: Localization of Exo70-GFP and Sec8-GFP in CDC42 and cdc42-6 strains. COOH terminally tagged GFP constructs were transformed into wild-type and cdc42-6 cells and examined by fluorescence microscopy. The results for wild-type (A and B) and cdc42-6 cells following a 1-h shift at 33°C (C and D) demonstrate that the polarization of the exocyst complex was unaffected in cdc42-6 cells. DIC and GFP fluorescence images were captured immediately following temperature shift. Similar were results were obtained by addition of formaldehyde directly to the media at the end of the temperature shift. Bar, 5 μm.
Mentions: One plausible explanation for the secretory defect of cdc42-6 cells would be a failure to localize the exocyst complex to the sites of exocytosis. However, using functional Sec8-GFP and Exo70-GFP constructs, we found that in living cells these exocyst components are correctly localized in cdc42-6 cells at the restrictive temperature. Sec3-GFP constructs were also tested and showed similar results for wild-type and mutant strains at all temperatures, but are not shown here because a low signal associated with the particular constructs resulted in low percentage of cells (20–30%) with polarized staining. In a wild-type population of cells grown at either permissive or restrictive temperature, ∼70% of small-budded cells showed polarized Sec8-GFP, and 51% showed polarized Exo70-GFP (Fig. 9 A and B). In cdc42-6 cells grown for 1 h at the restrictive temperature, 65% of small-budded cells showed polarized Sec8-GFP and 56% showed polarized Exo70-GFP (Fig. 9, C and D). Therefore, we conclude that the cdc42-6 mutant is not defective for the localization of the exocyst complex.

Bottom Line: The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells.Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis.Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Show MeSH
Related in: MedlinePlus