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A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling.

Fröhlich F, Moreira K, Aguilar PS, Hubner NC, Mann M, Walter P, Walther TC - J. Cell Biol. (2009)

Bottom Line: The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids.Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization.Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

View Article: PubMed Central - PubMed

Affiliation: Organelle Architecture and Dynamics, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

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NCE102 is required for normal plasma membrane organization. (a and b) Sur7-GFP (a) and Pma1-GFP (b) were expressed in wild-type (wt) or Δnce102 cells, and representative confocal top (bottom) and mid sections (top) are shown. (c) Wild-type and Δnce102 cells expressing Pil1-GFP (green) were pulse labeled with FM4-64 (red) for 1 min, and representative images are shown. Boxes indicate the area magnified in the bottom panels. (d) Numbers of FM4-64 intermediates per cell (n > 50) from images as in c are shown as a histogram. Bars, 5 µm.
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fig2: NCE102 is required for normal plasma membrane organization. (a and b) Sur7-GFP (a) and Pma1-GFP (b) were expressed in wild-type (wt) or Δnce102 cells, and representative confocal top (bottom) and mid sections (top) are shown. (c) Wild-type and Δnce102 cells expressing Pil1-GFP (green) were pulse labeled with FM4-64 (red) for 1 min, and representative images are shown. Boxes indicate the area magnified in the bottom panels. (d) Numbers of FM4-64 intermediates per cell (n > 50) from images as in c are shown as a histogram. Bars, 5 µm.

Mentions: Eisosomes organize MCCs, and mutation of PIL1 results in mislocalization of all tested MCC components (Walther et al., 2006; Grossmann et al., 2007, 2008). Because NCE102 deletion has a strong effect on eisosomes, we asked whether NCE102 is also required for plasma membrane organization. Indeed, compared with wild-type cells in which the MCC marker Sur7-GFP was organized in distinct domains, Δnce102 cells showed only few clusters and more uniform localization of Sur7-GFP throughout the plasma membrane (Fig. 2 a). Furthermore, we observed a consistent but less pronounced phenotype on the MCP marker Pma1, which localized more uniformly in Δnce102 cells compared with its normal localization in structured plasma membrane domains (Fig. 2 b).


A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling.

Fröhlich F, Moreira K, Aguilar PS, Hubner NC, Mann M, Walter P, Walther TC - J. Cell Biol. (2009)

NCE102 is required for normal plasma membrane organization. (a and b) Sur7-GFP (a) and Pma1-GFP (b) were expressed in wild-type (wt) or Δnce102 cells, and representative confocal top (bottom) and mid sections (top) are shown. (c) Wild-type and Δnce102 cells expressing Pil1-GFP (green) were pulse labeled with FM4-64 (red) for 1 min, and representative images are shown. Boxes indicate the area magnified in the bottom panels. (d) Numbers of FM4-64 intermediates per cell (n > 50) from images as in c are shown as a histogram. Bars, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712959&req=5

fig2: NCE102 is required for normal plasma membrane organization. (a and b) Sur7-GFP (a) and Pma1-GFP (b) were expressed in wild-type (wt) or Δnce102 cells, and representative confocal top (bottom) and mid sections (top) are shown. (c) Wild-type and Δnce102 cells expressing Pil1-GFP (green) were pulse labeled with FM4-64 (red) for 1 min, and representative images are shown. Boxes indicate the area magnified in the bottom panels. (d) Numbers of FM4-64 intermediates per cell (n > 50) from images as in c are shown as a histogram. Bars, 5 µm.
Mentions: Eisosomes organize MCCs, and mutation of PIL1 results in mislocalization of all tested MCC components (Walther et al., 2006; Grossmann et al., 2007, 2008). Because NCE102 deletion has a strong effect on eisosomes, we asked whether NCE102 is also required for plasma membrane organization. Indeed, compared with wild-type cells in which the MCC marker Sur7-GFP was organized in distinct domains, Δnce102 cells showed only few clusters and more uniform localization of Sur7-GFP throughout the plasma membrane (Fig. 2 a). Furthermore, we observed a consistent but less pronounced phenotype on the MCP marker Pma1, which localized more uniformly in Δnce102 cells compared with its normal localization in structured plasma membrane domains (Fig. 2 b).

Bottom Line: The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids.Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization.Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

View Article: PubMed Central - PubMed

Affiliation: Organelle Architecture and Dynamics, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

Show MeSH