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An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

Aghamohammadzadeh S, Smaczynska-de Rooij II, Ayscough KR - PLoS ONE (2014)

Bottom Line: This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function.This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites.These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

ABSTRACT
Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

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The Effect of Gene Deletions on Bulk Endocytosis in the presence and absence of 25 µM Lat-A.Different endocytic  mutant strains were grown to mid log phase and incubated with bulk endocytic markers in the absence of presence of 25 µM Lat-A. (A) FM4-64 uptake was assessed after 20 minutes incubation and categorized as being plasma membrane, endosomal or vacuolar. Error bars – std deviation. (B) Analysis of the status of LY puncta following incubation in the absence or presence of Lat-A. Categories determined (i) in the plane of the membrane, (ii) invaginated or (iii) successfully undergone scission. Number of vesicles counted ≥50 in ≥10 cells. Error is std deviation.
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pone-0103311-g003: The Effect of Gene Deletions on Bulk Endocytosis in the presence and absence of 25 µM Lat-A.Different endocytic mutant strains were grown to mid log phase and incubated with bulk endocytic markers in the absence of presence of 25 µM Lat-A. (A) FM4-64 uptake was assessed after 20 minutes incubation and categorized as being plasma membrane, endosomal or vacuolar. Error bars – std deviation. (B) Analysis of the status of LY puncta following incubation in the absence or presence of Lat-A. Categories determined (i) in the plane of the membrane, (ii) invaginated or (iii) successfully undergone scission. Number of vesicles counted ≥50 in ≥10 cells. Error is std deviation.

Mentions: Given that the presence of 25 µM Lat-A does not block uptake of bulk lipid or fluid. We next asked whether deletion of known components of the endocytic machinery compromise this observed uptake route. Cells with deletions in a number of genes for endocytic components including the actin binding protein Abp1, the amphiphysin Rvs167, the actin bundling protein Sac6, the adaptor protein Sla1, and the dynamin homologue Vps1 were analysed for uptake of FM4-64 in the presence and absence of 25 µM Lat-A. As shown in figure 3A mutants showed small or no significant differences from wild type cells in uptake nor subsequent trafficking of the dye. In the presence of Lat-A three mutants, abp1Δ, rvs167Δ and sac6Δ showed an inhibition in initial internalization of the dye. Surprisingly, the strongest defect in endocytosis in these conditions was in the cells lacking abp1 which, of the three mutations, causes the mildest effect on the classical endocytosis pathway.


An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

Aghamohammadzadeh S, Smaczynska-de Rooij II, Ayscough KR - PLoS ONE (2014)

The Effect of Gene Deletions on Bulk Endocytosis in the presence and absence of 25 µM Lat-A.Different endocytic  mutant strains were grown to mid log phase and incubated with bulk endocytic markers in the absence of presence of 25 µM Lat-A. (A) FM4-64 uptake was assessed after 20 minutes incubation and categorized as being plasma membrane, endosomal or vacuolar. Error bars – std deviation. (B) Analysis of the status of LY puncta following incubation in the absence or presence of Lat-A. Categories determined (i) in the plane of the membrane, (ii) invaginated or (iii) successfully undergone scission. Number of vesicles counted ≥50 in ≥10 cells. Error is std deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114835&req=5

pone-0103311-g003: The Effect of Gene Deletions on Bulk Endocytosis in the presence and absence of 25 µM Lat-A.Different endocytic mutant strains were grown to mid log phase and incubated with bulk endocytic markers in the absence of presence of 25 µM Lat-A. (A) FM4-64 uptake was assessed after 20 minutes incubation and categorized as being plasma membrane, endosomal or vacuolar. Error bars – std deviation. (B) Analysis of the status of LY puncta following incubation in the absence or presence of Lat-A. Categories determined (i) in the plane of the membrane, (ii) invaginated or (iii) successfully undergone scission. Number of vesicles counted ≥50 in ≥10 cells. Error is std deviation.
Mentions: Given that the presence of 25 µM Lat-A does not block uptake of bulk lipid or fluid. We next asked whether deletion of known components of the endocytic machinery compromise this observed uptake route. Cells with deletions in a number of genes for endocytic components including the actin binding protein Abp1, the amphiphysin Rvs167, the actin bundling protein Sac6, the adaptor protein Sla1, and the dynamin homologue Vps1 were analysed for uptake of FM4-64 in the presence and absence of 25 µM Lat-A. As shown in figure 3A mutants showed small or no significant differences from wild type cells in uptake nor subsequent trafficking of the dye. In the presence of Lat-A three mutants, abp1Δ, rvs167Δ and sac6Δ showed an inhibition in initial internalization of the dye. Surprisingly, the strongest defect in endocytosis in these conditions was in the cells lacking abp1 which, of the three mutations, causes the mildest effect on the classical endocytosis pathway.

Bottom Line: This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function.This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites.These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

ABSTRACT
Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

Show MeSH
Related in: MedlinePlus