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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 Lat-A on actin and endocytosis.(A) Lat-A was added at the levels indicated for 15 minutes, before cells were fixed and labelled with rhodamine phalloidin to visualize F-actin. Bar = 5 µm. (B) The fluid phase dye Lucifer yellow was added to cells in the presence of 0, 25 or 400 µM Lat-A. Uptake of Lucifer yellow was assessed after 90 minutes. Bar = 5 µm. (C) Cells were treated with Lat-A 25 µM or DMSO (control) for 20 minutes before incubating with FM4-64. The localization of the dye was categorized as plasma membrane (PM), endosomal (End) or vacuolar (vac). Shown is the mean±Std Dev of 3 experiments. An unpaired students t-test indicates that there is a significant increase in endosomal staining in the treated cells p<0.0001. Examples of cells stained with FM4-64 in the absence or presence of Lat-A are shown. Bar = 5 µm (D) Strains expressing integrated GFP-Snc1 were imaged in the presence or absence of 25 µM Lat-A. Green arrowheads indicate internalising or internalized material. Red arrowheads show puncta of GFP-Snc1 at the plasma membrane. Bar = 5 µm. (E) Wild type cells expressing Sla1-GFP were grown to mid-log phase, half the sample was treated with Lat-A for 20 minutes. Time lapse movies were recorded over 90 seconds and kymographs generated.
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pone-0103311-g002: The Effect of Lat-A on actin and endocytosis.(A) Lat-A was added at the levels indicated for 15 minutes, before cells were fixed and labelled with rhodamine phalloidin to visualize F-actin. Bar = 5 µm. (B) The fluid phase dye Lucifer yellow was added to cells in the presence of 0, 25 or 400 µM Lat-A. Uptake of Lucifer yellow was assessed after 90 minutes. Bar = 5 µm. (C) Cells were treated with Lat-A 25 µM or DMSO (control) for 20 minutes before incubating with FM4-64. The localization of the dye was categorized as plasma membrane (PM), endosomal (End) or vacuolar (vac). Shown is the mean±Std Dev of 3 experiments. An unpaired students t-test indicates that there is a significant increase in endosomal staining in the treated cells p<0.0001. Examples of cells stained with FM4-64 in the absence or presence of Lat-A are shown. Bar = 5 µm (D) Strains expressing integrated GFP-Snc1 were imaged in the presence or absence of 25 µM Lat-A. Green arrowheads indicate internalising or internalized material. Red arrowheads show puncta of GFP-Snc1 at the plasma membrane. Bar = 5 µm. (E) Wild type cells expressing Sla1-GFP were grown to mid-log phase, half the sample was treated with Lat-A for 20 minutes. Time lapse movies were recorded over 90 seconds and kymographs generated.

Mentions: While deletions and mutations in clathrin (heavy and light chains) and a number of other endocytic components including amphiphysins (Rvs161/Rvs167), Sla2/HIP1R and Sac6/fimbrin severely compromise endocytic uptake of cargoes including alpha factor receptor and GFP-Snc1 [23], [24], [25], [26], [27], uptake of FM4-64 often occurs relatively normally, albeit with some kinetic delay [22] and our unpublished observations). In previous work we demonstrated that addition of latrunculin-A (Lat-A), an actin monomer sequestering drug, disrupts actin organization and inhibits endocytosis [28]. Lat-A is routinely used at levels that result in the complete disassembly of cortical actin patches (200–400 µM). However further analysis of the effects of Lat-A has indicated that there are levels of this drug that do not appear to disrupt cortical actin patches and such levels can infact be remedial for cells with stabilized actin structures which themselves are linked with accumulation of reactive oxygen species [29]. To investigate the links between endocytosis and actin patches, wild type cells were grown to log phase and a range of levels of Lat-A (0, 25, 100, 200, 400 µM) were added for 10 minutes and the organization of F-actin analysed using rhodamine phalloidin staining. As shown, (figure 2A) at both 25 and 100 µM Lat-A, while actin cables appear to be disrupted, cortical actin sites remain intact, though the level of F-actin appears reduced in the cells incubated with 100 µM Lat-A. All actin structures were disrupted at levels of 200 and 400 µM Lat-A. The effect of 25 µM Lat-A on endocytosis was then determined. Four approaches were used; uptake of Lucifer yellow was used to determine any effects on fluid phase endocytosis; FM4-64 is a lipophylic dye used to determine membrane internalization; GFP-Snc1 is a reporter showing trafficking of a SNARE protein between the plasma membrane and endosomes; and analysis of Sla1-GFP allows the behaviour of individual endocytic sites to be assessed. As shown in figure 2B, in the presence of 25 µM Lat-A, Lucifer yellow was still internalized and trafficking to vacuoles was observed, while at 400 µM Lat-A uptake was completely abrogated. Analysis of lipid internalization using FM4-64 revealed that after 20 minutes incubation, 100% untreated cells internalized the dye and the majority of cells showed vacuolar staining. In the presence of 25 µM Lat-A 89±4% cells internalized the dye showing endocytosis was functioning, however there was a reduced number of cells with predominant vacuolar staining indicating a role for F-actin in post endosomal trafficking. A post-endosomal role for actin in yeast has previously been suggested [30]. These two approaches indicate that bulk endocytosis is not affected following addition of 25 µM Lat-A. GFP-Snc1 is a fluorescently tagged SNARE protein that has been used as a reporter for endosomal uptake and recycling [20], [23]. High throughput screens analysing uptake and recycling of the tagged SNARE GFP-Snc1 have indicated the importance of the recognized clathrin mediated endocytic pathway for its uptake into cells [26]. In wild type, untreated cells Snc1 is observed in puncta at the surface and also in discrete structures, presumed to be endosomes inside cells (Figure 2D). In the presence of 25 µM Lat-A, uptake is inhibited and localization is only seen at the cell surface. Inhibition of GFP-Snc1 uptake at levels of 25 µM Lat-A suggested that the CME pathway could be inhibited even when cortical patches are intact.


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 Lat-A on actin and endocytosis.(A) Lat-A was added at the levels indicated for 15 minutes, before cells were fixed and labelled with rhodamine phalloidin to visualize F-actin. Bar = 5 µm. (B) The fluid phase dye Lucifer yellow was added to cells in the presence of 0, 25 or 400 µM Lat-A. Uptake of Lucifer yellow was assessed after 90 minutes. Bar = 5 µm. (C) Cells were treated with Lat-A 25 µM or DMSO (control) for 20 minutes before incubating with FM4-64. The localization of the dye was categorized as plasma membrane (PM), endosomal (End) or vacuolar (vac). Shown is the mean±Std Dev of 3 experiments. An unpaired students t-test indicates that there is a significant increase in endosomal staining in the treated cells p<0.0001. Examples of cells stained with FM4-64 in the absence or presence of Lat-A are shown. Bar = 5 µm (D) Strains expressing integrated GFP-Snc1 were imaged in the presence or absence of 25 µM Lat-A. Green arrowheads indicate internalising or internalized material. Red arrowheads show puncta of GFP-Snc1 at the plasma membrane. Bar = 5 µm. (E) Wild type cells expressing Sla1-GFP were grown to mid-log phase, half the sample was treated with Lat-A for 20 minutes. Time lapse movies were recorded over 90 seconds and kymographs generated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103311-g002: The Effect of Lat-A on actin and endocytosis.(A) Lat-A was added at the levels indicated for 15 minutes, before cells were fixed and labelled with rhodamine phalloidin to visualize F-actin. Bar = 5 µm. (B) The fluid phase dye Lucifer yellow was added to cells in the presence of 0, 25 or 400 µM Lat-A. Uptake of Lucifer yellow was assessed after 90 minutes. Bar = 5 µm. (C) Cells were treated with Lat-A 25 µM or DMSO (control) for 20 minutes before incubating with FM4-64. The localization of the dye was categorized as plasma membrane (PM), endosomal (End) or vacuolar (vac). Shown is the mean±Std Dev of 3 experiments. An unpaired students t-test indicates that there is a significant increase in endosomal staining in the treated cells p<0.0001. Examples of cells stained with FM4-64 in the absence or presence of Lat-A are shown. Bar = 5 µm (D) Strains expressing integrated GFP-Snc1 were imaged in the presence or absence of 25 µM Lat-A. Green arrowheads indicate internalising or internalized material. Red arrowheads show puncta of GFP-Snc1 at the plasma membrane. Bar = 5 µm. (E) Wild type cells expressing Sla1-GFP were grown to mid-log phase, half the sample was treated with Lat-A for 20 minutes. Time lapse movies were recorded over 90 seconds and kymographs generated.
Mentions: While deletions and mutations in clathrin (heavy and light chains) and a number of other endocytic components including amphiphysins (Rvs161/Rvs167), Sla2/HIP1R and Sac6/fimbrin severely compromise endocytic uptake of cargoes including alpha factor receptor and GFP-Snc1 [23], [24], [25], [26], [27], uptake of FM4-64 often occurs relatively normally, albeit with some kinetic delay [22] and our unpublished observations). In previous work we demonstrated that addition of latrunculin-A (Lat-A), an actin monomer sequestering drug, disrupts actin organization and inhibits endocytosis [28]. Lat-A is routinely used at levels that result in the complete disassembly of cortical actin patches (200–400 µM). However further analysis of the effects of Lat-A has indicated that there are levels of this drug that do not appear to disrupt cortical actin patches and such levels can infact be remedial for cells with stabilized actin structures which themselves are linked with accumulation of reactive oxygen species [29]. To investigate the links between endocytosis and actin patches, wild type cells were grown to log phase and a range of levels of Lat-A (0, 25, 100, 200, 400 µM) were added for 10 minutes and the organization of F-actin analysed using rhodamine phalloidin staining. As shown, (figure 2A) at both 25 and 100 µM Lat-A, while actin cables appear to be disrupted, cortical actin sites remain intact, though the level of F-actin appears reduced in the cells incubated with 100 µM Lat-A. All actin structures were disrupted at levels of 200 and 400 µM Lat-A. The effect of 25 µM Lat-A on endocytosis was then determined. Four approaches were used; uptake of Lucifer yellow was used to determine any effects on fluid phase endocytosis; FM4-64 is a lipophylic dye used to determine membrane internalization; GFP-Snc1 is a reporter showing trafficking of a SNARE protein between the plasma membrane and endosomes; and analysis of Sla1-GFP allows the behaviour of individual endocytic sites to be assessed. As shown in figure 2B, in the presence of 25 µM Lat-A, Lucifer yellow was still internalized and trafficking to vacuoles was observed, while at 400 µM Lat-A uptake was completely abrogated. Analysis of lipid internalization using FM4-64 revealed that after 20 minutes incubation, 100% untreated cells internalized the dye and the majority of cells showed vacuolar staining. In the presence of 25 µM Lat-A 89±4% cells internalized the dye showing endocytosis was functioning, however there was a reduced number of cells with predominant vacuolar staining indicating a role for F-actin in post endosomal trafficking. A post-endosomal role for actin in yeast has previously been suggested [30]. These two approaches indicate that bulk endocytosis is not affected following addition of 25 µM Lat-A. GFP-Snc1 is a fluorescently tagged SNARE protein that has been used as a reporter for endosomal uptake and recycling [20], [23]. High throughput screens analysing uptake and recycling of the tagged SNARE GFP-Snc1 have indicated the importance of the recognized clathrin mediated endocytic pathway for its uptake into cells [26]. In wild type, untreated cells Snc1 is observed in puncta at the surface and also in discrete structures, presumed to be endosomes inside cells (Figure 2D). In the presence of 25 µM Lat-A, uptake is inhibited and localization is only seen at the cell surface. Inhibition of GFP-Snc1 uptake at levels of 25 µM Lat-A suggested that the CME pathway could be inhibited even when cortical patches are intact.

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