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Differential requirements for actin during yeast and mammalian endocytosis.

Aghamohammadzadeh S, Ayscough KR - Nat. Cell Biol. (2009)

Bottom Line: However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis.We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure.By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.

ABSTRACT
Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination.

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Related in: MedlinePlus

Alleviation of turgor pressure rescues a requirement for bundled actin during endocytosis(A) Wild-type yeast, or strains lacking either actin bundling protein Δsac6, or Δscp1 or both Δsac6Δscp1 were transformed with a marker of actin in endocytosis (GFP-Abp14). Sorbitol was added at either 0.25, 0.5, or 1 M to the cells for either 4 hours or 10 minutes and the effect on lifetimes of GFP-Abp1 measured. Number of patches assessed for each sample ≥30 in ≥4 cells. (B) Kymographs from these strains illustrating the effect of sorbitol on lifetime and behaviour of the patches. (C) The proportion of GFP-Abp1 patches showing inward movement was quantified for wild-type and the Δsac6Δscp1 strain. Number of patches assessed for each sample ≥45 in ≥8 cells (D) Actin-bundling mutants affect uptake of the fluid phase marker Lucifer yellow. Addition of sorbitol increases the proportion of cells showing uptake of the stain into vesicles (Ves) and endosomes in cells but few cells still show vacuolar (V) staining indicating a post-scission requirement for actin that is not affected by sorbitol. Bar = 5 μM. (E). The effect of increasing latrunculin-A concentration on endocytosis and partial rescue of the effect by sorbitol. Cells were treated with either 100 or 400 μM Latrunculin-A (or the control with DMSO alone), for 10 minutes, prior to addition of sorbitol for 10 minutes, and Lucifer yellow for 20 minutes. Spots that were visualized were categorised as indicated - (M) for spots still in the plane of the plasma membrane; (I) for spots that have moved out of the membrane but are still contiguous in terms of the lucifer yellow signal; (S) for spots that have successfully undergone scission and are at least 250 nm from the membrane. Number of spots assessed for each sample ≥140 in ≥60 cells.
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Figure 1: Alleviation of turgor pressure rescues a requirement for bundled actin during endocytosis(A) Wild-type yeast, or strains lacking either actin bundling protein Δsac6, or Δscp1 or both Δsac6Δscp1 were transformed with a marker of actin in endocytosis (GFP-Abp14). Sorbitol was added at either 0.25, 0.5, or 1 M to the cells for either 4 hours or 10 minutes and the effect on lifetimes of GFP-Abp1 measured. Number of patches assessed for each sample ≥30 in ≥4 cells. (B) Kymographs from these strains illustrating the effect of sorbitol on lifetime and behaviour of the patches. (C) The proportion of GFP-Abp1 patches showing inward movement was quantified for wild-type and the Δsac6Δscp1 strain. Number of patches assessed for each sample ≥45 in ≥8 cells (D) Actin-bundling mutants affect uptake of the fluid phase marker Lucifer yellow. Addition of sorbitol increases the proportion of cells showing uptake of the stain into vesicles (Ves) and endosomes in cells but few cells still show vacuolar (V) staining indicating a post-scission requirement for actin that is not affected by sorbitol. Bar = 5 μM. (E). The effect of increasing latrunculin-A concentration on endocytosis and partial rescue of the effect by sorbitol. Cells were treated with either 100 or 400 μM Latrunculin-A (or the control with DMSO alone), for 10 minutes, prior to addition of sorbitol for 10 minutes, and Lucifer yellow for 20 minutes. Spots that were visualized were categorised as indicated - (M) for spots still in the plane of the plasma membrane; (I) for spots that have moved out of the membrane but are still contiguous in terms of the lucifer yellow signal; (S) for spots that have successfully undergone scission and are at least 250 nm from the membrane. Number of spots assessed for each sample ≥140 in ≥60 cells.

Mentions: To test this we assessed the lifetime of GFP-Abp1 in patches at the plasma membrane in wild-type cells and in cells lacking either one (Δsac6, Δscp1) or both (Δsac6Δscp1) actin-bundling proteins. GFP-Abp1 is widely used as a reporter of mid to late stages of endocytosis and marks the stage at which actin is assembled at the endocytic site. To reduce the effects of turgor pressure, sorbitol was added to the medium. Using a range of sorbitol concentrations (0.25 - 1 M) and incubation times (10 minutes and 4 hours) we observed that additon of sorbitol could fully rescue the endocytic lifetime defects of both single mutants and substantially rescue the double mutant defect. Rescue was fully evident at just 10 minutes with 0.5 M sorbitol indicating that gene expression is not required for the effect. To demonstrate that reduction in lifetime also corresponded to a rescue in invagination, endocytic patches were studied using kymographs. Kymographs are generated by collating images of the same fluorescent spot taken in a time lapse series. Thus, they are able to provide information on spatial position over time. Time is represented in the x-axis and distance moved (i.e invagination) by any movement in the y-axis. As shown in figure 1B, inward movement of the patches can be clearly seen in both single and double mutants. Quantification of the rescue reveals a significant increase in the number of endocytic patches that are now able to internalise (figure 1C). Finally, the effect of sorbitol on fluid phase uptake of a fluorescent dye, Lucifer yellow was followed. Again the double mutant Δsac6Δscp1 shows almost no uptake of the dye. In the presence of sorbitol interestingly while uptake into vesicles can be observed, movement of these to the vacuoles appears inhibited (figure 1D). This indicates that while sorbitol can alleviate functions associated with turgor pressure at the plasma membrane, once vesicles have formed in the cell they continue to require bundled or crosslinked actin to move away from the membrane.


Differential requirements for actin during yeast and mammalian endocytosis.

Aghamohammadzadeh S, Ayscough KR - Nat. Cell Biol. (2009)

Alleviation of turgor pressure rescues a requirement for bundled actin during endocytosis(A) Wild-type yeast, or strains lacking either actin bundling protein Δsac6, or Δscp1 or both Δsac6Δscp1 were transformed with a marker of actin in endocytosis (GFP-Abp14). Sorbitol was added at either 0.25, 0.5, or 1 M to the cells for either 4 hours or 10 minutes and the effect on lifetimes of GFP-Abp1 measured. Number of patches assessed for each sample ≥30 in ≥4 cells. (B) Kymographs from these strains illustrating the effect of sorbitol on lifetime and behaviour of the patches. (C) The proportion of GFP-Abp1 patches showing inward movement was quantified for wild-type and the Δsac6Δscp1 strain. Number of patches assessed for each sample ≥45 in ≥8 cells (D) Actin-bundling mutants affect uptake of the fluid phase marker Lucifer yellow. Addition of sorbitol increases the proportion of cells showing uptake of the stain into vesicles (Ves) and endosomes in cells but few cells still show vacuolar (V) staining indicating a post-scission requirement for actin that is not affected by sorbitol. Bar = 5 μM. (E). The effect of increasing latrunculin-A concentration on endocytosis and partial rescue of the effect by sorbitol. Cells were treated with either 100 or 400 μM Latrunculin-A (or the control with DMSO alone), for 10 minutes, prior to addition of sorbitol for 10 minutes, and Lucifer yellow for 20 minutes. Spots that were visualized were categorised as indicated - (M) for spots still in the plane of the plasma membrane; (I) for spots that have moved out of the membrane but are still contiguous in terms of the lucifer yellow signal; (S) for spots that have successfully undergone scission and are at least 250 nm from the membrane. Number of spots assessed for each sample ≥140 in ≥60 cells.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Alleviation of turgor pressure rescues a requirement for bundled actin during endocytosis(A) Wild-type yeast, or strains lacking either actin bundling protein Δsac6, or Δscp1 or both Δsac6Δscp1 were transformed with a marker of actin in endocytosis (GFP-Abp14). Sorbitol was added at either 0.25, 0.5, or 1 M to the cells for either 4 hours or 10 minutes and the effect on lifetimes of GFP-Abp1 measured. Number of patches assessed for each sample ≥30 in ≥4 cells. (B) Kymographs from these strains illustrating the effect of sorbitol on lifetime and behaviour of the patches. (C) The proportion of GFP-Abp1 patches showing inward movement was quantified for wild-type and the Δsac6Δscp1 strain. Number of patches assessed for each sample ≥45 in ≥8 cells (D) Actin-bundling mutants affect uptake of the fluid phase marker Lucifer yellow. Addition of sorbitol increases the proportion of cells showing uptake of the stain into vesicles (Ves) and endosomes in cells but few cells still show vacuolar (V) staining indicating a post-scission requirement for actin that is not affected by sorbitol. Bar = 5 μM. (E). The effect of increasing latrunculin-A concentration on endocytosis and partial rescue of the effect by sorbitol. Cells were treated with either 100 or 400 μM Latrunculin-A (or the control with DMSO alone), for 10 minutes, prior to addition of sorbitol for 10 minutes, and Lucifer yellow for 20 minutes. Spots that were visualized were categorised as indicated - (M) for spots still in the plane of the plasma membrane; (I) for spots that have moved out of the membrane but are still contiguous in terms of the lucifer yellow signal; (S) for spots that have successfully undergone scission and are at least 250 nm from the membrane. Number of spots assessed for each sample ≥140 in ≥60 cells.
Mentions: To test this we assessed the lifetime of GFP-Abp1 in patches at the plasma membrane in wild-type cells and in cells lacking either one (Δsac6, Δscp1) or both (Δsac6Δscp1) actin-bundling proteins. GFP-Abp1 is widely used as a reporter of mid to late stages of endocytosis and marks the stage at which actin is assembled at the endocytic site. To reduce the effects of turgor pressure, sorbitol was added to the medium. Using a range of sorbitol concentrations (0.25 - 1 M) and incubation times (10 minutes and 4 hours) we observed that additon of sorbitol could fully rescue the endocytic lifetime defects of both single mutants and substantially rescue the double mutant defect. Rescue was fully evident at just 10 minutes with 0.5 M sorbitol indicating that gene expression is not required for the effect. To demonstrate that reduction in lifetime also corresponded to a rescue in invagination, endocytic patches were studied using kymographs. Kymographs are generated by collating images of the same fluorescent spot taken in a time lapse series. Thus, they are able to provide information on spatial position over time. Time is represented in the x-axis and distance moved (i.e invagination) by any movement in the y-axis. As shown in figure 1B, inward movement of the patches can be clearly seen in both single and double mutants. Quantification of the rescue reveals a significant increase in the number of endocytic patches that are now able to internalise (figure 1C). Finally, the effect of sorbitol on fluid phase uptake of a fluorescent dye, Lucifer yellow was followed. Again the double mutant Δsac6Δscp1 shows almost no uptake of the dye. In the presence of sorbitol interestingly while uptake into vesicles can be observed, movement of these to the vacuoles appears inhibited (figure 1D). This indicates that while sorbitol can alleviate functions associated with turgor pressure at the plasma membrane, once vesicles have formed in the cell they continue to require bundled or crosslinked actin to move away from the membrane.

Bottom Line: However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis.We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure.By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.

ABSTRACT
Key features of clathrin-mediated endocytosis have been conserved across evolution. However, endocytosis in Saccharomyces cerevisiae is completely dependent on a functional actin cytoskeleton, whereas actin appears to be less critical in mammalian cell endocytosis. We reveal that the fundamental requirement for actin in the early stages of yeast endocytosis is to provide a strong framework to support the force generation needed to direct the invaginating plasma membrane into the cell against turgor pressure. By providing osmotic support, pressure differences across the plasma membrane were removed and this reduced the requirement for actin-bundling proteins in normal endocytosis. Conversely, increased turgor pressure in specific yeast mutants correlated with a decreased rate of endocytic patch invagination.

Show MeSH
Related in: MedlinePlus