Differential requirements for actin during yeast and mammalian endocytosis

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

An Abp1-Dependent Route of Endocytosis Functions when the Classical Endocytic Pathway in Yeast Is Inhibited

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

The Effect of Gene Deletions on Bulk Endocytosis in the presence and absence of 25 µM Lat-A.

<p>Different endocytic null 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.</p

Alternative endocytic components and routes in wild type cells.

<p>(A) Cells co-expressing Sla1-mRFP and Sla2-GFP (KAY1734) were imaged and composition of spots was analysed. Kymographs show examples of puncta that show the two proteins together and with Sla2-GFP alone. Kymographs are from full movie (120 seconds) (B) Cells expressing Sla1-GFP (KAY733) were incubated with FM4-64FX for 5 minutes and imaged. Examples of both GFP only and FM4-64 only puncta invaginating are shown in kymographs. Kymographs are from full movie (120 seconds). The images are stills taken from supplemental movie at the times indicated. Scale bar = 2 µm.</p

The Effect of Lat-A on actin and endocytosis.

<p>(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.</p

The Effect of Abp1 mutations on Bulk Endocytosis.

<p>(A) Schematic of full length Abp1 and the different mutants analysed in this study. (B) Wild type, <i>abp1</i>Δ and <i>abp1Δ</i> cells expressing wild type <i>ABP1</i> or mutant versions were grown to mid log phase before treating with Lat-A and following uptake of FM4-64. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error bars are standard deviation. (C) The effect of Abp1 mutations on actin organization as assessed by rhodamine phalloidin staining. (D) A number of proteins have been shown to bind to the SH3 domain of Abp1. Deletions in genes encoding 3 of these proteins, Ark1, Prk1 and Srv2/CAP were analysed to determine whether any of these interactions is potentially required for the Abp1 dependent endocytic pathway in the presence of 25 µM Lat-A. Cells were classified according to the most prominent staining after 20 minutes FM4-64 incubation. Error is Std Deviation.</p

The effect of abp1 deletion on the uptake and trafficking of Lucifer yellow.

<p>(A) Wild type and <i>abp1</i>Δ cells were grown to mid log phase. Half of the cells were incubated with 25 µM Lat-A. LY was added for 90 minutes as described. Localization of stain was categorized as being at the plasma membrane, endosomes or vacuoles. Error bars are std deviation. (B) Representative images from the cells visualized. Bar = 5 µm.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis

Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline–arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type ISH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast