Differential requirements for actin during yeast and mammalian endocytosis

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.

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.

Figure 2. Increases in turgor pressure are detrimental to endocytic invagination

(A). Deletion of the type I phosphatase genes ppz1 and ppz2 have been linked to an increase in turgor pressure. Strains lacking either or both ppz1 and ppz2 were transformed with GFP-Abp1 and the lifetimes of GFP in the endocytic patch complexes measured. Because we were unable to generate a viable Δppz1Δppz2 double mutant in the Euroscarf strain used for our other experiments, this mutant was obtained from L.Yenush and compared directly to its W303 background parent. Because the timings of W303 and BY4741 wild-type strains are different for endocytic markers, the timings here are relative to each parental strain. Differences between wild-type cells and all mutants are statistically relevant. In t-test comparisons: wild-type to Δppz1, P value 0.02; wt to Δppz2, P value 0.005; and wild type (W303) to Δppz1Δppz2, P value ≤0.0001. Number of patches assessed for each sample ≥30 in ≥4 cells. (B) Kymographs from wild-type and Δppz1Δppz2 strains illustrating the marked effect of their deletion on endocytic invagination. (C) A model to illustrate the findings. (i) In wild-type cells in exponential phase the inward force generation by actin in association with its bundling proteins is sufficient to overcome the outward force of turgor pressure. (ii) in actin-bundling mutants little invagination is observed due to the inability of a sufficiently strong actin meshwork to support the required inward force. (iii) Addition of sorbitol effectively balances out cell turgor pressure and now the requirement for actin during invagination is substantially alleviated. Actin - red; endocytic coat - blue; turgor - black arrows; external osmotic force - green arrows.