Role of turgor pressure in endocytosis in fission yeast

Abstract

Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane.

FIGURE 1:

Addition of sorbitol to the media accelerates initial events of endocytosis. (A) Three fission yeast cells expressing endocytic patch markers sla1-GFP (adaptor protein, green) and crn1-Tomato (coronin; red; FC2589). Medial focal plane confocal image. (B) Time-lapse images of a single endocytic patch marked sla1-GFP and crn1-Tomato in wild-type cell. The onset of inward movement is designated as time 0. Images are shown at 1-s intervals. (C) Behavior of patches in wild-type cells at indicated sorbitol concentrations. For each condition n = 56 patches. (D) Average residence time of sla1-GFP and crn1-Tomato at the cortex before ingression (at t = 0) in wild-type (FC2589) and gpd1∆ (FC2592) cells in the indicated sorbitol concentrations. These are temporal maps of patch behavior, where green is the period during which the patch contains sla1-GFP without crn1-Tomato, and yellow denotes when both proteins are present. For each condition n = 20 patches. (E) Track of a representative sla1-GFP patch in a wild-type cell. Images were acquired every 100 ms in a single confocal plane, and positions of the patches were determined with subpixel resolution (Materials and Methods). Time 0 is onset of detectable movement into the cell. Note that there are at least two phases of inward movement with different rates, followed by a transition to more random movement, which may represent time of scission. (F) Average distance traveled inward from the plasma membrane by sla1-GFP patches in wild-type cells in 0 (blue) and 0.2 M (red) sorbitol. n = 20 and 19 patches, respectively. Error bars, SD.

FIGURE 2:

Sorbitol rescues endocytic ingression defects of cells treated with a low dose of latrunculin A. (A) Time-lapse images of a single patch containing sla1-GFP and crn1-Tomato (FC2589) after treatment with 2 μM LatA. (B) Time-lapse images of a single patch after treatment with 2 μM LatA and 0.2 M sorbitol. Images acquired at 1-s intervals. (C) Behavior of patches in wild-type cells treated with 2 μM LatA and at indicated sorbitol concentrations. The behavior of each patch was categorized as those that remain stationary at the cortex (as in A), those that move inward but bounce back (as in B), and those that internalize successfully. For each condition n = 56 patches. (D) Tracks of individual sla1-GFP patches in wild-type cells in 2 μM LatA and 0 (blue) or 0.2 M (red) sorbitol. (E) Average distances of patches from the cortex, as described in D. n = 18 and 15 patches, respectively. Error bars, SD.

FIGURE 3:

Sorbitol rescues a subset of endocytic mutants. (A) Top, time-lapse images of an individual patch containing sla1-GFP and crn1-Tomato in wsp1∆ (FC2587) at 0 and 0.1 M sorbitol. Images are shown at 1-s intervals. Graph shows behavior of patches. For each condition n = 47 patches. (B) Top, time-lapse images of an individual patch in myo1∆ (FC2659) at 0 and 0.2 M Sorbitol and graph of patch behavior. For each condition n = 43 patches. (C) Average residence times of sla1-GFP and crn1-Tomato at the cortex in arp2-1 cells (FC2660) at indicated sorbitol concentrations. For each condition n = 20 patches. Note that all patches eventually ingress in this mutant. Bars, SD. (D) Behavior of patches in fim1∆ (FC2591). For each condition n = 48 patches. (E) Behavior of patches in end4∆ (FC2590). For each condition n = 70 patches.

FIGURE 4:

Endocytosis in the absence of the cell wall. (A) Time-lapse images of a protoplast expressing sla1-GFP (green) and crn1-Tomato (red) (FC2589). Arrowheads follow actin patches during endocytosis. (B) Average resident time of sla1-GFP and crn1-Tomato at the cortex of walled cells and protoplasts before invagination. For each condition n = 10 patches. (C) Average distance traveled inward by sla1-GFP patches in protoplasts and normal cells. Time 0 is beginning of inward movement. n = 16 and 18 patches, respectively. **p < 0.005 and *p < 0.05 in comparison with times at 0 M sorbitol. Error bars, SD.