Plasma membrane tension regulates eisosome structure and function

Abstract

Eisosomes are membrane furrows at the cell surface of yeast that have been shown to function in two seemingly distinct pathways, membrane stress response and regulation of nutrient transporters. We found that many stress conditions affect both of these pathways by changing plasma membrane tension and thus the morphology and composition of eisosomes. For example, alkaline stress causes swelling of the cell and an endocytic response, which together increase membrane tension, thereby flattening the eisosomes. The flattened eisosomes affect membrane stress pathways and release nutrient transporters, which aids in their down-regulation. In contrast, glucose starvation or hyperosmotic shock causes cell shrinking, which results in membrane slack and the deepening of eisosomes. Deepened eisosomes are able to trap nutrient transporters and protect them from rapid endocytosis. Therefore, eisosomes seem to coordinate the regulation of both membrane tension and nutrient transporter stability.

FIGURE 1:

Eisosomes respond differently to various stress conditions. (A) Wild type expressing Nce102-mCherry (AMY4) was monitored by fluorescence microscopy for changes in localization of Nce102. The pictures show the projection of 10 optical sections representing the bottom half of the cell. Cells were maintained in a microfluidics system under optimal growth conditions (SD_comp medium at 30°C) (1) and then shifted to different stress conditions for 15 min. To disrupt the proton gradient, 50 mM Tris buffer, pH 8, was added to conditioned growth medium (medium obtained by centrifugation of the starting culture, which is identical to the medium of the control cells), which changed the pH from 4 to 7.5 (2). For glucose and leucine starvation, SD_comp medium buffered to pH 4 was used lacking either glucose or leucine (3 and 4). For the hypoosmotic shock (1 M > 0 M sorbitol) (5), cells were grown in SD_comp + 1 M sorbitol and shifted to conditioned SD_comp lacking sorbitol (this medium was obtained from a parallel culture that grew to the same cell density in absence of sorbitol). (B) Quantification of eisosomes using a 2D-polygon analysis (see Materials and Methods). The error bars indicate the SD of four to five analyzed microscopy pictures containing on average ∼40 cells each. (C) Example of the 2D polygon analysis used to quantify changes in Nce102 localization (B). The microscopy pictures are identical to those shown in A2. Red outlines mark the by the algorithm identified objects. The line graph shows the intensity profile along the line indicated in the micrographs. The intensity profiles were standardized to the last peak (set to 1.0). (D) Wild-type cells expressing Nce102-mCherry (AMY4) were grown in SD_comp + 1 M sorbitol, shifted for 3 min to conditioned medium lacking sorbitol, and then shifted back for 3 min to conditioned medium containing 1 M sorbitol. (E) Wild-type, end3-1, and rsp5-1 cells expressing Nce102-mCherry (AMY4, DAY20, DAY21) grown in SD_comp were treated with 50 mM Tris buffer, pH 8, in conditioned medium either in the presence or in the absence of 1 M sorbitol.

FIGURE 2:

Plasma membrane morphology observed by TEM. (A) The micrographs show representative cells either before or 15 min after Tris treatment. Black arrowheads indicate invaginations at the plasma membrane. The calculation of the number of invaginations per µm² was based on the assumption that the invaginations represent membrane furrows (eisosomes) with a length of 300 nm (the number of invaginations found in the 60 nm-thin sections was corrected by dividing by 5). The numbers represent the mean ± SD. (B) The changes in cell size after chemical fixation was determined using fluorescence microscopy of cells expressing Fur4-GFP. Cells were imaged in a microfluidics system and the cell diameter of the same cells before and after 15 min of fixation was measured.

FIGURE 3:

Loss of proton gradient causes flattening of eisosomes. (A) Superresolution microscopy of live yeast cells in a microfluidics system. The pictures show a single optical section of 50 nm through the center of the cells. Wild-type or end3-1 cells expressing Pil1-mCherry and Fur4-GFP (AMY6 pJK19, DAY27 pJK19) were grown in SD_comp-ura and shifted to conditioned medium containing 50 mM Tris, pH 8. To determine the effect of glucose starvation, cells expressing a stabilized, N-terminally deleted Fur4-GFP, Fur4(∆N)-GFP (AMY6 pJK30; the N-terminal deletion prevents ubiquitination and thus impairs down-regulation of Fur4 [ Keener and Babst, 2013]) were shifted to SD_comp-ura medium adjusted to pH 4 and lacking glucose. Because of the rapid endocytic response, after 15 min glucose starvation, wild-type Fur4-GFP was no longer present at the plasma membrane. (B) Fluorescence microscopy of wild type expressing Pil1-mCherry and Fur4-GFP (AMY6 pJK19). The picture shows a single optical section comparable with the superresolution pictures shown in A. (C) Quantification of Fur4-Pil1 colocalization of over 400 eisosomes (numbers of eisosomes in the control cells indicated; 30–40 cells). Colocalization was defined by <25 nm distance between the centers of the Fur4 and Pil1 signals.

FIGURE 4:

Effect of different stresses on Fur4 localization and on cell surface area. (A) Fluoresence microscopy of wild-type, end3-1, and rsp5-1 cells expressing Fur4-GFP (SEY6210 pJK19, BWY1346 pJK19, MYY880 pJK19). The pictures show a single optical section through the center of the cells. Cells were grown in SD_comp-ura or in the case of the hypoosmotic shock (-sorbitol) in SD_comp-ura +1 M sorbitol. These cells were shifted into medium either containing 50 mM Tris, pH 8, containing 1 M sorbitol (+sorbitol), lacking glucose (-glucose), or lacking sorbitol (-sorbitol). (B) Quantification of the cell surface area change of 50 cells after treatment in the microfluidics system (15 min treatment if not indicated otherwise). The black line indicates the median of each data set.

FIGURE 5:

Fur4 uracil import activity under different stress conditions and the effect of heat-shock on Nce102 localization. (A) Uracil import assays of wild type expressing Fur4(∆N)-GFP (N-terminal deletion that lacks the ubiquitination sites; SEY6210 pJK88). Cells were grown in SD_comp-ura or SD_comp-ura + 1 M sorbitol (for the hypoosmotic shock) and treated for 10 min as indicated. Uracil (20 μg/ml) was added for 10 min, and the cells were harvested and analyzed for the uracil content using normal-phase chromatography. The bar graph shows the average and standard deviations of three measurements. The data were standardized relative to the control samples (= 100%). (B) Fluorescence microscopy of wild-type cells expressing Nce102-mCherry (AMY4). The cells were grown at 25°C and imaged in the microfluidics system at 25°C (control). The temperature of the microfluidics chamber was rapidly increased to 35°C by switching to a heated objective. After imaging the heat-shocked cells (8 min), the growth medium was switched to a medium containing 1 M sorbitol. The bar graph illustrates the quantification of Nce102 objects performed by 2D polygon analysis and standardized to starting conditions (100% = 1854 objects).

FIGURE 6:

Loss of proton gradient causes relocalization of Slm1. (A) Wild type expressing Slm1-GFP and Pil1-mCherry (LGY31) was analyzed by fluorescence microscopy using a microfluidics device. The pictures show projections of 10 optical sections representing the bottom half of the cells. The cells were grown in SD_comp and shifted to conditioned SD_comp +50 mM Tris, pH 8. (B, D) Quantification of Slm1 localization relative to start conditions. Wild-type and end3-1 mutant cells (LGY31, DAY28) were treated either with 50 mM Tris, pH 8, or by moving the culture from growth in the presence of 1 M sorbitol to conditioned medium without sorbitol. The number above the bar graph indicates the total number of Slm1-GFP structures counted in the control cells (= 100%; ∼50 cells analyzed). (C) Quantification of the Slm1-GFP distribution (cell surface/total) in LGY31 cells before and 15 min after the addition of 50 mM Tris, pH 8 (same data set as used for quantification in B). (E) Fluorescence microscopy of wild-type cells expressing Slm1-GFP and Avo3-mCherry (DAY53). The pictures show a projection of 10 optical sections (250 nm each) representing the bottom half of the cells. The bar graph represents the quantification of 30 cells, determining the number Slm1-GFP only, Avo3-mCherry only, and colocalizing structures per half-cell. The quantification was performed manually. Small structures >4 pixels were omitted.

FIGURE 7:

Membrane stress-induced changes in Orm2 phosphorylation. (A, B) Cells expressing wild-type or the S46,47,48A mutant FLAG-Orm2 in an orm2∆ background (YWY005 pOS129, YWY005 pYW001) were grown in SD medium adjusted to pH 4 and were treated with 50 mM Tris, pH 8 (+ Tris), in the presence or absence of 1 M sorbitol (+ sorbitol), and samples were taken at different time points. As a control, a sample was prepared from the 1 h + Tris cells that were treated for an additional 1 h with 2.5 µM myriocin (+ myr). The cell lysates were separated by Phostag Gel or standard SDS–PAGE, and proteins were detected by Western blot (Pgk1 served as a loading control).

FIGURE 8:

Eisosome mutations do not impair endocytosis or cell integrity. (A) Wild-type and mutant cells were grown in SD_comp + 1 M sorbitol. The cells of the midlog yeast cultures were moved into SD_comp pH 4 medium and after 10 min the cells were stained with PI and analyzed by flow cytometry. (B) Microscopy pictures of Fur4-GFP expressing cells (WT, SEY6210 pJK19; nce102∆, AMY41 pJK19) in the presence or absence of 50 mM Tris, pH 8, were quantified by determining the signal at the plasma membrane of Fur4 relative to the total signal. The graph shows the result of ∼50 cells for each strain and condition. The black line indicates the median of the data set.

FIGURE 9:

Eisosomes deepen after a hyperosmotic shock or glucose starvation. (A) Wild-type cells expressing Pil1-mCherry and Fur4-GFP (AMY6 pJK19) were treated with 50 mM Tris, pH 8 (+ Tris), with 1 M sorbitol (+ sorbitol) or combined with Tris and sorbitol. For the glucose starvation (- glucose) cells were transferred from the growth medium (SD_comp-ura) to growth medium lacking glucose. (B) Wild-type cells expressing Pil1-GFP (AMY6) were analyzed by fluorescence microscopy before (control) and after 3 h of glucose starvation. Before microscopy the cells were stained with FM4-64 (2 µg/ml) for 5 min at room temperature.

FIGURE 10:

Glucose starvation causes shrinking of the cells. Wild-type cells (BY4741) were glucose starved for 3 or 24 h and analyzed by TEM. Deep invaginations of the plasma membrane are marked by arrowheads.

FIGURE 11:

Model of the response of eisosomes to high membrane tension. Under optimal growth conditions, eisosomes harbor inactive APC-type nutrient transporters that are in an equilibrium with active transporters outside of eisosomes. The tetraspan protein Nce102 and the membrane stress sensor Slm1 both localize to eisosomes. Loss of the proton gradient causes swelling of the cell and a rapid endocytic response. The resulting increase in membrane tension flattens the eisosomes. As a consequence, Nce102, APC transporters and Slm1 move out of eisosomes. Outside of eisosomes the APC transporters can be targeted for ubiquitination, endocytosis, and degradation. Similar to high membrane tension caused by high extracellular pH, heat-shock conditions also cause eisosome flattening and trigger degradation of APC transporters. Therefore, eisosomes seem to respond to increased fluidity of the membrane.