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Distribution of cortical endoplasmic reticulum determines positioning of endocytic events in yeast plasma membrane

PLoS ONE (2012) 7(4)
DOI: 10.1371/journal.pone.0035132
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Abstract

In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis. © 2012 Stradalova et al.

Figures

  • Figure 1. Endocytosis is initiated in the ER-free zones of the plasma membrane. Mutual localization was performed for Ede1-GFP, a marker of early stages of endocytosis, and cortical ER visualized by ss-dsRed-HDEL. Only rare colocalization events were detected (A). Similarly, the cortical ER network (B) and the initiation sites of endocytosis (C) were not colocalized with MCC domains marked with Sur7-GFP and Sur7-mRFP, respectively. Tangential confocal sections of BY4741 cells expressing fluorescently labeled proteins are presented. The fluorescence intensity profiles along the numbered arrows were scaled to the same range in the red and green channels. Bar: 5 mm. doi:10.1371/journal.pone.0035132.g001
  • Table 1. Quantification of mutual localization of MCC, Ede1 sites and cortical ER.
  • Figure 2. Endocytic events are randomly distributed within ERfree PM areas. In tangential confocal sections of individual W303 cells expressing Ede1-GFP and ss-dsRed-HDEL (A), the minimal distance of endocytic sites from the cortical ER boundary was measured. The histogram of the measured distances (full bars in B; 906 sites in 200 cells were analyzed) was compared to the distribution of the distances of model foci randomly positioned in the plasma membrane (empty bars in B; 320 foci in 100 cells; see Methods for details). In order to maximize the accuracy of the distance measurements, for all the measurements we chose only the foci located to easily discernible ER holes positioned in central parts of the tangential confocal sections, so that the entire borders of the holes could be traced. Bar: 1 mm. doi:10.1371/journal.pone.0035132.g002
  • Figure 3. Differential cortical ER coverage defines micron-scale PM domains. The dynamics of cortical ER was followed in time-lapse series of 20 tangential confocal sections of BY4741 cells expressing ss-GFP-HDEL together with Sur7-mCherry (rate: 10 s/frame). Raw data after a 363 mean filtration (A) and binarized cortical ER pattern (B) of the first and the last three frames in the series are presented. For better lucidity, the red fluorescence channel (MCC/Sur7-mCherry) is not shown. In order to visualize the local dynamics of cortical ER, all twenty binarized frames were superimposed. Three out of 33 cells analyzed are presented in false colors denoting the number of frames in the series in which cortical ER was detected (C–E). Bar: 1 mm. doi:10.1371/journal.pone.0035132.g003
  • Figure 4. Lateral mobility of cortical ER determines the positioning of endocytic events. Initiation of endocytic events in cells coexpressing Ede1-GFP and ss-dsRed-HDEL was monitored in a time-lapse experiment (20 frames, 30 s/frame). Superposition of all the frames is presented. The 4th column represents the superimposed binarized ER signals from 20 consecutive frames in a false-color blue-to-red scale to highlight the dynamics of the cortical ER network (see Fig. 3 legend for an explanation). The column on the far right shows this superposition of binarized ER signals in red overlaid by the green channel, in which the positions of the maxima of the Ede1 sites in the 20 frames were marked by round spots. Bar: 1 mm. doi:10.1371/journal.pone.0035132.g004
  • Figure 5. Defect in MCC integrity results in alteration of the cortical ER pattern. Tangential confocal sections of BY4741, pil1D and nce102D cells expressing ss-GFP-HDEL and Sur7-mCherry markers are presented (A). Statistical analysis of cortical ER pattern in all strains (n.30) revealed that the cortical ER network in pil1D cells exhibits fewer (B) but larger (C) holes. Importantly, no difference in total cortical ER area with respect to the individual tested strains was detected. Mean values (6 standard deviation) are compared and the significance of detected effects as revealed by Student T test is denoted (* p,0.05;***p,0.001). Bar: 5 mm. doi:10.1371/journal.pone.0035132.g005
  • Figure 7. Distinct localization of MCC marker Sur7 and cortical ER distributions is maintained in MCC-defective strains. The mutual position of fluorescence signals in tangential confocal sections of BY4741, pil1D and nce102D cells co-expressing Sur7-GFP and ssdsRed-HDEL (n.140) was analyzed. Relative numbers (mean 6 standard deviation) of Sur7-GFP domains localized outside the ssdsRed-HDEL (cortical ER) pattern are compared. doi:10.1371/journal.pone.0035132.g007
  • Figure 6. Electron microscopic analysis of the MCC-specific alterations of cortical ER pattern. The length and distribution of cortical ER cisternae (arrows) on thin sections of BY4741 (A), pil1D (B) and nce102D cells (C) were compared. No difference in total length of the cortical ER structures with respect to the individual tested strains was detected. Bar: 1 mm. doi:10.1371/journal.pone.0035132.g006

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CITATION STYLE

APA

Stradalova, V., Blazikova, M., Grossmann, G., Opekarová, M., Tanner, W., & Malinsky, J. (2012). Distribution of cortical endoplasmic reticulum determines positioning of endocytic events in yeast plasma membrane. PLoS ONE, 7(4). https://doi.org/10.1371/journal.pone.0035132

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