Tricalbin-Mediated Contact Sites Control ER Curvature to Maintain Plasma Membrane Integrity

Abstract

SummaryMembrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) play fundamental roles in all eukaryotic cells. ER-PM MCS are particularly abundant in Saccharomyces cerevisiae, where approximately half of the PM surface is covered by cortical ER (cER). Several proteins, including Ist2, Scs2/22, and Tcb1/2/3 are implicated in cER formation, but the specific roles of these molecules are poorly understood. Here, we use cryo-electron tomography to show that ER-PM tethers are key determinants of cER morphology. Notably, Tcb proteins (tricalbins) form peaks of extreme curvature on the cER membrane facing the PM. Combined modeling and functional assays suggest that Tcb-mediated cER peaks facilitate the transport of lipids between the cER and the PM, which is necessary to maintain PM integrity under heat stress. ER peaks were also present at other MCS, implying that membrane curvature enforcement may be a widespread mechanism to regulate MCS function.

Reliable estimation of membrane curvature for cryo-electron tomography

“…PyCurv was already applied in a cryo-ET study in yeast proposing that cER membrane curvature plays a key role in the regulation of ER-to-PM lipid homeostasis at membrane contact sites [16]. Moreover, the analysis of data generated by MRI and light microscopy shows that PLOS COMPUTATIONAL BIOLOGY our method can be applied to any segmented membrane compartments or other volumes from which a surface can be extracted, originating from any 3D imaging technique.…”

“…As real-world test input files for PyCurv, in this study we used membrane segmentations from in situ cryo-ET data collected from vitrified cells: a human HeLa cell [17], yeast Saccharomyces cerevisiae (EMD-10767 and EMD-10765) and a primary mouse neuron (EMD-10766). The cells were milled down to 150-250 nm thick lamellas using cryo-focused ion beam [16,54] and imaged using a Titan Krios cryo-electron microscope (FEI), equipped with a K2 Summit direct electron detector (Gatan), operated in dose fractionation mode. Tilt series were recorded using SerialEM software [55] at magnifications of 33,000 X (pixel size of 4.21 Å) for the HeLa cell and the mouse neuron and 42,000 X (pixel size of 3.42 Å) for yeast, typically from -50˚to +60˚with increments of 2˚.…”

“…AVV and SSVV proved most robust to quantization noise in synthetic surfaces. To evaluate their performance on real cryo-ET data, we used a cER compartment segmentation that contains several high curvature regions or peaks [16].…”

“…We have recently employed cryo-ET to visualize peaks of extreme curvature on the cortical endoplasmic reticulum (cER) membrane facing the plasma membrane (PM). These high curvature structures are formed by Tcb proteins and help to maintain PM integrity under heat stress [16]. We have also used cryo-ET to show that polyQ-expanded huntingtin exon I fibrils induce high curvature in the endoplasmic reticulum (ER) membrane, perhaps leading to ER membrane disruption [17].…”

Osh6 requires Ist2 for localization to the ER-PM contacts and efficient phosphatidylserine transport

“…1A). Among the proteins that co-purified with Osh6 (Table S1), we identified Ist2, an important yeast ER-PM tether (Manford et al, 2012;Wolf et al, 2012;Hoffmann et al, 2019;Collado et al, 2019). Due to their over-lapping localization, Ist2 seemed a good candidate for mediating Osh6 targeting to the cortical ER.…”