Molecular Architecture of the ER-Mitochondria Encounter Structure

Membrane contact sites (MCS) form between all organelles of eukaryotic cells and facilitate interdependent processes between unique subcellular compartments. In budding yeast, MCS between the endoplasmic reticulum (ER) and mitochondria are mediated by a protein complex known as the ER-mitochondria encounter structure (ERMES). ERMES is important for the maintenance of mitochondrial morphology and is implicated in the process of mitochondrial fission. ERMES organises as diffraction limited foci between the ER and mitochondria; however, the molecular architecture of ERMES components within these foci is unknown. Using integrative approaches including correlative light and electron microscopy (CLEM), live-cell and quantitative fluorescence imaging, as well as cryo-CLEM and subtomogram averaging, this work investigates the role of ERMES in mitochondrial fission within yeast and provides a model for the molecular architecture of ERMES from in situ observations. Live-cell imaging experiments reveal that the shape and number of mitochondria per cell are synergistically controlled by ERMES and the availability of the mitochondrial fission protein Dnm1. Both overexpression and deletion of Dnm1 in cells without ERMES affects mitochondrial shape and number, suggesting that ERMES is important for mitochondrial fission but not necessary. CLEM on resin-embedded cells at ERMES and Dnm1 sites shows that mitochondria are more constricted when Dnm1 is absent of ER-mitochondria MCS. ER- mitochondria MCS were also found without ERMES signal, an observation that highlights the importance of CLEM to identify ERMES-mediated ER-mitochondria MCS with ET. Furthermore, I used focussed ion beam milling, which maintains the highest level of cellular preservation amongst current sample thinning techniques, to prepare thin lamellae of vitreous yeast for cryo-ET. A post-milling cryo-fluorescence step was used in this study to identify ERMES within lamellae. Cryo-ET of ER-mitochondria MCS marked by ERMES reveals rod-like protein structures that bridge the ER and mitochondria. Subtomogram averaging of these rod-like proteins yields a density map with three distinct portions which are consistent with the known length of the lipid binding domains of the two ERMES components for which a structure is known. Separately, quantitative live-cell fluorescence microscopy was used to determine the total number and stoichiometry of ERMES components within contact sites. With these results I propose an integrative model of ERMES as rod-like structures which are three-lipid binding domains in length and are arranged in a linear orientation, spanning the distance between the ER and mitochondria.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Wozny, Michael

Advisors dc:contributor.advisor

• Kukulski, Wanda
• Miller, Elizabeth

• cryo-EM
• cryo-ET
• mitochondria
• ER
• membrane contact sites
• ERMES

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.83895

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/336477