Title: Linking membrane architecture to function by correlative microscopy
Funder: Medical Research Council

Abstract: Cellular membranes act as dynamic boundaries that delimit compartments and at the same time host an enormous range of vital, membrane-dependent functions. Through local association with protein complexes, membranes are involved in numerous metabolic processes, control the exchange of water, nutrients and signals, perform intracellular trafficking and communication tasks, facilitate biogenesis of cellular components, and provide structural integrity to organelles and cells. Many membrane-bound processes require a fine interplay between protein function and local changes in membrane shape, curvature and topology. While a lot is known about protein function and dynamics associated with these processes, linking these data to changes of the underlying membrane shape is challenging. Therefore, the role of membrane architecture in many cellular processes has remained elusive. Our goal is to understand the interplay of compositional and structural aspects of functional membrane architectures. We have developed a correlative light and electron microscopy procedure that allows precisely locating fluorescent signals in electron tomograms. This method allows us to directly link presence or absence of protein of interest to membrane shape. It also enables acquisition of large, multi-dimensional datasets that can be subjected to quantitative analysis. We also expand this experimental concept by combining it with electron cryo tomography, which allows us to visualize assemblies of protein structures at cellular membranes. Therefore, we develop approaches that combine cryo fluorescence microscopy with electron cryo tomography of vitreous sections, and with cryo-FIB milling of vitrified cells. Our projects address questions linking membrane dynamics with compartmentalization. Contact sites between organelles are paradigms for functional domains within cellular membranes that compartmentalise many physiological processes including lipid transport and signalling. We aim at understanding the interdependence of architecture and function at organelle contacts sites, with a current focus on the mechanisms of lipid transfer at interfaces between endoplasmic reticulum and plasma membrane, as well as between two lipid droplets. We also study the mechanisms of mitochondrial fission and removal of mitochondria through mitophagy in mammalian cells. These processes are part of the cell’s quality control for maintaining a pool of healthy mitochondria. Along the same lines, we study the changes of mitochondrial membrane structure that play a pivotal role during programmed cell death (apoptosis), when mitochondrial proteins are released into the cytoplasm to act as key apoptotic factors. We want to understand the mechanisms by which mitochondrial membranes are perforated and reorganised. We currently use budding yeast and mammalian cell cultures as model systems. In the long term, we are planning to extend our approach to multi-cellular organisms, to eventually address the roles of membrane ultrastructure in tissue physiology and development.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE