Supra-molecular structures at the cytoplasmic periphery of the plasma membrane

Abstract

The self-assembly of biomolecules into supra-molecular structures is an important principle underlying cellular organization. These structures define spatial environments for diverse biological processes. In particular, at the cytoplasmic periphery of the plasma membrane (PM) a rich variety of supra-molecular structures are found, relying on lipid-lipid, lipidprotein and protein-protein interactions. Examples include protein-lipid nano-clusters, membrane scaffolds or membrane-membrane contact points. Investigation of such fragile structures is challenging and requires minimally invasive methods. In this work, modern fluorescence microscopy techniques were applied in combination with physical unroofing of cells. This allows for imaging at high signal-to-noise ratio and access to the cytoplasmic leaflet of the basal PM, while preserving the integrity of the supra-molecular structures. This study provides a detailed analysis of two exemplary supra-molecular structures: the syntaxin cluster and ER-PM membrane contact sites (MCSs). <br /> Syntaxin, a transmembrane protein important for synaptic vesicle exocytosis, can be found in local accumulations of presumably 75 molecules within a diameter of 50 - 60nm. In this work, the impact of cytoplasmic protein-protein interactions on the inner architecture of the cluster was studied. Analysis by high resolution microcopy revealed overall conserved physical dimensions of protein clusters even if major parts of the cytosolic protein domains were deleted or the transmembrane region was exchanged for a lipid anchor. Mobility measurements revealed that the N-terminal part of the SNARE motif conveys attraction into the clustered state. Deletion of this protein domain entailed the loosening of clusters as indicated by packing sensitive probes. From these results, it is postulated that there is a basic clustering reaction mediated by general attractive forces acting on the TM segment. For the formation of tight intermolecular packing, however, specific protein domains are necessary. <br /> As a second example, ER-PM membrane contact sites (MCS) were chosen. It has been debated whether lipids are able to spontaneously move between two opposing membranes. For analyzing lipid transfer at the contact sites, metabolic labeling of the membrane lipid phosphatidylcholine (PC) in combination with "click chemistry” was applied. This successfully enabled the visualization of the lipid dynamics, but did not provide an indication for spontaneous lipid transfer (SLT) between the opposing membranes. Similar observations were made for cholesterol. It can be concluded that MCSs do not per se constitute hot spots for SLT of PC and cholesterol. This renders the here developed preparation method attractive for the study of protein-mediated lipid transfer in absence of other lipid transfer mechanisms such as SLT or vesicular transfer. <br /> This work was laid out to characterize two multifaceted supra-molecular structures at the cytoplasmic periphery of the PM. In this context, experimental strategies were developed to probe features of the supra-molecular structures. The here presented results give detailed insights into very specific aspects of these structures. Future investigation could focus on unraveling further aspects of the inner architecture of nano-clusters or clarify the role of proteins associated with lipid transfer at ER-PM MCSs.