Abstract

Molecule clustering is an important mechanism underlying cellular self-organization. In the cell membrane, a variety of fundamentally different mechanisms drive membrane protein clustering into nanometre-sized assemblies. To date, it is unknown whether this clustering process can be dissected into steps differentially regulated by independent mechanisms. Using clustered syntaxin molecules as an example, we study the influence of a cytoplasmic protein domain on the clustering behaviour. Analysing protein mobility, cluster size and accessibility to myc-epitopes we show that forces acting on the transmembrane segment produce loose clusters, while cytoplasmic protein interactions mediate a tightly packed state. We conclude that the data identify a hierarchy in membrane protein clustering likely being a paradigm for many cellular self-organization processes.

Highlights

  • Molecular components of cells are organized on the large scale through compartmentalization, and on the nm- to mm-scale via self-organization of molecules into clusters

  • In addition to live cells, we studied the constructs in isolated basal plasma membrane sheets

  • We comparatively studied transfected cells by microscopy and western blot: while in microscopy epitope accessibility scales with packing density, for western blot analysis cells are lysed, wherefore it is insensitive to differences in packing density

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Summary

Introduction

Molecular components of cells are organized on the large scale through compartmentalization, and on the nm- to mm-scale via self-organization of molecules into clusters. For the clustering of cytosolic or membrane components, interactions must be strong enough to mediate clustering, but at the same time they should be weak enough to prevent irreversibly forming, cytotoxic aggregates. A prominent example of such self-organization occurs in the cell membrane, where membrane proteins form microdomains. The identified mechanisms underlying clustering are mostly based on forces mediated by the membrane leaflets. Such include hydrophobic mismatch, lipid wetting, protein-lipid ionic sequestering and depletion attraction forces (for review see Destainville et al, 2016; Recouvreux and Lenne, 2016). Highly specific proteinprotein interactions must contribute to the clustering process

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