LS-2-P-6117 Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

Most proteins have uneven distributions in the plasma membrane¹. This may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. To find out if a general mechanism exists behind this observation, we introduced several imaging approaches that aim to investigate all proteins in the plasma membrane simultaneously rather than focusing on interactions of individual protein species. This was achieved by large-scale metabolic labeling of proteins in mammalian cells through extended incorporation of non-canonical amino acid analogues, followed by fluorescent tagging via click chemistry². By combining this direct labeling method with super-resolution stimulated emission depletion (STED) microscopy, we studied the protein patterning in plasma membranes of living cells, as well as in membrane sheets. We found that a general mosaic-like pattern governs the protein organization³. Multiple proteins were heterogeneously gathered into protein-rich domains surrounded by a protein-poor background. We termed these long-lived high-abundance domains “protein assemblies” and examined the contributions of different factors to their formation and maintenance. We identified cholesterol as the main organizer of the assembly pattern and the actin cytoskeleton as a secondary factor that borders and separates the assemblies. To understand the relation of this mesoscale arrangement to the nanoclusters of individual protein species, we analyzed distributions of specific proteins with respect to the protein assemblies. All of the specific proteins we analyzed were enriched in the assemblies, but they displayed differential enrichment profiles. Many proteins were preferentially located in particular areas within the assemblies, such as their edges or centers. Functionally related protein groups showed similar preferences, suggesting that functional protein-protein interactions create specialized subdomains within the assemblies. We conclude that the assemblies constitute a fundamental principle of the mesoscale membrane organization, which affects the nanoscale patterning of most membrane proteins, and possibly also their activity.

References:

1. Lang T & Rizzoli SO. Membrane protein clusters at nanoscale resolution: more than pretty pictures. Physiology 25, 116–124 (2010).

2. Dieterich DC et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13, 897–905 (2010).

3. Saka SK et al. Multi-protein assemblies underlie the mesoscale organization of the plasma membrane. Nat Commun 5, 4509 (2014).