Colocalization controls

Self-organisation of membrane domains

Biological membranes form selective barriers between cellular compartments. The plasma membrane (PM) in particular plays an essential role as the site for cellular homeostasis and as a signal-processing hub. Perturbations in PM organization contribute to the development of diseases. A detailed understanding of the mechanisms that mediate biological membrane organization is therefore of general relevance. It is generally thought that many membrane components organize into specific domains, although the mechanisms responsible are still a matter of intense debate. Recently, we have used a combination of Total Internal Reflection Fluorescence Microscopy (TIRFM) and deconvolution1 to demonstrate that all proteins in the yeast PM are organized into distinct domains and that they exhibit unusually slow diffusion2. Importantly, we found that PM proteins with similar transmembrane (TM) domains co-segregated, and that domain patterns were strongly influenced by the cellular lipid composition. Our findings indicate that the yeast PM self-organizes into a patchwork of overlapping protein domains that likely result from weak interactions between the diverse lipid and protein constituents typical for biological membranes.

We now want to use a combination of experimental and numerical approaches to investigate if a specific "transmembrane code" is responsible for lateral positioning of integral proteins in the PM. To this end we will characterize localization profiles of GFP fusions to various endogenous, mutated, chimeric or synthetic TM sequences using TIRFM. We will measure protein mobility by fluorescence recovery after photobleaching and autocorrelation analysis. We will then vary lipid composition in cells and in the PM through genetic modifications and drug treatments. To better resolve domain boundaries, we will use structured illumination microscopy (SIM) and stochastic localization techniques such as PALM and STORM. We have also established chemical labeling strategies in yeast cells (SNAP/CLIP or ACP/MCP) which will allow us to use chemical fluorophores with their superior photo stability and switching kinetics.

  1. Visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy
    Spira F, Dominguez-Escobar J, Muller N, Wedlich-Soldner R. . J Vis Exp. 63, e3982 (2012)
  2. Patchwork organization of the yeast plasma membrane into numerous coexisting domains
    Spira F, et al. Nat Cell Biol. 14, 640-8 (2012)