Nanoscopic organization of the plasma membrane by inositol lipids
The inositol lipids interact with and regulate all sorts of protein complexes on the plasma membrane. Prominent examples include nascent endocytic compartments and focal adhesions, growth factor receptors, small G-proteins and even ion channels. Inositol lipids such as PtdIns(4,5)P2 or PtdIns(3,4,5)P3 can either recruit or directly activate these complexes – but the mechanistic details of how this happens in space and time are still hazy; are the complexes regulated by acute changes in the local concentrations of the lipids, triggering their assembly and/or activation? Or do local “hot spots” of concentrated lipids acts as pre-defined hubs where recruitment/activation occurs? Conversely, do the lipids even serve as localization factors at all, or do protein-protein interactions amongst the complex components instead concentrate the lipids for the lifetime of the complex?
TIRF (left hand images) and STORM super-resolution images (on the right) of GFP-clathrin and endogenous AP-2 in a COS-7 cell. The enlarged region is 2.2 µm across.
Answering these questions is central to determining how specificity emerges from protein-lipid interactions, and consequently to understand how specific interactions fail in disease – and how therapeutic interventions might one day be tailored to specifically target aberrant interactions. A prominent example is the growth factor signaling pathways that are up-regulated in most cancers, which trigger changes to membrane organization and polarity necessary for both constitutive growth and survival, as well the loss of polarity and a more motile phenotype associated with the epithelial to mesenchymal transition. Selective disruption of these inositol lipid-dependent processes in cancer cells must be balanced against maintaining the essential housekeeping transport and signaling functions occurring in the neighboring healthy tissue – and can only be achieved by understanding how specificity is achieved in building the protein-lipid complexes that mediate both disease-associated and healthy cellular function.
Tackling these questions requires approaches than can (i) resolve individual molecular complexes within the crowded molecular milieu of the plasma membrane, (ii) localize the lipids themselves in the plane of the membrane, and (iii) track both sets of components’ dynamic interaction in real time. For this we primarily turn to advanced optical imaging-based single molecule approaches. The lab is equipped with a Nikon TIRF microscope with latest generation sCMOS camera for multi-color single molecule imaging for dynamic real-time measurements of protein complexes and fluorescent lipids and lipid reporters. An A1R dual resonant/galvo driven confocal scan head allows supporting measurement of molecular population dynamics through fluorescence bleaching and activation experiments. Through close collaboration with the Center for Biologic Imaging, we also have access to super-resolution imaging techniques (such as STORM) for high resolution, static imaging of membrane organization.