Active Patterning of Cell Surface Molecules from Nanoscale Clusters to Mesoscale Membrane Mosaics Dictated by Dynamic Actin

The spatial distribution and remodeling dynamics of nanoscale clusters of outer-leaflet GPI-anchor proteins (GPI-APs), glycolipids and inner-leaflet Ras proteins are regulated by the activity of the underlying cortical acto-myosin machinery. Recently, we proposed a theoretical framework based on active hydrodynamics of short polar filaments interacting with myosin motors to explain the nanoclustering of the GPI-APs. This framework involves the coupling of cell-surface molecules to these dynamic filaments of actin at the inner leaflet, this in turn leads to their transient clustering. We predicted and experimentally confirmed that transmembrane proteins with cytoplasmic domains capable of directly binding filamentous actin (TM-ABD), would be organized both at nanoscale (<20 nm) and mesoscale (300-1000 nm) by the same dynamic actin, akin to the GPI-APs. We expect the two molecules (GPI-AP and TM-ABD) to have varying affinity (or strength of coupling) towards the dynamic filaments owing to differences in their coupling mechanism with the actin, and our theoretical analysis shows that this will have consequences on their cluster remodeling dynamics and spatial segregation as well as sorting of these molecules. Here, we explore the differences in the dynamics of nanoclustering between the two molecules, using a novel multi-point photolysis tool. Moreover, using Homo-FRET imaging, we show how the clusters of these two molecules are spatially segregated at both nanoscale and mesoscale to form distinct domains. Using polarity sensitive membrane probes we study the local lipid environment of the clustered regions of the two probes. Our results show how an actin-based mechanism can drive the active phase segregation of membrane domains in a living context. These studies will eventually contribute significantly in building up a generalized paradigm for understanding molecular organization and its spatio-temporal regulation on the plasma membrane.