February 2, 2017 at 2.30 pm in AG-80
Revealing the Nanoscale Order of Dynamic Molecules Within Microscale Assemblies (Part-I)
Living systems differ from inanimate ones by their ability to create and sustain ordered assemblies of molecules at the expense of chemical energy. The ‘parts list’ of biomolecular assemblies is being discovered at a rapid pace, but how these parts come together to form functional cellular mechanisms remains an outstanding question in many fields of biology. For example, the principal components of the cellular contractile machinery that shapes, divides, and moves cells have been known for a long time, viz., actomyosin network, plasma membrane, and adhesion complexes. But the dynamic architecture of this machinery remains challenging to measure, especially in three-dimensional (3D) (patho)physiological environments.
Our recent work has led to fluorescence-based computational microscopy assays that reveal nanoscale architecture of molecules within the context of microscale assemblies. We exploit intrinsic polarization of fluorescence to measure sub-resolution orientation and alignment of molecules. We have developed an instantaneous fluorescence polarization microscope (instantaneous fluorescence PolScope) to analyze the dynamic changes in concentration, position, and orientation of molecules (Mehta et al., PNAS 2016). Instantaneous fluorescence PolScope acquires four polarization-resolved images of dynamic molecules with single molecule sensitivity. The image data is then computationally translated into orientation, concentration, and kinetics of the cytoskeletal networks. This computational microscopy approach revealed nanoscale orientation of actin filaments relative to the retrograde flow of the network at the leading edge of cells migrating on 2D surfaces. Analysis of actin filament orientation at the leading edge has been possible only in fixed cells with electron microscopy. Further, in a multi-institutional collaboration (Swaminathan et al., bioRxiv 2016 and Nordenfelt et al., bioRxiv 2016), synergistic use of fluorescence polarization microscopy and computational analysis revealed that integrin transmembrane receptors are ‘actively aligned’ by their engagement with retrograde flow and extracellular ligand. The active alignment of integrin receptors may be a general mechanism used by cells to sense directional cues within extracellular matrix and is uniquely accessible with fluorescence polarization microscopy in live cells.