Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.276824v1?rss=1 Authors: Leyden, F., Uthistran, S., Moorthi, U. K., York, H. M., Patil, A., Gandhi, H., Petrov, E. P., Arumugam, S. Abstract: Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 control the membrane protrusions that rise and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold number of molecules for sustained actin polymerization leading to ruffling. Our experiments also suggest that Rac1 activation leads to actin assembly at the cell membrane that are intrinsically linear, giving rise to the lamellar nature of the protrusions. Deactivating PA Rac1 leads to complex contractile processes leading to formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically enriches in the ruffles. Overall, a biphasic mechanism can be envisioned; a polymerization phase driven by activated Rac1 resulting in growth of membrane protrusions and subsequent deactivation of Rac1 leading to contractile collapse and engulfment. The morphological dynamics and presence of Myo1e suggests that the general architecture and molecular compositions may be conserved across lamellar membrane protrusion machinery in living cells. Copy rights belong to original authors. Visit the link for more info