Optochemical Control of Protein Localization and Activity within Cell-like Compartments

Abstract

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.

Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions

“…The microscopy-based analysis is amenable to automation and customization in mediumthroughput assays in multi-well plate format, and temporally precise induction using lightresponsive proteins (Glantz et al, 2018) or photocaged ligands (Caldwell et al, 2018). Recently, we used this platform to establish the relative lipid-binding selectivity and signaling structure-function of BcLOV4, a natural photosensory protein that binds anionic membrane phospholipids through a directly light-regulated electrostatic interaction (Glantz et al, 2018); when expressed in cells, this protein is also useful as a single-component system for optogenetic membrane recruitment of fused proteins.…”

“…The method reported here for creating synthetic cell-like emulsion droplets (Figure 4) is adapted from work by others in cell-free signaling (Caldwell et al, 2018;Good, 2016;Good et al, 2013). We first solubilize the lipids in chloroform to facilitate dispensing, and then the organic solvent is evaporated to generate a lipid film that is subsequently resuspended in decane oil and blended with other lipids to the desired relative composition.…”

Chemical modification of proteins with photocleavable groups

“…) stimuli. (Caldwell et al, 2018;He et al, 2017;Hernot & Klibanov, 2008;Kost, Wolfrum, & Langer, 1987;Nappini, Al Kayal, Berti, Norden, & Baglioni, 2011;Pinheiro, Baptista, & Lima, 2008;Wu, Deiters, Cropp, King, & Schultz, 2004) Light is a useful trigger for biological molecules, because light itself is relatively easy to manipulate in space and time.…”

“…Cellular processes such as protein localization and protein-protein interactions have also been studied using this approach. (Caldwell et al, 2018;Priestman & Lawrence, 2010) In our lab, we have used light to control the release of therapeutic proteins from injectable dermal depots.(P. K. Jain, D. Karunakaran, & S. H. Friedman, 2013;K.…”

Aptamer-based optical manipulation of protein subcellular localization in cells

“…While methods using light-responsive small molecules have been reported for manipulating protein translocation activity 6,7 , their applications were limited by severe off-target effects. By combining techniques from optics and genetics, optogenetics showed feasible for providing both high spatiotemporal resolution and high molecular specificity for controlling protein translocation activity [8][9][10][11] . On the other hand, proteins were endowed with photoresponsive directionality through the sitespecific genetic fusion of photoreceptors or molecular tags, which could alter natural protein structure.…”