Mumford, T. R. | bioRxiv Science

bioRxiv2022

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor

Optogenetic clustering is a versatile method to control protein activity in living cells, tissues, and organisms. Here we show that the BcLOV4 photoreceptor both clusters and translocates to the plasma membrane in response to blue light, representing a new class of light-dependent behavior. We demonstrate that dual translocation and clustering can be harnessed for novel single-component optogenetic tools, including for activation of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are causally linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~3-4-fold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

bioengineering↗

bioRxiv2022

Visual detection of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.

Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity, called CluMPS (Clusters Magnified by Phase Separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context.

bioengineering↗