Rapid optogenetic clustering of a cytoplasmic BcLOV4 variant.

Protein clustering is a powerful form of optogenetic control, yet there is currently only one protein -Cry2-whose light-induced clustering has been harnessed for these purposes. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple clustering to membrane binding, allowing us to engineer a variant of BcLOV4 that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant, BcLOVclust, clustered over many cycles with dramatically faster clustering and de-clustering kinetics compared to Cry2. The magnitude of BcLOVclust clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by optimizing the fluorescent protein to which it was fused. BcLOVclust retained the temperature sensitivity of BcLOV4 such that light induced clustering was transient, and the rate of spontaneous declustering increased with temperature. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. Thus BcLOVclust provides an alternative to Cry2 for optogenetic clustering and a method for multiplexed clustering. While its usage is currently suited for organisms that can be cultured below ~30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.