Curated Optogenetic Publication Database

Abstract: The behavior of stem cells is regulated by mechanical cues in their niche that continuously vary due to extracellular matrix (ECM) remodeling, pulsated mechanical stress exerted by blood flow, and/or cell migration. However, it is still unclear how dynamics of mechanical cues influence stem cell lineage commitment, especially in a 3D microenvironment where mechanosensing differs from that in a 2D microenvironment. In the present study, we investigated how temporally varying mechanical signaling regulates expression of the early growth response 1 gene (Egr1), which we recently discovered to be a 3D matrix-specific mediator of mechanosensitive neural stem cell (NSC) lineage commitment. Specifically, we temporally controlled the activity of Ras homolog family member A (RhoA), which is known to have a central role in mechanotransduction, using our previously developed Arabidopsis thaliana cryptochrome-2-based optoactivation system. Interestingly, pulsed RhoA activation induced Egr1 upregulation in stiff 3D gels only, whereas static light stimulation induced an increase in Egr1 expression across a wide range of 3D gel stiffnesses. Actin assembly inhibition limited Egr1 upregulation upon RhoA activation, implying that RhoA signaling requires an actin-involved process to upregulate Egr1. Consistently, static-light RhoA activation rather than pulsed-light activation restricted neurogenesis in soft gels. Our findings indicate that the dynamics of RhoA activation influence Egr1-mediated stem cell fate within 3D matrices in a matrix stiffness-dependent manner.

Abstract: The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A. thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.