An Optogenetic Approach to Delineate Spatial Regulation of Plexin Signaling

Abstract

Plexins are primary receptors of Semaphorins, and transduce guidance signals during embryonic development and tissue remodeling. Previous studies using soluble Semaphorins provide a limited understanding of how intracellular signaling events downstream of Plexins are spatially regulated. Here we developed an optogenetic approach, namely optoPlexin, based on the plant cryptochrome‐2 to specifically activate Plexins at precise subcellular locations with light. Using Plexin‐B1 as an example, we examined the spatiotemporal dynamics of Semaphorin‐4D signaling in osteoblastic MC3T3‐E1 cells. We found that activation of optoPlexin in lamellipodial protrusions led to rapid, local recruitment of PDZ‐containing RhoGEFs, including PDZ‐RhoGEF and LARG (Leukemia associated RhoGEF), subsequent accumulation of myosin and finally, collapse of these protrusions. Biosensors of Rho GTPases identified the specific induction of RhoA activity upon optoPlexin activation. Activation of RhoA pathway required both the presence of a PDZ binding motif in optoPlexin and the interaction of optoPlexin with Rnd1 or Rac1 on the membrane, preserving the specificity of Plexin‐B1 signaling. Activation of optoPlexin also induced redistribution of β‐Pix from sites of illumination to distal regions and concomitant production of new protrusions. We observed activation of Cdc42, rather than Rac1, in these distal protrusions. The optoPlexin‐induced coordination among Rho GTPases was dependent on ROCK kinase activity and myosin‐dependent contractility. These results revealed spatially distinct effects of Plexin‐B1 signaling in regulating Rho GTPases and cell migration, and highlighted the utility of optogenetic approaches in studying spatiotemporal regulation of cellular signaling.