One of the technical challenges in spatial control is that existing ligand-based methods may not be optimal for localized stimulation. corresponding author upon request. Abstract During bone remodelling, osteoclasts induce chemotaxis of osteoblasts and yet maintain spatial segregation. We show that osteoclasts express the repulsive guidance factor Semaphorin 4D and induce contact inhibition of locomotion (CIL) in osteoblasts through its receptor Plexin-B1. To examine causality and elucidate how localized Plexin-B1 stimulation may spatiotemporally coordinate its downstream targets in guiding cell migration, we develop an optogenetic tool for Plexin-B1 designated optoPlexin. Precise optoPlexin activation at the leading edge of migrating osteoblasts readily induces local retraction and, unexpectedly, distal protrusions to steer cells away. These morphological changes are accompanied by reorganization of Myosin II, PIP3, adhesion and active Cdc42. We attribute the resultant repolarization to RhoA/ROCK-mediated redistribution of -Pix, which activates Cdc42 and promotes protrusion. Thus, our data demonstrate a causal role of Plexin-B1 for CIL in osteoblasts and reveals a previously unknown effect of Semaphorin signalling on spatial distribution of an activator of cell migration. In multicellular organisms, migrating cells respond to attractive or repulsive cues to precisely control velocity and directionality, and reach their destination with spatial and temporal accuracy. Contact inhibition of locomotion (CIL) explains the repulsive effect on a migrating cell upon contact with another cell1. CIL has been implicated in many physiological and pathological phenomena such as embryonic development, tissue patterning, collective migration and cancer metastasis. Although CIL has long been observed semaphorins are often present as directional cues for cell migration, and thus experimental perturbations with spatial control at the subcellular scale can offer unique Rabbit polyclonal to FDXR insight into their signalling mechanisms. One of the technical challenges in spatial control is usually that existing ligand-based methods may not be optimal for localized stimulation. Here we describe a novel approach to precisely control the location and time of Plexin-B1 activation with light. We validate this new optogenetic reagent by tracking its binding with two known interacting RhoGEFs, PRG and LARG, and by visualizing its activation of RhoA. We named the new tool optoPlexin, following the naming convention of many optogentic reagents developed in recent years46,48,49. To our knowledge, optoPlexin is the first optogenetic tool Ademetionine disulfate tosylate for the receptors of repulsive guidance molecules. The optogenetic module Cry2 has two impartial50 modes of action, that is, inducible translocation mediated by CIB1 (ref. 31) and homo-oligomerization32, which benefitted optoPlexin design to reduce dark background. We found that mere oligomerization (by omitting CIB-CAAX) is not Ademetionine disulfate tosylate sufficient for inducing binding of PRG, a GEF for RhoA (Fig. 3d,e) or RhoA activation (Fig. 3g). It is likely to be that additional factors such as Rnd1 or Rac1 around the plasma membrane may participate in recruiting PRG20,51,52. In addition, the slow off-rate of Cry2-CIB1 binding helped to maintain the Plexin-RhoGEF complex locally, mimicking sustained contact between osteoblasts and osteoclasts. Using optoPlexin, we exhibited spatial modulation of different regulators of cell migration to understand the signalling mechanism involved in CIL between osteoclasts and osteoblasts. As plexins share high sequence homology in their intracellular regions53, optoPlexin design should be extendable to other plexins as well. Upon Sema4D stimulation ErbB2 associates with and is transactivated by Plexin-B1 and regulates RhoA activation through phospholipase C- (PLC-)37,38. Given that the conversation with ErbB2 is usually mediated by the extracellular domain name of Plexin-B1, it is unlikely to be that optoPlexin can associate with or activate ErbB2, which could be a limitation of optoPlexin. However, clustering of the cytosolic domain name of Plexin-B1 around the membrane was shown to be sufficient for RhoA activation17. Our results also exhibited that optoPlexin sufficiently recruits RhoGEF and activates RhoA. Mutations designed to abrogate PLC- association (optoPlexin-YF) did not affect RhoGEF recruitment, RhoA activation or the repulsion phenotype induced by optoPlexin. It is possible Ademetionine disulfate tosylate that ErbB2-mediated tyrosine phosphorylation of Plexin-B1 and consequent.