However, it did not confer responsiveness to PlxnA2 (Fig 7DC7H). Open in a separate window Fig 7 Cell-contraction induced by PlxnA2-Sema6A interaction depends on Abl signaling.(A-F) NIH3T3 cells expressing GFP (A and D), Sema6A-FL (B and E), and Sema6A-?Abl (C and F) were treated with purified AP-Fc (AP; a-c) or PlxnA2-EC-Fc (PlxnA2-EC; D-F). test.(TIF) pone.0158686.s002.tif (246K) GUID:?BD640F95-5D93-4C2A-8AA7-60756E403150 S3 Fig: PD-1-IN-1 Co-expression of PlxnA2 or Sema6A does not affect expression levels. PD-1-IN-1 Immunoblots of total lysates from transfected COS-7 cells expressing PlxnA2, Sema6A or PlxnA2 and Sema6A shows roughly comparable levels of expression of the two proteins and no change in expression LRCH4 antibody levels when they are co-expressed.(TIF) pone.0158686.s003.tif (257K) GUID:?4FFF48B5-FC82-4E0C-A123-EBAFB4CCE3BC S4 Fig: Sema6A monomers can still interact with PlxnA2 in 0.05; one-way ANOVA followed by Bonferroni multiple comparison test.(TIF) pone.0158686.s006.tif (1.2M) GUID:?80C9FE2D-E0AA-49B6-9B2A-5E4A5AB91ED1 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The transmembrane semaphorin, Sema6A, has important roles in axon guidance, cell migration and neuronal connectivity in multiple regions of the nervous system, mediated by context-dependent interactions with plexin receptors, PlxnA2 and PlxnA4. Here, we demonstrate that Sema6A can also signal cell-autonomously, in two modes, constitutively, or in response to higher-order clustering mediated by either PlxnA2-binding or chemically induced multimerisation. Sema6A activation stimulates recruitment of Abl to the cytoplasmic domain of Sema6A and phos?phorylation of this cytoplasmic tyrosine kinase, as well as phosphorylation of additional cytoskeletal regulators. Sema6A reverse signaling affects the surface area and cellular complexity of non-neuronal cells and aggregation and neurite formation of primary neurons but not vice versa. These experiments reveal the complex nature of Sema6A biochemical functions and the molecular logic of the context-dependent interactions between Sema6A and PlxnA2. Introduction Morphogenesis of the nervous system requires the migration of myriad cell types to their preordained positions, the guidance of growing axons along stereotyped pathways to their final target regions and the pairing of pre- and postsynaptic cellular partners. Semaphorins comprise a large family of proteins with important roles in many of these processes, coordinated with the families of their receptor proteins, neuropilins and plexins . The transmembrane Semaphorin-6 subclass consists of four members, which interact directly with members of the Plexin-A subclass. Of these, Semaphorin-6A (Sema6A) and Semaphorin-6B (Sema6B) form a cognate subgroup with Plexin-A2 (PlxnA2) and Plexin-A4 (PlxnA4). Genetic studies have revealed context-dependent interactions between members of this group, which control processes of cell migration, axon guidance and neuropil organization in various parts of the developing nervous system . Sema6A signals cell-non-autonomously via PlxnA2 on responding cells to initiate a switch in migratory mode in cerebellar granule cells [2, 3] and to restrict motor neuron cell bodies from exiting the ventral nerve root in the spinal cord [4, 5]. Sema6A signals via PlxnA4, to confine corticospinal projections [6, 7] and to establish laminar connectivity in the retina [8, 9]. In the developing hippocampus, signals from both Sema6A and Sema6B restrict mossy fibre projections via PlxnA4 [10, 11]. In addition to trans interactions across cells, interactions are also important. In the hippocampus, PlxnA2 co-expression in the target zone antagonizes Sema6A-PlxnA4 signaling and defines a permissive zone for mossy fibre projection and synapse formation [10, 11]. interactions between Sema6A and PlxnA2 may also be important in controlling dendritic arborization of retinal ganglion cells . Direct binding in has PD-1-IN-1 also been demonstrated between Sema6A and PlxnA4. In sensory neurons, co-expression of Sema6A inhibits the response of PlxnA4 to Sema6A in trans, thus making these neurons insensitive to this cue, unlike sympathetic neurons, which normally express only PlxnA4 . The fact that the four proteins in this cognate group are often co-expressed suggests that such interactions may contribute significantly to the combinatorial logic of their functions in other contexts. Signaling may also occur in a bidirectional manner between these proteins. Interactions in the canonical forward direction, with Sema6A or Sema6B as the ligand and PlxnA2 and/or PlxnA4 in the responding cells, cannot readily account for all the phenotypes observed in animals with mutations in these genes, many of which are apparent in axons normally expressing Sema6A [14, 15] or co-expressing Sema6A and PlxnA2 . Moreover, there are several known examples of related transmembrane semaphorins that can also signal in the reverse direction, mediating cell-autonomous responses to exogenous cues. In Drosophila, Sema1a, the orthologue of the Sema6 family, acts as a receptor for.