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. 2021 Jan 29;4:145. doi: 10.1038/s42003-021-01667-4

Fig. 7. Plexin-B2 signaling engages the Ras-GAP domain.

Fig. 7

a Cartoon of Plexin-B2 domain structure. Cell membrane shown as a gray bar; dashed line indicates proteolytic cleavage into α and β chains. Sema, Sema domain; PSI, plexin-semaphorin-integrin domain; IPT, Ig-like fold shared by plexins and transcription factors; RBD, Rho-binding domain; GAP, GTPase activating protein; VTDL, PDZ-domain binding site formed by four C-terminal amino acids for docking of PDZ-Rho-GEF or LARG proteins. b Diagrams of lentiviral vectors encoding cDNA of wild-type or signaling mutants of Plexin-B2. PB2* is CRISPR-resistant cDNA, due to synonymous mutations (X) at sgRNA target site. dEcto lacks extracellular domain, mGAP has mutated GTPase activation domain, mRBD has mutated Rho-binding domain, and dVTDL has deleted PDZ-binding motif. c Cell aggregation assay in hanging drops used for Plexin-B2 (PB2) rescue experiments. Photos and enlargements show aggregates formed from GSCs with the indicated Plexin-B2 mutations in hanging drops after 96 h. Quantifications of aggregate numbers and sizes in hanging drops at 24 or 96 h are shown below. The mGAP and dEcto Plexin-B2 mutants failed to rescue the KO phenotype (marked as blue). n = 5–11 drops per group; one-way ANOVA with Dunnett’s multiple comparison test of each group against SD2-Ctrl; *P < 0.05, ***0.001. d Left, representative micrographs of cell dispersion assay with GSCs expressing mutant Plexin-B2 rescue constructs. Dispersed cells from aggregates were visualized at 4 h with DAPI nuclear stain. Bottom, quantifications show the number of nuclei detached from spheres normalized to mean in the control condition. The PB2 mGAP mutant failed to rescue the KO phenotype (marked as blue). Box plots and whiskers indicate quartiles, center lines indicate median. n = 18–34 spheres per group; one-way ANOVA with Dunnett’s multiple comparison test of each group against control; *P < 0.05, ***P < 0.001. e Model depicting Plexin-B2 signaling through Ras-GAP domain to affect cell biomechanics during GBM invasion.