Figure 9.
Combining parallel Shp2-mediated pathways to reconstruct cell morphodynamics. (A) Schematic representation of individual signaling pathways mediated by N-SH2 domain of Shp2 and Shp2 signaling in focal adhesions. Protein-protein interactions mediated by the N-SH2 domain of Shp2 are sufficient to induce ROCK II-dependent cell migration following activation of Shp2 (Left). Shp2 activation in focal adhesion complexes results in downstream activation of ERK and ERK dependent spreading in the absence of N-SH2-mediated protein-protein interactions (Middle). Combined signaling through local Shp2 activation in focal adhesion complexes and N-SH2 domain-mediated protein-protein interactions reconstitutes both cell spreading and cell migration (right). (B–E) Created with biorender.com. HeLa cells transiently expressing R138L RapR-Shp2-Cerulean-flag and FAK(397)FRB-mVenus were stained with CellMask Deep Red membrane dye and imaged live. CellMask images were used for analysis. N = 17 cells, three independent experiments. (B) Changes in cell area induced by activation of R138L RapR-Shp2 in focal adhesion complexes binned over 1 h intervals (F[3,1] = 51.08, P < 0.001). (C) Changes in cell migration shown as path length over 1 h intervals (F[3,1] = 14.21, P < 0.001). (D and E) Effect of R138L RapR-Shp2 activation in focal adhesion complexes on Protrusive (D) and Retraction (E) activity binned over 10 min intervals with mean activity at each 2 min timepoint overlayed as a solid line, error bars show 90% CI. Activation of R138 L RapR-Shp2 in complex with FAK induced significant changes in protrusion (F[23,1] = 10.10, P < 0.001) from time points 10–40 min and significant changes in retraction (F[23,1] = 2.53, P < 0.001) from time point 50–60 min. (B–E) Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction relative to the basal (−60 to 0, or 0 min) time-frame post hoc. (B and C) Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean.

Combining parallel Shp2-mediated pathways to reconstruct cell morphodynamics. (A) Schematic representation of individual signaling pathways mediated by N-SH2 domain of Shp2 and Shp2 signaling in focal adhesions. Protein-protein interactions mediated by the N-SH2 domain of Shp2 are sufficient to induce ROCK II-dependent cell migration following activation of Shp2 (Left). Shp2 activation in focal adhesion complexes results in downstream activation of ERK and ERK dependent spreading in the absence of N-SH2-mediated protein-protein interactions (Middle). Combined signaling through local Shp2 activation in focal adhesion complexes and N-SH2 domain-mediated protein-protein interactions reconstitutes both cell spreading and cell migration (right). (B–E) Created with biorender.com. HeLa cells transiently expressing R138L RapR-Shp2-Cerulean-flag and FAK(397)FRB-mVenus were stained with CellMask Deep Red membrane dye and imaged live. CellMask images were used for analysis. N = 17 cells, three independent experiments. (B) Changes in cell area induced by activation of R138L RapR-Shp2 in focal adhesion complexes binned over 1 h intervals (F[3,1] = 51.08, P < 0.001). (C) Changes in cell migration shown as path length over 1 h intervals (F[3,1] = 14.21, P < 0.001). (D and E) Effect of R138L RapR-Shp2 activation in focal adhesion complexes on Protrusive (D) and Retraction (E) activity binned over 10 min intervals with mean activity at each 2 min timepoint overlayed as a solid line, error bars show 90% CI. Activation of R138 L RapR-Shp2 in complex with FAK induced significant changes in protrusion (F[23,1] = 10.10, P < 0.001) from time points 10–40 min and significant changes in retraction (F[23,1] = 2.53, P < 0.001) from time point 50–60 min. (B–E) Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction relative to the basal (−60 to 0, or 0 min) time-frame post hoc. (B and C) Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean.

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