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Figure 2. BcLOV4 toolbox for Rho-family GTPase signaling activation. (a) Nodal activation of Rho-family signaling and actin polymerization by GTPases and GEFs. (b) Summary of BcLOV4-derived tools for enhanced signaling of Cdc42, Rac1, and RhoA initiated at the GEF and GTPase level. (c) Schematized induction of filopodia formation by opto-Cdc42 activation. (d) Exemplar images of opto-Cdc42-induced filopodia formation with pulsatile patterned stimulation (1.6% duty ratio), visualized by mCherry fluorescence. White box = blue-light illumination field. Scale = 10 μm. (e) Percent change in miRFP703-LifeAct fluorescence intensity within the stimulated region, compared to BcLOV4-mCherry control. Center line, median; “X”, mean; box limits, upper and lower quartiles; whiskers, 1.5× interquartile ranges. Mann−Whitney U test, uncorrected for multiple comparisons. (**) = p < 0.01. d > 0.5. N = 18 independent videos per condition. (f) Schematized induction of lamellipodia formation by opto- Tiam1 activation. (g) Exemplar images of opto-Tiam1-induced lamellipodia formation with pulsatile patterned stimulation (1.6% duty ratio), visualized by mCherry fluorescence. White box = illumination field. Scale = 10 μm. (h) Percent change cell area within the stimulated region, compared to BcLOV4-mCherry control. Center line, median; “X”, mean; box limits, upper and lower quartiles; whiskers, 1.5× interquartile ranges. Mann−Whitney U test, uncorrected for multiple comparisons. (*) = p < 0.05. d > 0.5. N = 9 independent videos per condition.

Journal: ACS synthetic biology

Article Title: Designing Single-Component Optogenetic Membrane Recruitment Systems: The Rho-Family GTPase Signaling Toolbox.

doi: 10.1021/acssynbio.1c00604

Figure Lengend Snippet: Figure 2. BcLOV4 toolbox for Rho-family GTPase signaling activation. (a) Nodal activation of Rho-family signaling and actin polymerization by GTPases and GEFs. (b) Summary of BcLOV4-derived tools for enhanced signaling of Cdc42, Rac1, and RhoA initiated at the GEF and GTPase level. (c) Schematized induction of filopodia formation by opto-Cdc42 activation. (d) Exemplar images of opto-Cdc42-induced filopodia formation with pulsatile patterned stimulation (1.6% duty ratio), visualized by mCherry fluorescence. White box = blue-light illumination field. Scale = 10 μm. (e) Percent change in miRFP703-LifeAct fluorescence intensity within the stimulated region, compared to BcLOV4-mCherry control. Center line, median; “X”, mean; box limits, upper and lower quartiles; whiskers, 1.5× interquartile ranges. Mann−Whitney U test, uncorrected for multiple comparisons. (**) = p < 0.01. d > 0.5. N = 18 independent videos per condition. (f) Schematized induction of lamellipodia formation by opto- Tiam1 activation. (g) Exemplar images of opto-Tiam1-induced lamellipodia formation with pulsatile patterned stimulation (1.6% duty ratio), visualized by mCherry fluorescence. White box = illumination field. Scale = 10 μm. (h) Percent change cell area within the stimulated region, compared to BcLOV4-mCherry control. Center line, median; “X”, mean; box limits, upper and lower quartiles; whiskers, 1.5× interquartile ranges. Mann−Whitney U test, uncorrected for multiple comparisons. (*) = p < 0.05. d > 0.5. N = 9 independent videos per condition.

Article Snippet: Nature 2009, 461 (7260), 99−103. (29) Byrne, K. M.; Monsefi, N.; Dawson, J. C.; Degasperi, A.; Bukowski-Wills, J. C.; Volinsky, N.; Dobrzynski, M.; Birtwistle, M. R.; Tsyganov, M. A.; Kiyatkin, A.; Kida, K.; Finch, A. J.; Carragher, N. O.; Kolch, W.; Nguyen, L. K.; von Kriegsheim, A.; Kholodenko, B. N. Bistability in the Rac1, PAK, and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches.

Techniques: Activation Assay, Derivative Assay, Control, MANN-WHITNEY