Journal: bioRxiv

Article Title: Reconstitution of Ras-PI3Kγ membrane communication and feedback using light-induced signaling inputs

doi: 10.64898/2026.02.17.706477

Figure Lengend Snippet: (a) Schematic cartoon showing his 10 -mNG-iLID tethered to a supported lipid bilayer (SLB) via a NiNTA lipid. In the dark, its binding partner, AF647-SspB, is predominantly in solution. A digital micromirror device (DMD) can be used to spatially control the light-induced activation of membrane-tethered iLID and AF647-SspB membrane recruitment. (b) SspB variants (nano, micro, and milli) have distinct membrane absorption properties in the absence of 488 nm light and responses to activating light. All SspB variants exponentially dissociate from the membrane in the dark. (c) Representative TIRF-M images showing SLB localization of iLID and AF647-SspB(micro) in the absence and presence of uniform 488 nm light. (d) TIRF-M image showing DMD-generated patterns of 488 nm-excited iLID on SLBs. (e) TIRF-M images showing DMD-generated shapes (100, 25, 4, and 1 µm 2 ) of 488 nm-excited iLID. (f) Montage of TIRF-M images showing the time-dependent change in AF647-SspB(micro) membrane localization following DMD-guided activation of iLID. (g) Kinetic traces showing AF647-SspB(micro) membrane recruitment and dissociation measured over a range of LED powers (0-50 μW, see Supplemental Fig. 2a ). (h) Representative kinetic trace of AF647-SspB(micro) membrane recruitment and dissociation. Fold membrane enrichment was calculated using (basal recruitment + 488 nm light recruitment)/basal recruitment. Fitting to non-linear one phase decay curves (dashed line) were used to calculated signal decay halftime. (i) Quantification of AF647-SspB(nano, micro, and milli) fold membrane enrichment as a function of LED power. (j) Quantification of AF647-SspB(nano, micro, and milli) signal decay halftime as a function of LED power. (b-j) Membrane composition: 94% DOPC, 2% NiNTA (+iLID conjugated), 2% MCC-PE, and 2% PIP 2 lipids.

Article Snippet: Purified SNAP-RBD and Btk(PH-TH,R49S/K52S)-SNAP were labeled in vitro by combining 1.5x molar excess of SNAP-Surface Alexa546 dye (NEB, Cat# S9132S) or SNAP-Surface Alexa647 dye (NEB, Cat# S9136S) with purified proteins.

Techniques: Binding Assay, Control, Activation Assay, Membrane, Generated

Journal: bioRxiv

Article Title: Reconstitution of Ras-PI3Kγ membrane communication and feedback using light-induced signaling inputs

doi: 10.64898/2026.02.17.706477

Figure Lengend Snippet: (a) Kinetics of membrane-anchored Ras activation in the presence of 50 nM RasGEF. Ras activation was monitored with 50 nM AF546-labeled Ras binding domain (RBD). Representative images show bulk membrane localization and specificity of AF546-RBD on bilayers with inactive Ras(GDP) and active Ras(GTP). (b) Cartoon schematic showing how light-induced activation of iLID drives membrane recruitment of AF647-GEF-SspB to locally activate membrane-anchored Ras. Global GAP inhibition reverses light-mediated activation of Ras. (c) Representative TIRF-M montages showing the time-dependent change in AF647-GEF-SspB(micro) membrane localization and Ras activation following local iLID activation with patterned 488 nm light (dashed blue circle). Images are separated by 25 seconds. (d) Kinetic traces showing membrane recruitment and dissociation of AF647-GEF-SspB(micro) and AF546-RBD following a 50 ms 488 nm light pulse. (e) Varying RasGAP concentrations (0-3200 nM) modulate the initial steady-state fraction of activated Ras and response to local light-induced activation by GEF-SspB(micro). (f) Quantification of the light-induced fold increase in Ras(GTP) as a function of global RasGAP concentration in (e) . Fold activation was calculated using (steady-state light-independent Ras activation + peak 488 nm light dependent activation)/steady-state light-independent activation. (c-f) Membrane composition: 92% DOPC, 2% NiNTA (+iLID bound), 2% MCC-PE (+Ras bound), and 4% PIP 2 lipids. For all reactions, GEF refers to the catalytic domain of SOS1 (i.e. SosCat).

Article Snippet: Purified SNAP-RBD and Btk(PH-TH,R49S/K52S)-SNAP were labeled in vitro by combining 1.5x molar excess of SNAP-Surface Alexa546 dye (NEB, Cat# S9132S) or SNAP-Surface Alexa647 dye (NEB, Cat# S9136S) with purified proteins.

Techniques: Membrane, Activation Assay, Labeling, Binding Assay, Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Reconstitution of Ras-PI3Kγ membrane communication and feedback using light-induced signaling inputs

doi: 10.64898/2026.02.17.706477

Figure Lengend Snippet: (a) Diagram of light-inducible Ras activation module. Global RasGAP activity prevents Ras activation until light-induced recruitment of GEF-SspB generates a transient pool of Ras(GTP), which can be visualized via an AF546-RBD biosensor. This reaction is amplified by a positive feedback loop mediated by GEF(fb). (b) Hyperbolic Ras activation kinetics in the presence of 5 nM GEF and sigmoidal Ras activation kinetics in the presence of 1 nM GEF(fb). (c) Representative TIRF-M image showing Ras(GTP) (AF647-RBD) patterns spontaneously formed in the presence of GEF(fb) and RasGAP in the absence of 488 nm activating light. (d) Ras activation requires a threshold density of AF647-GEF-SspB to trigger GEF-mediated positive feedback. Representative TIRF-M images showing the region of iLID emission following excitation with patterned 488 nm light (left), AF647-GEF-SspB membrane localization with corresponding fold enrichment (middle), and the resulting Ras activation over time (right). (e) Change in the fraction of locally activated Ras over time for the experiment shown in (d). (f) Graph showing the change in the radii of the regions of AF546-RBD fluorescence over time for the experiment shown in (d). (b) Membrane composition: 98% DOPC and 2% MCC-PE (+Ras bound) lipids. (c-f) Membrane composition: 92% DOPC, 2% NiNTA (+iLID bound), 2% MCC-PE (+Ras bound), and 4% PIP 2 lipids.

Article Snippet: Purified SNAP-RBD and Btk(PH-TH,R49S/K52S)-SNAP were labeled in vitro by combining 1.5x molar excess of SNAP-Surface Alexa546 dye (NEB, Cat# S9132S) or SNAP-Surface Alexa647 dye (NEB, Cat# S9136S) with purified proteins.

Techniques: Activation Assay, Activity Assay, Amplification, Membrane, Fluorescence