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Figure 2.1: Comparison of FRET Signal Measurement in Intramolecular and Intermolecular GTPase Activity FRET Sensors.

(A) Single-chain probe design: a schematic representation of the intramolecular FRET activity biosensor. GTP: guanosine triphosphate, GDP: guanosine diphosphate, CFP: cyan fluorescent protein, YFP: yellow fluorescent protein.

(B) Dual-chain probe design: a schematic representation of the intermolecular FRET activity biosensor. GTP: guanosine triphosphate, GDP: guanosine diphosphate, CFP: cyan fluorescent protein, YFP: yellow fluorescent protein.

(C and D) Representative images and quantitation of effects of binding domain truncation on the FRET/CFP emission ratio (RhoA activity) in both single-chain and dual-chain sensors. Red outline in panel at far right indicates location of cell. Heat map indicating dynamic range scaling is shown for each cell. Data are represented as means  95% confidence intervals at α = 0.05.

(E) Representative images of the FRET ratio, total bleedthrough corrected FRET signal, and CyPet-Rac1 or CyPet-Rac1/K-Ras4b modified as acquired by widefield

epifluorescence or total internal reflection microscopy. Red lines indicate linescans used for analysis in panel (F).

(F) Representative linescans of the normalized intensities for FRET ratio, total bleedthrough corrected FRET signal, and CyPet-Rac1 or CyPet-Rac1 (K-Ras4b modified) as acquired by widefield epifluorescence or total internal reflection microscopy.

Figure 2.2: Comparison of Sensitivity of Intramolecular and Intermolecular GTPase Activity FRET Sensors.

(A) Quantitation of raw FRET emission intensity in dual-chain versus single-chain RhoA sensors as assessed in adherent mouse embryonic fibroblasts at equivalent expression levels.

(B) Quantitation of detection of a 20% increase in RhoA FRET signal at the leading edge of protrusions compared to cell center in migratory mouse embryonic fibroblasts

expressing varying concentrations (as assessed by CFP Intensity) of a dual-chain or single-chain RhoA sensor. Black data points indicate presence of a 20% increase, red data points indicate absence of a 20% increase. Solid red lines indicate means of CFP intensity where there was absence of a 20% increase, with dashed red lines indicating confidence intervals at α = 0.05.

(C) Quantitation of FRET efficiency as determined by fluorescence lifetime microscopy (FLIM) in Cos-7 cells expressing either a dual-chain Rac1 sensor or a single-chain RhoA sensor in either non-stimulated (“Free”) or stimulated (“Bound”) states. Data are represented as means.

Figure 2.3: Comparison of C-terminal Modifications in GTPase Activity FRET Sensors.

(A) Alignment of C-termini of Rac1 components used to generate wild-type, no-tail, and K-Ras-modified dual chain probes. K-Ras sequence appended to Rac1 is highlighted in red.

(B) Representative images of effects of C-terminal tail modification in Rac1 dual-chain sensors on FRET/CFP emission ratio (Rac1 activity, left panel) and localization (right panel). Heat map indicating dynamic range scaling is shown for each cell.

(C) Representative images of effects of C-terminal tail modification of a Rac1 dual-chain sensor on localization to membranes (a), with quantitation of relative intensity of internal membranes and cytosol (cell center) versus the plasma membrane (b). Data are represented as means  95% confidence intervals at α = 0.05.

(D) Effects of C-terminal tail modification of a Rac1 dual-chain sensor on normalized FRET/CFP emission ratios representing Rac1 activity. Biosensor responses were assessed with wild type and K-Ras modified dual-chain Rac1 biosensors in 293 cell suspension assays in the presence of the GEF Vav2, control DNA, or RhoGDI-1. Data are represented as means  95% confidence intervals at α = 0.05.

(E) Representative images of effects of C-terminal tail modification of a RhoA dual- chain sensor on localization to membranes and the golgi (indicated by the red arrow in the wild-type condition).

Figure 2.4: Rational Development and Improvement of Intramolecular and Intermolecular GTPase Activity FRET Sensors.

(A) Effects of linker length on normalized FRET/CFP emission ratios representing Rac1 activity. Biosensor responses were assessed with a wild type single-chain Rac1 biosensor in 293 cell suspension assays. Biosensor response was assessed in the presence or

absence of the GEF Vav2 or RhoGDI-1. Data are represented as means  95% confidence intervals at α = 0.05.

(B) Representative images of effects of linker length on Rac1 single-chain sensor localization. Left panel illustrates continued membrane localization in the presence of GDI with a 1L linker, but increasing to a 4L linker causes all Rac1 to be cytosolic in the presence of GDI (middle panel). Right panel illustrates membrane localization of sensor in the presence of the GEF Vav2.

(C) Effects of assay type on normalized FRET/CFP emission ratios representing Rac1 activity. Biosensor responses were assessed with a wild type single-chain Rac1 biosensor in 293 cell suspension assays and in adherent Cos-7 cells in the presence or absence of constitutively active Tiam-1. Data are represented as means  95% confidence intervals at α = 0.05.

(D) Effects of fluorophore positioning on normalized FRET/CFP emission ratios representing RhoA activity. Biosensor responses were assessed with a wild type dual- chain RhoA biosensor in 293 cell suspension assays in the presence of a RhoA binding domain from Rhotekin tagged at either the N- or C-terminus with YPet. Data are represented as means  95% confidence intervals at α = 0.05.

chain RhoA biosensor in 293 cell suspension assays in the presence of RhoA binding domains from Rhotekin of increasing length tagged at the C-terminus with YPet. Data are represented as means  95% confidence intervals at α = 0.05.

(F) Effects of increasing the number of acceptor fluorophores on normalized FRET/CFP emission ratios representing RhoA activity. Biosensor responses were assessed with a wild type dual-chain RhoA biosensor in 293 cell suspension assays in the presence of RhoA binding domains from Rhotekin tagged at the C-terminus with either one or two YPet acceptors. Data are represented as means  95% confidence intervals at α = 0.05.

Figure 2.5: Generation of Orthogonal Red-Shifted FRET Probes for use with CFP/YFP FRET Probes.

(A) Dual-chain probe design: a schematic representation of the intermolecular FRET activity biosensor. GTP: guanosine triphosphate, GDP: guanosine diphosphate, mKO: monomeric Kusabira orange fluorescent protein, mCherry: red fluorescent protein. (B) Emission intensity scans from 293 cell suspension assays with cells expressing a CFP/YFP RhoA single-chain sensor or a red-shifted single-chain RhoA sensor in the presence of either the catalytic DH/PH domain of the GEF Tim (active) or RhoGDI-1 (inactive). Emission intensities are normalized to the CFP or mKO peak at a value of 1.0 for comparison. FRET quantity is indicated by the second, right-most peak in each scan (~525 nm for CFP/YFP and ~ 610 nm for mKO/mCherry). Data are represented as an average of three experiments.

(C) Emission intensity scans from 293 cell suspension assays with cells expressing either a CFP/YFP dual-chain Rac1 sensor or a red-shifted tandem-mCherry acceptor dual-chain Rac1 sensor in the presence of either the catalytic DH/PH domain of the GEF Vav2 (active) or RhoGDI-1 (inactive). Emission intensities are normalized to the CFP or mKO peaks at a value of 1.0 for comparison. FRET quantity is indicated by the second, right- most peak in each scan at ~ 525 for CFP/YFP and ~610 nm for mKO/mCherry). Data are represented as an average of three separate experiments.

Figure 2.6: Generation of a Novel Expression Cassette for Dual-Chain FRET Sensors.

(A) Dual-chain probe design: a schematic representation of the intermolecular FRET Rac1 activity biosensor. GTP: guanosine triphosphate, GDP: guanosine diphosphate, CyPet: cyan fluorescent protein, YPet: yellow fluorescent protein.

(B) Dual-chain autocleavable probe design: a schematic representation of the intermolecular FRET Rac1 autocleavable activity biosensor. GTP: guanosine

triphosphate, GDP: guanosine diphosphate, CyPet: cyan fluorescent protein, YPet: yellow fluorescent protein, A2 sequence: autocleavable motif.

(C) Representative western blot illustrating appropriate cleavage and expression of dual- chain autocleavable Rac1 activity biosensor. Wild-type or mutated versions of the A2 sequence lacking autocleavable function are shown demonstrating that lack of a functional A2 sequence leads to expression of a single higher molecular weight band corresponding to uncleaved product.

(D) Quantitation of CFP:YFP expression level ratio in adherent Cos-7 cells expressing the autocleavable Rac1 dual-chain sensor or selected manually for imaging from a population of Cos-7 cells transfected with both components of the dual-chain sensor. Solid black lines represent means, and dashed black lines indicate 95% confidence intervals at α = 0.05.

(E) Representative pseudocolored images of differential localization of the CFP and YFP components (CyPet-Rac1, YPet-PBD) of the Rac1 dual-chain autocleavable sensor (mutant, left panel and wild-type, right panel) demonstrating differential localization. (F) Representative linescans across the cell body demonstrating the localization of the

autocleavable sensor taken from the images in panel (E) with either the wild-type P2A sequence, or the AA mutant.

(G) Representative images of FRET/CFP emission ratio from wild-type and autocleavable dual-chain Rac1 activity sensors. Images are scaled so that regions of intense GTPase activity are shown in white. Heat map indicating dynamic range scaling is shown for each cell.

Supplemental Figure 2.1, Related to Figure 2.1: Comparison of FRET Signal and Measurement in Intramolecular and Intermolecular Rho GTPase Activity Sensors.

(A) Response of RhoA.SC RBD truncation mutant sensor to regulation by GEFs, GAPs, and GDI-1 as assessed by changes in normalized FRET/CFP emission ratios representing RhoA activity. Biosensor responses were assessed with an RBD-truncated and wild-type single-chain RhoA biosensor in 293 cell suspension assays in the presence or absence of regulators of RhoA. Data are represented as means  95% confidence intervals at α = 0.05. Asterisks indicate p < 0.05 compared to wild-type (RhoA.SC RBD truncation) or GEF (Wild-type RhoA.SC).

(B) Representative images of RhoA activity in mouse embryonic fibroblasts and HeLa cells acquired using RhoA.SC and RhoA.DC sensors illustrating differences in resultant FRET signal from both probes in different cell lines.

(C) Representative images of Cdc42 activity (left panel) and Cdc42 protein localization (right panel) using a constitutively active Cdc42 (Q61L) illustrating areas of non-uniform FRET activity (black arrows) in the cytoplasm.

Supplemental Figure 2.2, Related to Figure 2.2: Analysis of Perturbation of Downstream Signaling by Overexpression of Intramolecular and Intermolecular Rho GTPase FRET Biosensors.

Representative western blot illustrating the effects of overexpression of Rac1 and RhoA dual- chain and single-chain sensors on cofilin phosphorylation in the presence or absence of serum stimulation after 20 minutes. Top-most cofilin and phospho-cofilin bands correspond to experiments performed with the Rac1 dual-chain and single-chain sensors. Lower cofilin and phospho-cofilin bands correspond to experiments performed with the RhoA dual-chain and single-chain sensors. Phospho-MAPK and total MAPK levels are given to show that serum stimulation was effective. Short and long exposures for GFP indicate the expression of RhoA dual-chain and single-chain sensors at low and high levels. Actin expression levels are given as a loading control.

Supplemental Figure 2.3, Related to Figure 2.4: Validation of Updated or Newly Generated GTPase FRET Probes.

Validation of various dual-chain and single-chain GTPase sensors, as indicated, by assessing normalized FRET/CFP emission ratios representing activity in the presence or absence of regulatory molecules, as indicated. Biosensor responses were assessed in 293 cell suspension assays in the presence of GEFs, GAPs, and GDIs, as indicated. Data are represented as means.

CHAPTER THREE: RHOA, RAC1, AND CDC42 GTPASE DYNAMICS AT THE CELLULAR LEADING EDGE