Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation

Area of Science:

• Cell biology
• Biochemistry
• Molecular imaging

Background:

• Biosensors reporting endogenous protein activity in vivo are crucial for understanding cellular processes.
• Environment-sensing fluorescent dyes are often used, but typically show intensity changes, limiting precise quantitation.
• Ratiometric imaging requires dyes with shifts in excitation or emission maxima, which are rare.

Purpose of the Study:

• To develop a novel ratiometric biosensor for measuring Cdc42 (a key signaling protein) activity in vivo.
• To introduce mero199, an environment-sensing dye with a significant solvent-dependent excitation shift.
• To enable simultaneous monitoring of Cdc42 and Rac1 activities in live cells.

Main Methods:

• Development of mero199, a fluorescent dye exhibiting a 33 nm excitation shift with solvent polarity.
• Creation of CRIB199, a ratiometric biosensor for Cdc42 activation using the mero199 dye.
• Simultaneous imaging of CRIB199 (for Cdc42) and a FRET sensor (for Rac1) in cellular protrusions.
• Utilizing a novel program, EdgeProps, to correlate protein activity with cell edge dynamics.

Main Results:

• The mero199 dye enabled ratiometric sensing of Cdc42 activity without additional fluorophores.
• Simultaneous observation revealed that Rac1 activity, not Cdc42, was reduced during cell tail retraction.
• Specific protrusions showed reduced Cdc42 activity, and EdgeProps analysis indicated Rac1 reduction during retraction.

Conclusions:

• Mero199 is a versatile dye for creating ratiometric biosensors for intracellular protein activity.
• Cdc42 and Rac1 play distinct roles in cell protrusion dynamics, with Rac1 being critical for retraction.
• The developed biosensors and analysis tools offer new capabilities for studying Rho GTPase signaling in vivo.