Pinkin, N. K. | bioRxiv Science

bioRxiv2026

Tracking the conformational dynamics of single molecules in live cells; GTPase cycling in Cdc42 gradients and microclusters

The Rho family of GTPases play a critical role in many cellular processes by acting as molecular switches relying on conformational changes to transition between active and inactive states. The spatial distribution and kinetics of these conformational changes are complex and vary across the cell. We describe here biosensors based on environment-responsive fluorescent dyes, capable of reporting the conformational changes of individual GTPase molecules as they traffic within living cells (SPARC Cdc42 and SPARC Rac1, 'Single Particle Activation Reporter Constructs'). We use SPARC Cdc42 to determine the rates of different steps in GTPase regulation, activation/inactivation and membrane insertion, that together generate localized gradients of Cdc42 activity driving cell motility. The biosensors also revealed heterogeneously distributed clusters of Cdc42, the mechanisms of their activation, and potential roles in polarized motility.

cell biology↗

bioRxiv2020

Using SNAP-tag for facile construction of dye-based biosensors in living cells

Fluorescent biosensors based on environment-sensitive dyes have important advantages over alternative methodologies such as FRET, including the potential for enhanced brightness, elimination of bleaching artifacts, and more possibilities for multiplexing. However, such biosensors have been difficult to use because they required proteins to be covalently labeled and reintroduced into cells. Recent development of self-labeling enzymes that covalently react with membrane-permeable dyes (e.g. SNAP-tag) provide an opportunity to easily generate dye-based biosensors within cells. Here, we generate a new biosensor for Cdc42 activation by positioning SNAP-tag between Cdc42 and a peptide that binds selectively to active Cdc42. We generate a membrane-permeable Nile Red derivative that exhibits 50-fold fluorescence enhancement upon covalent labeling of the biosensor, then optimize the biosensor so the dye undergoes a 20 nm emission shift upon Cdc42 activation, enabling ratiometric imaging with a single dye. The biosensor, named SNAPsense Cdc42, is validated by examining its response to known regulatory proteins and studying Cdc42 activation during protrusion in living cells. Variants using other dyes are also presented.

cell biology↗