Biology subjects

Pimenta, F. M.

Publications and source records attributed to Pimenta, F. M..

3 recordsLinked to original sources

bioRxiv2026

RhoG, Rac1 and Cdc42 cooperation in cell protrusion revealed bymultiplexed optogenetics and biosensor imaging

The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoGs effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.

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↗

bioRxiv2019

VIEW-MOD: A Versatile Illumination Engine With a Modular Optical Design for Fluorescence Microscopy

We developed VIEW-MOD (Versatile Illumination Engine With a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.

bioengineering↗