Biology subjects

Benaissa, H.

Publications and source records attributed to Benaissa, H..

3 recordsLinked to original sources

bioRxiv2024

Multiplexed in vivo imaging with fluorescence lifetime modulating tags

Fluorescence lifetime imaging opens new dimensions for highly multiplexed imaging in live cells and organisms using differences in fluorescence lifetime to distinguish spectrally identical fluorescent probes. Here, we describe a set of fluorescence-activating and absorption-shifting tags (FASTs) capable of modulating the fluorescence lifetime of embedded fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives. We show that changes in the FAST protein sequence can vary the local environment of the chromophore and lead to significant changes in fluorescence lifetime. These fluorescence lifetime modulating tags enabled multiplexed imaging of up to three targets in one spectral channel using a single HBR derivative in live cells and live zebrafish embryo. The combination of fluorescence lifetime multiplexing with spectral multiplexing allowed us to successfully image six targets in live cells, opening great prospects for multicolor fluorescence lifetime multiplexing.

cell biology↗

bioRxiv2023

A tripartite chemogenetic fluorescent reporter for imaging ternary protein interactions

Most cellular processes are carried out by multiprotein assemblies. Although various molecular tools exist to visualize binary protein interactions in live cells, the visualization of multiprotein complexes remains a challenge. Here, we report the engineering of a complementation-based approach allowing one to visualize the interaction of three proteins through effective proximity-induced complementation of three fragments of pFAST, a chemogenetic fluorescent reporter that binds and stabilizes the fluorescent state of fluorogenic chromophores (so-called fluorogens). This tripartite-split-pFAST allowed the observation of dynamic ternary protein complexes in the cytosol, at the plasma membrane, in the nucleus and at the junction of multiple organelles, opening great prospects to study the role and function of multiprotein complexes in live cells and in various biologically relevant contexts.

cell biology↗

bioRxiv2021

An engineered multifunctional protein tag for advanced fluorescence imaging

Biocompatible fluorescent reporters with spectral properties spanning the entire visible spectrum are indispensable tools for imaging the biochemistry of living cells and organisms in real time. Here, we present the engineering of a fluorescent chemogenetic reporter with tunable optical and spectral properties. A collection of live-cell compatible fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design. We showed that the ability to tune the fluorescence color and properties through simple molecular modulation provides an unprecedent experimental versatility for imaging proteins in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms. The ability to tune the spectral properties and fluorescence performance enables to match the spectral specifications and requirements of the most advanced imaging techniques, and allowed us to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

molecular biology↗