Biology subjects

El Hajji, L.

Publications and source records attributed to El Hajji, L..

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↗

bioRxiv2024

A tunable and versatile chemogenetic near infrared fluorescent reporter

Near-infrared (NIR) fluorescent reporters provide additional colors for highly multiplexed imaging of cells and organisms, and enable imaging with less toxic light and higher contrast and depth. Here, we present the engineering of nirFAST, a small tunable chemogenetic NIR fluorescent reporter that is brighter than top-performing NIR fluorescent proteins in cultured mammalian cells. nirFAST is a small genetically encoded protein of 14 kDa that binds and stabilizes the fluorescent state of synthetic, highly cell-permeant, fluorogenic chromophores (so-called fluorogens) that are otherwise dark when free. Engineered to emit NIR light, nirFAST can also emit far-red or red lights through change of chromophore. nirFAST allows the imaging of proteins in live cultured mammalian cells, chicken embryo tissues and zebrafish larvae. Its near infrared fluorescence provides an additional color for high spectral multiplexing. We showed that nirFAST is well-suited for stimulated emission depletion (STED) nanoscopy, allowing the efficient imaging of proteins with subdiffraction resolution in live cells. nirFAST enabled the design of a chemogenetic green-NIR fluorescent ubiquitination-based cell cycle indicator (FUCCI) for the monitoring of the different phases of the cell cycle. Finally, bisection of nirFAST allowed the design of a fluorogenic chemically induced dimerization technology with NIR fluorescence readout, enabling the control and visualization of protein proximity.

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↗