Biology subjects

El Hajji, L.

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

9 recordsLinked to original sources

bioRxiv2026

Chemogenetic timestamping for the precise tracing of cell history into protein assemblies

Self-assembling protein fibers enable to record events in single cells, bypassing the need for long-term time-lapse imaging. Fluorescent marks introduced within the growing fiber at user-defined times provide timestamps, giving access to the temporal dynamics of the recorded event. Here, we introduce CATCHFiber, a single-color timestamping strategy for tracing cellular events with high temporal resolution into self-assembling protein fibers. Relying on chemically-induced dimerization to precisely and rapidly control the incorporation of fluorescent proteins into the fiber, CATCHFiber allows the introduction of short 30-min spaced timestamps, significantly increasing the precision of event timings compared to existing methods. This increase in temporal resolution expands the use of fiber-based recorders beyond transcriptional activity, allowing to trace the kinetics of faster processes such as protein degradation, protein neosynthesis and kinase activity, and to determine the timing of cell cycle steps.

cell biology↗

bioRxiv2026

Non-covalent reversibly photoconvertible fluorescent tags for wash-free protein labeling

Reversibly photoswitchable fluorophores are widely used in advanced bioimaging but their design remains demanding. Here, we introduce a new series spanning the whole visible range, which results from combining a large set of fluorogens with the FAST protein scaffold. We first demonstrate that these well-established labeling fluorescent protein tags turn into negative reversible photoswitchers upon decreasing the fluorogen concentration and increasing light intensity. We then show that using not anymore one but two fluorogens adds new responses to illumination. Thus, we obtain positive reversible photoswitchers, that increase their brightness under illumination. We also generate a palette of non-covalent reversibly photoconvertible fluorescent proteins changing their fluorescence color upon illumination, a reversible behavior that still remains absent in regular fluorescent proteins. This light-induced color change opens the possibility to discriminate six spectrally similar FAST variants in live cells upon demonstrating the superiority of using multiple spectral channels for exploiting the time dependence of the fluorescence response to illumination.

biophysics↗

bioRxiv2025

Early HIV-1 maturation drives Env clustering and fusion competence

Human immunodeficiency virus type 1 (HIV-1) particles are initially released from the cell in an immature state that cannot cause infection. The proteolytic processing of the Gag structural polyprotein into its individual components triggers a dramatic structural reorganisation that produces fully mature and infectious virions. HIV-1 maturation remodels the Gag lattice to enable membrane fusion, but how early proteolytic steps regulate the envelope glycoprotein (Env) organisation is unknown. Although cryo-electron tomography has defined structural intermediates of maturation, it has not resolved how Env conformers relate to specific early maturation states. Here, we show that partial Gag cleavage, prior to capsid formation, allosterically reorganises the membrane-proximal lattice, promoting Env clustering and fusion-competent conformations. Using native virus preparations combined with cryogenic fluorescence-lifetime imaging correlated to cryo-electron tomography, we map Env conformational signatures onto defined Gag-processing intermediates. This reveals an unrecognised early maturation stage in which incomplete Gag processing primes Env for entry, shifting current models by positioning functional Env activation upstream of core maturation. These results establish that early lattice remodelling is a key determinant of HIV-1 fusion competence and expose a previously inaccessible, intervention-sensitive step with implications for therapeutic and vaccine strategies targeting maturation-entry coupling.

microbiology↗

bioRxiv2025

Photoejection turns non-covalent fluorescent tags into negative reversible photoswitchers

Reversibly photoswitchable fluorophores have enabled a broad range of applications in advanced fluorescence bioimaging. Here, we introduce RSpFAST, a new class of reversibly photoswitchable fluorescent labels that combine a biomolecular host (pFAST protein tag) with a reversibly photoisomerizable guest (fluorogen), allowing fluorescence brightness to be modulated through illumination and molecular complexation. We combine thermokinetic, photochemical, and structural investigations to obtain a comprehensive mechanistic and kinetic understanding of RSpFAST. Building on this theoretical framework, we demonstrate in both live and fixed cells that RSpFAST exhibits an unprecedented dual behavior: a stable and wash-free fluorescent labeling tag turns into a negative reversible photoswitcher by lowering the fluorogen concentration and increasing light intensity. In this photoejection-driven kinetic regime, RSpFAST is shown to be an efficient marker for dynamic contrast and super-resolution microscopy.

biochemistry↗

bioRxiv2025

Chemogenetic modulation of luciferase emission color for imaging and sensing

Bioluminescent luciferases have emerged as powerful tools for bioimaging, enabling to image biological systems without external excitation light, reducing thus phototoxicity and eliminating background autofluorescence. Advanced imaging requires luciferases that deliver high photon output for enhanced sensitivity, tunable emission colors for multicolor imaging, and red-shifted emission for effective deep tissue imaging. Here, we introduce LumiFAST, a small tunable luciferase engineered by fusing the bright blue-light emitting NanoLuc with the tunable chemogenetic fluorescent reporter pFAST. pFAST binds and stabilizes the fluorescent state of a variety of synthetic fluorogenic chromophores (also called fluorogens). Its proximity with NanoLuc leads to efficient bioluminescence resonance energy transfer (BRET), enabling customizable red-shifted emission. Thanks to the small size of pFAST, LumiFAST maintains a compact structure, while its modular design allows emission color to be tuned from cyan to green, yellow, orange and red simply by changing the fluorogen. Systematic optimization of the fusion topology and linker length yielded an optimal variant with apparent BRET efficiencies reaching up to 90 %. The red-shifted emission of LumiFAST enables dual-color microscopy imaging when used alongside NanoLuc and allows imaging through thick scattering media. Beyond imaging, our insights into the structural factors governing efficient BRET allowed us to engineer biosensors based on NanoLuc and pFAST for the visualization of protease activity and protein-protein interactions in live cells.

cell biology↗

bioRxiv2025

Peptide-functionalized fluorescent polymeric nanoparticles: polysarcosine length defines stealth properties

Stealth properties of nanoparticles are essential for their proper functionalization in biological systems. To address limitations of polyethylene glycol (PEG), commonly used for this purpose, we explore the potential of polysarcosine (PSar) as stealth shell in peptide-functionalized dye-loaded polymeric NPs. To this end, polymeric NPs loaded with rhodamine dye with bulky hydrophobic counterion and bearing azide groups at their surface were grafted with PSar of different lengths ranging from 5 to 19 sarcosine units using strain-promoted cycloaddition. The obtained peptide-functionalized NPs showed remarkable colloidal stability in physiological media. The length of PSar showed a profound effect on stealth properties of NPs. The increase in the length of grafted PSar lead to decrease in the negative surface charge to nearly neutral values and decreased protein adsorption according to fluorescence correlation spectroscopy. The NPs with 19mer PSar showed minimal interactions with live cells and glass surfaces in a complex biological medium, in contrast to its shorter PSar analogues. These stealth NPs bearing HaloTag ligand enabled specific targeting of proteins at the cell surface. The obtained results show that a relatively short PSar peptide can be used for achieving stealth properties in polymeric NPs, allowing specific protein targeting with minimized non-specific interactions. The obtained PSar-functionalized polymeric NPs appear as a powerful platform for the fabrication of the next generation of nanomaterials for bioimaging and biosensing applications.

pharmacology and toxicology↗

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↗