Biology subjects

Khan, A. U. M.

Publications and source records attributed to Khan, A. U. M..

3 recordsLinked to original sources

bioRxiv2025

Photoclickable HaloTag Ligands for Spatiotemporal Multiplexed Protein Labeling on Living Cells

Precise spatiotemporal control over fluorescence labeling is a powerful approach for selective marking and tracking of proteins of interest within living systems. Here, we report a photoclickable labeling platform based on the 2,3-diaryl-indanone epoxide (DIO) photoswitch scaffold and the self-labeling protein HaloTag. Upon illumination, the protein-bound DIO undergoes reversible photoisomerization to form a metastable oxidopyrylium ylide (PY) that reacts with ring-strained dipolarophiles via [5+2] cycloaddition, enabling covalent spatiotemporal labeling. We synthesize and characterize a library of DIO-HaloTag and DIO-SNAP- tag ligands, systematically examining the effects of linker architecture and scaffold substitution on the photoswitching and photoclick reactivity in vitro and on living cells. We identify a naphthyl-substituted DIO ligand exhibiting superior photoswitching and photoclick efficiency, allowing robust and selective labeling of HaloTag on the surface of living cells using visible light activation. Using this system, we achieve two- and three-color labeling of defined cell surface regions with excellent spatial and temporal precision, additionally allowing combinatorial labeling. Together, this work establishes a versatile framework for multiplexed, light- directed protein labeling compatible with living systems, with promising future applications in long-term tracking and cellular barcoding.

biochemistry↗

bioRxiv2025

Adhesion-driven tissue rigidification triggers epithelial cell polarity

The active regulation of tissue material properties via phase transitions is central in morphogenesis. Transitions abruptly occur at critical points in diverse control parameters, including cell density, shape or adhesion. Whether these parameters are interdependent, performing redundant or distinct functions, is unknown. Here we show that co-regulation of multiple control parameters impacts not only tissue deformability, but also cell polarization. We theoretically define a new phase diagram capturing the material states of zebrafish pluripotent tissues and show that they cross simultaneously critical points in cell density, connectivity and adhesion strength. Combining optogenetics, biophysical measurements and quantitative morphometrics, we independently modulate each parameter, identifying adhesion as the main determinant of tissue rheology. Unexpectedly, uncoupling adhesion-driven from density-driven rigidification in amorphous tissues triggers epithelial organization via tricellular junction formation, followed by luminogenesis and apicobasal polarization. Altogether, this work reveals the non-linear dynamics of emergent tissue mechanics as instructive mechanisms of tissue organization.

biophysics↗

bioRxiv2024

A photoswitchable HaloTag for spatiotemporal control of fluorescence in living cells

Photosensitive fluorophores, which emission can be controlled using light, are essential for advanced biological imaging, enabling precise spatiotemporal tracking of molecular features, and facilitating super-resolution microscopy techniques. While irreversibly photoactivatable fluorophores are well established, reversible reporters which can be re-activated multiple times remain scarce, and only few have been applied in living cells using generalizable protein labelling methods. To address these limitations, we introduce chemigenetic photoswitchable fluorophores, leveraging the self-labelling HaloTag protein with fluorogenic rhodamine dye ligands. By incorporating a light-responsive protein domain into HaloTag, we engineer a tunable, photoswitchable HaloTag (psHaloTag), which can reversibly modulate the fluorescence of a bound dye-ligand via a light-induced conformational change. Our best performing psHaloTag variants show high performance in vitro and in living cells, with large, reversible, far-red fluorescence turn-on upon 450 nm illumination across various biomolecular targets. Together, this work establishes the chemigenetic approach as a versatile platform for the design of photoswitchable reporters, tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging.

biochemistry↗