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A selection of articles by Francisco Mesquita (March 2019)

Site-specific protein labelling helps tracking intracellular membrane dynamics
From Nature Chemical Biology (2019)

In recent years, several methods for protein labelling have been developed to overcome the limitations of large fluorescent protein tags. One approach involves labelling proteins with functionalised small-molecule fluorophores using bioorthogonal chemistry. This requires introducing chemical functionality into proteins, which can be achieved by incorporating bioorthogonal unnatural amino acids that have minimal impact in protein structure and allow position-specific labelling. Previous studies highlighted the potential of such strategy, but mostly focusing on surface proteins. In 2016, Howard Hang’s group from the Rockefeller University, successfully optimized the conditions for site-specific labelling and imaging of interferon-inducible transmembrane protein 3 (IFITM3), an immune modulator that protects against various viral infections. Protection requires S-palmitoylation of IFITM3. However, since palmitoylation does not affect IFITM3 steady state levels nor localisation, the details of such protective mechanism remain unknown. In the latest issue of Nature Chemical Biology, the same group bypassed this problem and demonstrated how palmitoylation of site-specific-labelled IFITM3 promotes fusion of IFITM3-positive endosomes with viral particles, enhancing their delivery to lysosomes. This study, further showcases the importance of site-specific labelling techniques to unravel the dynamics of intricate cell biology processes.

Spence J.S. et al, “IFITM3 directly engages and shuttles incoming virus particles to lysosomes”, Nature Chemical Biology, 15, 259–268, 2019.

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Novel probes for live imaging of cholesterol dynamics
From Cell Chemical Biology (2018)

Many challenges emerge when attempting to visualise the cellular cholesterol distribution and dynamics. Current approaches involving fluorescent cholesterol-binding compounds, bacterial-derived biosensors, fluorescent analogues or fluorophore-labelled cholesterol derivatives have indeed provided significant contributions to our understanding of cholesterol biology, yet not without certain limitations. Such strategies which often display low photostability may be limited to the accessible fraction of cholesterol, compromise cholesterol distribution, and rely on inefficient delivery protocols that affect native compartmentalization and are not suitable for live imaging experiments. With this mind, the Glorious group from the University of Munster aimed to generate more sophisticated cholesterol-based probes that display efficient membrane penetration without disruption, high stability and are amenable for subsequent biochemical engineering. By combining cholesterol with imidazolium salts, Rakers et al. generated tailor-made analogues with cholesterol-like membrane incorporation and biophysical properties in simple bilayer systems. More importantly these analogues efficiently incorporated into different cell types and could be subsequently fluorescently labelled by click chemistry. This made it possible to observe, in live cells, how such probes reflect cholesterol-like distribution and dynamics. Future analysis will further elucidate their behaviour in complex membrane systems and likely retrieve important findings regarding cholesterol compartmentalisation and dynamics in live-cell models.

Rakers et al., “Addressable cholesterol analogs for live imaging of cellular membranes”, Cell Chemical Biology, 25, 952–961, 2018.

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