UMR 8640 : Chimie Biophysique

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Actin-Network Architecture Regulates Microtubule Dynamics

Curr Biol. (16) 2018 : 2647-2656

 

In Brief Colin et al. show that branched actin networks block microtubule growth and trigger microtubule disassembly using Xenopus egg extracts and in vitro reconstituted systems. This demonstrates the role of actin-network architecture in regulating microtubule dynamics. 

 

Highlights 

·     Branched actin networks block microtubule growth and trigger their disassembly 

·     Unbranched actin networks do not interfere with microtubule growth 

·     Branched actin networks perturb meiotic spindle assembly in Xenopusegg extracts

 

Circularly permuted fluorogenic proteins for the design of modular biosensors

ACS Chem. Biol. 2018

Fluorescent reporters are essential components for the design of optical biosensors able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca2+in living mammalian cells in real-time.

Fluorogenic Probing of Membrane Protein Trafficking

Bioconjugate Chem. 2018

 

Methods to differentially label cell-surface and intracellular membrane proteins are indispensable for understanding their function and the regulation of their trafficking. We present an effi cient strategy for the rapid and selective fluorescent labeling of membrane proteins based on the chemical-genetic fl uorescent marker FAST (fluorescence activating and absorption-shifting tag). Cell-surface FASTtagged proteins could be selectively and rapidly labeled using fluorogenic membrane-impermeant 4-hydroxybenzylidene rhodanine (HBR) analogs. This approach allows the study of protein trafficking at the plasma membrane with various fluorometric techniques, and opens exciting prospects for the high-throughput screening of small molecules able to restore disease-related trafficking defects.

Resonant out-of-phase fluorescence microscopy and remote imaging overcome spectral limitations

Nature Communications 8, 969 (2017)

 

We present speed out-of-phase imaging after optical modulation (OPIOM), which exploits reversible photoswitchable fluorophores as fluorescent labels and combines optimized periodic illumination with phase-sensitive detection to specifically retrieve the label signal. Speed OPIOM can extract the fluorescence emission from a targeted label in the presence of spectrally interfering fluorophores and autofluorescence. Up to four fluorescent proteins exhibiting a similar green fluorescence have been distinguished in cells either sequentially or in parallel. Speed OPIOM is compatible with imaging biological processes in real time in live cells. Finally speed OPIOM is not limited to microscopy but is relevant for remote imaging as well, in particular, under ambient light. Thus, speed OPIOM has proved to enable fast and quantitative live microscopic and remote-multiplexed fluorescence imaging of biological samples while filtering out noise, interfering fluorophores, as well as ambient light.

 

Programmed Self-Assembly of a Biochemical and Magnetic Scaffold to Trigger and Manipulate Microtubule Structures

Scientific REPOrtS | 7: 11344 | 2017

 

Artificial bio-based scaffolds offer broad applications in bioinspired chemistry, nanomedicine, and material science. One current challenge is to understand how the programmed self-assembly of biomolecules at the nanometre level can dictate the emergence of new functional properties at the mesoscopic scale. Here we report a general approach to design genetically encoded protein-based scaffolds with modular biochemical and magnetic functions. By combining chemically induced dimerization strategies and biomineralisation, we engineered ferritin nanocages to nucleate and manipulate microtubule structures upon magnetic actuation. Triggering the self-assembly of engineered ferritins into micrometric scaffolds mimics the function of centrosomes, the microtubule organizing centres of cells, and provides unique magnetic and self-organizing properties. We anticipate that our approach could be transposed to control various biological processes and extend to broader applications in biotechnology or material chemistry.