Biology subjects

Goutou, A.

Publications and source records attributed to Goutou, A..

4 recordsLinked to original sources

bioRxiv2023

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called ACTIF that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells. HIGHLIGHTSO_LIEngineering of a recombinant actin-vimentin crosslinker called ACTIF with the plectin intermediate filament binding domain (IFBD), calponin homology domains that mediate actin binding, and a coiled-coil linker. C_LIO_LIACTIF crosslinks F-actin and vimentin and mediates their co-localization in vitro. C_LIO_LIACTIF has a binding affinity for vimentin that is about 500 times higher than for F-actin. C_LIO_LIACTIF forms composite bundles of F-actin and vimentin filaments. C_LIO_LIComposite F-actin/vimentin networks stiffen upon crosslinking with ACTIF. C_LIO_LIThe design of actin-vimentin crosslinker is modular, as other IFBDs like APCn2 can also be used. C_LI

biochemistry↗

bioRxiv2021

Using a Quadruplet Codon to Expand the Genetic Code of an Animal

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.

synthetic biology↗

bioRxiv2021

Generation of photocaged nanobodies for in vivo applications using genetic code expansion and computationally guided protein engineering

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

synthetic biology↗

bioRxiv2020

Optically splitting symmetric neuron pairs in C. elegans

Synthetic strategies for optically controlling gene expression may enable the precise spatiotemporal control of genes in any combination of cells that cannot be targeted with specific promoters. We develop an efficient genetic code expansion system in C. elegans and use it to create a photo-activatable Cre recombinase. We laser-activate Cre in single neurons within a bilaterally symmetric pair to selectively switch on expression of a loxP controlled optogenetic channel in the targeted neuron. We use the system to dissect, in freely moving animals, the individual contributions of the mechanosensory neurons PLML/PLMR to the C. elegans touch response circuit, revealing distinct and synergistic roles for these neurons. We thus demonstrate how genetic code expansion and optical targeting can be combined to break the symmetry of neuron pairs and dissect behavioural outputs of individual neurons that cannot be genetically targeted.

neuroscience↗