Reconstitution of cytolinker-mediated crosstalk between actin and vimentin

Isturiz Petitjean, I.; Tran, Q. D.; Goutou, A.; Kabir, Z.; Wiche, G.; Leduc, C.; Koenderink, G. H.

doi:10.1101/2023.10.27.564417

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bioRxiv · 10.1101/2023.10.27.564417

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin

Isturiz Petitjean, I.·Tran, Q. D.·Goutou, A.·Kabir, Z.·Wiche, G.·Leduc, C.·Koenderink, G. H.

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Abstract

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

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Isturiz Petitjean, I., Tran, Q. D., Goutou, A., Kabir, Z., Wiche, G., Leduc, C., Koenderink, G. H.. 2023-10-28. Reconstitution of cytolinker-mediated crosstalk between actin and vimentin. https://doi.org/10.1101/2023.10.27.564417

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