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.

Generation of Photocaged Nanobodies for Intracellular Applications in an Animal 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.

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.

Precise optical control of gene expression in using improved genetic code expansion and Cre recombinase.

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 improved genetic code expansion system in and use it to create a photoactivatable 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.