SSA - Revealing IGFN1 functional roles through in vivo CRISPR/CAS targeting and in vitro mechanical protein unfolding

Background. Cytoskeletal crosslinkers have been recently placed at the forefront of mechanotransduction. They act as sensors of mechanical strain by triggering a novel inducible autophagy mechanism when they unfold. IGFN1 is a novel muscle-specific protein conserved in mammals and a strong candidate for a mechanotransduction sensor in skeletal muscle.

Objectives. The role of IGFN1 will be evaluated by: 1) Generating an Igfn1 loss of function allele in single adult muscles by CRISPR/CAS mediated genome editing. Using a highly efficient electroporation method, loss of function mutations will be generated in hindlimb muscles of adult mice in vivo; 2) Measuring the loss of function effect on muscle structure and function by histology, immunofluorescence and electron microscopy; and 3) Measuring the elastic properties of purified IGFN1 by atomic force microscopy. Cytoskeletal sensors must extend and contract in order to maintain the sarcomeric integrity during muscle contraction and relaxation. The force vs. extension curves obtained will inform about the maximum length of unfolded IGFN1, which can be related to the physical dimensions of the Z-disc, and the domain composition in this crosslinker protein.

Novelties. Genome editing of adult muscle in vivo is a novel approach for mechanistic studies. Successful genome editing of EDL muscle will be evaluated at genomic level. If IGFN1 is required for normal physiological activity, elimination of functional alleles in the majority of myonuclei will result in recognizable myopathic changes. These will be assessed at the single fibre level.

Timeliness. The scope of chaperone-assisted selective autophagy in muscle mechanotransduction is not established. This mechanism has only been partially elucidated in smooth muscle cells, but not in skeletal or heart muscle. This project will provide mechanistic insights into the role of IGFN1 as a structural stabilizer and client of chaperone-assisted autophagy using state-of-the-art genome editing and single-molecule biophysical tools.