Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation.

Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration in vivo . Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.

Immunofluorescence Labeling of Skeletal Muscle in Development, Regeneration, and Disease.

Skeletal muscle is composed of long multinucleated cells, termed myofibers, that are formed through the activation and differentiation of resident muscle stem cells, called satellite cells. In healthy individuals, skeletal muscle enables voluntary locomotion while also playing a role in energy metabolism and thermoregulation. As skeletal muscle is integral to everyday processes, perturbations to skeletal muscle function can have devastating consequences. Here we describe an integral tool in biomedical research of skeletal muscle regeneration and disease, the immunofluorescence staining of myogenic cells. We highlight useful techniques for immunostaining myogenic cells, and we list validated antibodies for the staining of muscle proteins across different species and multiple developmental time points. This includes methods for unmasking antigens following formaldehyde fixation (using myosin heavy chain staining as an example) and practices for preserving endogenous fluorescent proteins by cardiac perfusion fixation.