The MYO1F interactome reveals ASAP1, CD2AP and SH3KBP1 as novel adaptor proteins in podosomes and phagosomes

MYO1F, a long-tailed myosin of class I, is selectively expressed in immune cells and upregulated in microglia associated with neurodegenerative pathogenesis. Myosin motor functions are regulated by adaptor proteins that mediate cargo attachment and motor recruitment. To define the MYO1F interactome, we used in situ proximity labelling and proteomics in human myeloid cells. We identified a distinct SH3-domain-dependent adaptor module comprising CD2AP, ASAP1, SH3BP2, and SH3KBP1 (CASS group of proteins). Interestingly, CD2AP is an AD risk gene upregulated in patient microglia implicated in phagocytic responses to amyloid-β. Structural modelling and mutagenesis confirmed multivalent proline-rich motif interactions between the CASS group of proteins and the MYO1F SH3 domain. Additional binding partners associate with the MYO1F pleckstrin homology (PH) domain. Immunofluorescence revealed co-localization of MYO1F and the CASS group of proteins at actin-rich podosomes and phagocytic cups in macrophages and microglia. Functional assays demonstrated that MYO1F recruitment to the phagocytic cup requires motor activity and intact PH and SH3 domains. We provide the first MYO1F interactome identifying adaptor proteins for MYO1F in podosomes and during phagocytosis offering new insights into its function in disease-associated microglia during neurodegeneration.