Arden, S. D.; Pennink, E.; Lakatos, A.; Griffiths, G. M.; Lippert, A. H.; Buss, F.

MYO1F, a long-tailed myosin of class I, is selectively expressed in immune cells and upregulated in microglia associated with neurodegenerative pathogenesis. The intracellular functions of myosin motors involve adaptor proteins, which regulate cargo attachment and intracellular motor recruitment. To define the MYO1F interactome, we performed an in situ proximity labelling-based proteomic analysis in human myeloid U937 cells. We identified a distinct SH3-domain-dependent adaptor module comprising CD2AP, ASAP1, SH3BP2, and SH3KBP1 (CASS complex), which localizes with MYO1F in podosomes and phagocytic cups. Structural modelling and mutagenesis confirmed multivalent proline-rich motif interactions of the CASS complex with the MYO1F SH3 domain. Further deletions revealed a second group of membrane-associated adaptor proteins that bind to the MYO1F pleckstrin homology (PH) domain. Immunofluorescence in macrophages and microglia confirmed the conserved localization of MYO1F and its adaptors at actin-rich podosomes and phagocytic cups. Functional assays demonstrated that MYO1F recruitment to the phagocytic cup requires motor activity and intact PH and SH3 domains. This study provides the first comparative interactome of MYO1F and its paralogue MYO1E and supports a role for MYO1F in podosomes and during phagocytosis in both peripheral and brain-resident myeloid cells.