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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 (herein termed the CASS group of proteins). Interestingly, CD2AP is an Alzheimer's disease (AD) risk gene upregulated in the microglia of individuals with AD, which are implicated in phagocytic responses to amyloid-\u03b2. 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 colocalisation 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.<\/jats:p>","DOI":"10.1242\/jcs.264357","type":"journal-article","created":{"date-parts":[[2025,11,10]],"date-time":"2025-11-10T08:34:12Z","timestamp":1762763652000},"source":"Crossref","is-referenced-by-count":2,"title":["The MYO1F interactome reveals ASAP1, CD2AP and SH3KBP1 as novel adaptor proteins in podosomes and phagosomes"],"prefix":"10.1242","volume":"138","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-5235-6512","authenticated-orcid":false,"given":"Susan D.","family":"Arden","sequence":"first","affiliation":[{"name":"Cambridge Institute for Medical Research 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]},{"name":"University of Cambridge 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-9080-2906","authenticated-orcid":false,"given":"Eva","family":"Pennink","sequence":"additional","affiliation":[{"name":"Cambridge Institute for Medical Research 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]},{"name":"University of Cambridge 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]},{"name":"University of Cambridge 2 John van Geest Centre for Brain Repair, Department of Clinical Neurosciences , , Cambridge CB2 0PY ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1301-2292","authenticated-orcid":false,"given":"Andr\u00e1s","family":"Lakatos","sequence":"additional","affiliation":[{"name":"University of Cambridge 2 John van Geest Centre for Brain Repair, Department of Clinical Neurosciences , , Cambridge CB2 0PY ,","place":["UK"]},{"name":"MRC-WT Cambridge Stem Cell Institute 3 , Cambridge Biomedical Campus, Cambridge CB2 0AW ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0434-5842","authenticated-orcid":false,"given":"Gillian M.","family":"Griffiths","sequence":"additional","affiliation":[{"name":"Cambridge Institute for Medical Research 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]},{"name":"University of Cambridge 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0771-8491","authenticated-orcid":false,"given":"Anna H.","family":"Lippert","sequence":"additional","affiliation":[{"name":"Cambridge Institute for Medical Research 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]},{"name":"University of Cambridge 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]},{"name":"Julius-Maximilians University W\u00fcrzburg 4 Institute for Systems Immunology , , 97078 W\u00fcrzburg ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4457-3479","authenticated-orcid":false,"given":"Folma","family":"Buss","sequence":"additional","affiliation":[{"name":"Cambridge Institute for Medical Research 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]},{"name":"University of Cambridge 1 , Department of Clinical Biochemistry , , Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge CB2 0XY ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2025,12,22]]},"reference":[{"key":"2025123006213163200_JCS264357C77","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"2025123006213163200_JCS264357C1","doi-asserted-by":"publisher","first-page":"545","DOI":"10.1016\/j.bbamcr.2018.12.009","article-title":"Variations on the theme of podosomes: a matter of context","volume":"1866","author":"Alonso","year":"2019","journal-title":"Biochim. 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