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Many extravasating cells, including T lymphocytes and hematopoietic stem cells, migrate upstream against the direction of shear flow on ICAM-1. Upstream migration has been shown to depend on intracellular signaling through Crk family adaptor proteins following engagement of lymphocyte function-associated antigen-1 (LFA-1). Because upstream migration is an important illustration of leukocyte mechanotransduction, we sought to identify key intracellular regulators of upstream migration using CRISPR\/Cas9 gene editing and the KG-1a stem cell line. We made targeted deletions of molecules that function downstream of LFA-1 to regulate actin polymerization, link integrin signaling to the actin cytoskeleton, or form scaffolding complexes with Crk proteins. We found that CrkL, CasL (also known as HEF1 or NEDD9), L-plastin (LCP1) and RIAM (APBB1IP) are crucial for dynamic F-actin-rich lamellipodia and upstream migration. Although WASP (WAS), Hem1 (NCKAP1L), EVL and vinculin were found to be dispensable for upstream migration, pan-formin inhibition with SMIFH2 revealed a role for formins in upstream migration. This study illustrates the role of LFA-1 mechanotransduction in driving F-actin assembly at the leading edge to drive upstream migration.<\/jats:p>","DOI":"10.1242\/jcs.265036","type":"journal-article","created":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T09:41:48Z","timestamp":1791970908000},"source":"Crossref","is-referenced-by-count":1,"title":["Mechanotransduction through LFA-1 controls upstream migration in the KG-1a stem cell line"],"prefix":"10.1242","volume":"139","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-9000-1422","authenticated-orcid":false,"given":"Ai","family":"Mochida","sequence":"first","affiliation":[{"name":"University of Pennsylvania 1 Department of Bioengineering , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-2714-6637","authenticated-orcid":false,"given":"Emily","family":"Valerio","sequence":"additional","affiliation":[{"name":"University of Pennsylvania 2 Department of Chemical and Biomolecular Engineering , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8176-1375","authenticated-orcid":false,"given":"Janis K.","family":"Burkhardt","sequence":"additional","affiliation":[{"name":"Children's Hospital of Philadelphia and Perelman School of Medicine, University of Pennsylvania 3 Department of Pathology and Laboratory Medicine , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3522-3154","authenticated-orcid":false,"given":"Daniel A.","family":"Hammer","sequence":"additional","affiliation":[{"name":"University of Pennsylvania 1 Department of Bioengineering , , Philadelphia, PA 19104 ,","place":["USA"]},{"name":"University of Pennsylvania 2 Department of Chemical and Biomolecular Engineering , , Philadelphia, PA 19104 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2026,10,14]]},"reference":[{"key":"2026101405414291500_JCS265036C1","doi-asserted-by":"publisher","first-page":"1043","DOI":"10.1002\/eji.201445125","article-title":"Cell surface levels of endothelial ICAM-1 influence the transcellular or paracellular T-cell diapedesis across the blood-brain barrier","volume":"45","author":"Abadier","year":"2015","journal-title":"Eur. 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