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Use of crispr-based editing to elucidate molecular mechanisms that control directional migration of amoeboid cells under flow Ai Mochida
- Format:
- Book
- Thesis/Dissertation
- Author/Creator:
- Mochida, Ai, author.
- Language:
- English
- Subjects (All):
- Bioengineering.
- Cellular biology.
- Biochemistry.
- 0202.
- 0379.
- 0487.
- Local Subjects:
- Bioengineering.
- Cellular biology.
- Biochemistry.
- 0202.
- 0379.
- 0487.
- Genre:
- Academic theses
- Physical Description:
- 1 online resource (138 pages)
- Contained In:
- Dissertations Abstracts International 87-12B
- Place of Publication:
- Ann Arbor : ProQuest Dissertations and Theses, 2026
- Language Note:
- English
- Summary:
- Amoeboid migration is fundamental to the immune system and relies on force generation through actin polymerization coupled to myosin-based contractility. During immune cell recruitment to sites of inflammation or infection, leukocytes migrate on endothelial surfaces in postcapillary venules prior to exiting towards tissues. Many extravasating immune cells, including T lymphocytes and hematopoietic stem cells, migrate upstream against the direction of shear flow on endothelial surfaces mediated by differential signaling through the integrin LFA-1, which binds to ICAM-1. In this thesis, we explore the molecules involved in the LFA-1 mechanotransduction that control upstream migration using CRISPR/Cas9 gene editing. We used the human KG-1a cell line, which demonstrates robust upstream migration on ICAM-1, to make targeted deletions of molecules downstream of LFA-1 that regulate actin polymerization, link integrin signaling to the actin cytoskeleton, or form scaffolding complexes with Crk family proteins and characterized the migratory behavior of the knockouts on ICAM-1 with and without shear flow. We found additional LFA-1 intermediates to be involved in mediated upstream migration, including actin bundling molecule L-plastin, scaffolding molecules CrkL and CasL, and Rap1 effector RIAM. However, actin regulatory molecules WASP, Hem1, and EVL and the cytoskeletal adaptor protein vinculin were found to be dispensable for KG-1a upstream migration. Pan-formin inhibition with SMIFH2 induced KG-1a cells to lose the directional preference to migrate upstream on ICAM-1, suggesting formin-mediated linear actin assembly mediates directional migration. Lifeact-GFP imaging showed that knockout cells with deletions that disrupted upstream migration appeared to have a loosely adhered leading edge with few protrusion/retraction cycles and a strongly adhered uropod. Finally, we found that KG-1a NT, CrkL KO, and L-plastin KO cells maintained their directional migration preference on HUVEC monolayers. Together, this work highlights the importance of adaptor molecules and mechanotransducers in controlling the direction of cell migration under shear flow through LFA-1 signaling and more broadly, in immune cell recruitment
- Notes:
- Source: Dissertations Abstracts International, Volume: 87-12, Section: B.
- Advisors: Hammer, Daniel A. Committee members: Bugaj, Lukasz; Burkhardt, Janis K.; Momin, Noor
- Ph.D. University of Pennsylvania 2026
- Vendor supplied data
- Local Notes:
- School code: 0175
- ISBN:
- 9798247979814
- Access Restriction:
- Restricted for use by site license
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