Upstream Migration Under Shear Flow: Force Generation and Molecular Regulation

The ability of leukocytes to migrate persistently against the direction of blood flow -- upstream migration -- is a mechanically anomalous behavior implicated in hematopoietic stem cell trafficking and immune cell surveillance. Despite the centrality of mechanical force to this process, the forces upstream-migrating cells generate at the cell-substrate interface have not been directly measured during sustained upstream migration, and the molecular machinery responsible for producing them remains uncharacterized. This dissertation addresses both gaps through quantitative traction force microscopy, molecular perturbation, and computational bond mechanics. Using KG1a hematopoietic progenitor cells on ICAM-1-functionalized polyacrylamide hydrogels under physiological shear flow, an integrated platform was developed for traction force measurement during sustained upstream migration. Upstream-migrating cells generate approximately two-fold higher average and peak traction forces than cells migrating randomly under static conditions, yet the spatial distribution of forces across the cell footprint is statistically indistinguishable between conditions. Upstream migration is therefore characterized by selective amplification of contractile output without reorganization of the underlying force architecture. To identify molecular regulators of this force program, CRISPR/Cas9 knockout of CrkL, an upstream LFA-1 signaling adaptor, and L-plastin, a hematopoietic actin crosslinker, was performed in KG1a cells. Both knockouts abolished upstream migration while leaving all ten quantified traction force metrics unchanged, including under multivariate permutational analysis. Force generation is robustly buffered against single-protein perturbation at both the signaling and cytoskeletal levels, consistent with a collectively buffered force-generation architecture rather than a simple linear hierarchy. Finally, Adhesive Dynamics simulations demonstrate that two individually slip-classified bonds in series can exhibit emergent catch bond-like behavior — pseudo-catch — when the apparent spring constant of the series complex falls below the transition state threshold. This provides a mechanical criterion for assessing whether molecular force probe configurations report faithfully on intrinsic receptor-ligand kinetics or generate artifactual catch-like behavior through measurement geometry alone. Together, these results establish upstream migration as a force-intensive, redundantly regulated mode of motility whose quantitative characterization demands careful attention to the physics of measurement at every scale.

Author dc:creator

• Lee, Dong Hun

Advisor dc:contributor.advisor

• Hammer, Daniel, A.

• Biochemistry, Biophysics, and Structural Biology
• Engineering
• Immunology and Infectious Disease

Language dc:language.iso

en

Repository record dc:identifier.uri

https://repository.upenn.edu/handle/20.500.14332/62722

OAI identifier oai:identifier

oai:repository.upenn.edu:20.500.14332/62722