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Combining gene editing with traction force microscopy to achieve a comprehensive understanding of mechanotransduction in leukocyte motility

Award Number: R35GM161553
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 05/07/2026
PERIOD OF PERFORMANCE END DATE: 02/28/2031

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Combining gene editing with traction force microscopy to achieve a comprehensive understanding of mechanotransduction in leukocyte motility - Summary Leukocyte motility is critical for immunology, inflammation, and hemostasis. Immune cells exchange molecular information by direct contact, enabled by motility within secondary lymphoid organs. In inflammation, neutrophils crawl into sites of infection after adhering to blood vessel walls. Hematopoietic stem and progenitor cells (HSPCs) migrate into to bone marrow after adhesion to the blood vessel wall under flow. In work largely funded by NIGMS, the Hammer laboratory has worked to understand the chemo- mechanics of leukocyte migration for over two decades. We have used traction force microscopy (TFM), in which we measure the forces exerted by cells during motility by monitoring the defection of fiduciary beads embedded within an elastic polyacrylamide gel. We have used this method to measure the traction forces of neutrophils and macrophages, as well as many other cell types. With the technique of TFM in hand, we are now positioned for significant breakthrough in our molecular understanding of traction stresses during leukocyte motility, owing to the development of methods to delete or alter intracellular components within a cell. Of specific interest is the fascinating phenomenon of upstream migration, in which leukocytes migrate against the direction of flow on surfaces presenting intercellular adhesion molecule-1 (ICAM-1). Using CRISPR-Cas9, we now are able do a directed screen of molecules that have been implicated in cell migration, and specifically, upstream migration, to understand precisely how these molecules contribute to the generation of traction forces in leukocytes. We will use two cultured cells lines – KG1a cells (a model HSPCs) and HL-60 cells (a model neutrophil) – that allow us to compare the role of different intracellular effector molecules in cell motility to establish universal mechanisms. We will conduct a directed screen of a limited but important set of effector molecules which have been implicated in upstream migration and leukocyte motility, such as cytoskeletal regulators and Rho-GTPases. Our elucidation of traction stresses will be complemented by immuno-fluorescent staining of the spatial distribution of adhesion receptors and actin cytoskeleton to provide information about cell organization. This MIRA is organized in three projects. In Project 1, we will perform traction mapping of KG1a cells during upstream migration. We will then screen candidate controllers of upstream migration using CRISPR-Cas9 and then measure how deletion affects both directional motility and traction stresses. In Project 2, we will use CRSIPR-Cas9 to screen a family of motility modulators and study their effect on HL-60 cell chemokinesis and chemotaxis. In Project 3, we will use TFM to measure the traction stresses of HL-60 cells during upstream migration and when upstream migration is reversed through CRISPR-Cas9 deletion. Then, we will measure the correlation between upstream migration of HL-60 cells and trans-endothelial migration on HUVEC monolayers.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $438,609 )
2026	2026	TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, THE	3451 WALNUT ST	PHILADELPHIA	PA	19104	PHILADELPHIA	USA	Biomedical Research and Research Training	000	1	5/7/2026	NEW	$438,609
														Subtotal = $438,609

Grand Total All Awards = $438,609

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Combining gene editing with traction force microscopy to achieve a comprehensive understanding of mechanotransduction in leukocyte motility

Award Number: R35GM161553

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 05/07/2026

PERIOD OF PERFORMANCE END DATE: 02/28/2031

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