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Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling.

Frontiers in immunology (2021-06-05)
Janine J G Arts, Eike K Mahlandt, Lilian Schimmel, Max L B Grönloh, Sanne van der Niet, Bart J A M Klein, Mar Fernandez-Borja, Daphne van Geemen, Stephan Huveneers, Jos van Rijssel, Joachim Goedhart, Jaap D van Buul
ABSTRACT

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Actin antibody, Mouse monoclonal, clone AC-40, purified from hybridoma cell culture
Sigma-Aldrich
Anti-HA antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Monoclonal Anti-RHOJ antibody produced in mouse, clone 1E4, purified immunoglobulin, buffered aqueous solution
SAFC
Trypsin-EDTA Solution 10X