Thompson, Paul William; (2002) Analysis of ICAM-1 function in endothelial cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The cellular mechanisms that control endothelial responses to leukocyte adhesion, such as signaling from leukocyte receptors and cytoskeletal rearrangements, are poorly understood. The aim of this project was to investigate further the roles of ICAM-1 and ICAM-2 during leukocyte adhesion, and their interactions with components of the actin cytoskeleton. TNF-α primes endothelial cells for leukocyte adhesion. ICAMs were found to colocalize with ERM (ezrin, radixin, and moesin) proteins, which act as cytoskeleton/plasma membrane linkers, and F-actin in the microvilli of resting and TNF-α-stimulated cells. ERM activation has been previously linked to the RhoA GTPase, which regulates actin stress fibre assembly in endothelial cells. TNF-α caused ERM activation but failed to activate total cellular RhoA. TNF-α also induced transient increases in ICAM-2, RhoA, RhoE, and c-fos mRNA and protein levels. To mimic leukocyte binding, monoclonal antibodies were used to cross-link ICAMs. Cross-linking of ICAM-1 induced co-clustering of moesin, whereas ICAM-2 cross- linking caused no rearrangements of these proteins. ICAM-1, but not ICAM-2, cross- linking activated RhoA and induced stress fibre formation. ICAM-1 clustering also induced the upregulation of c-fos mRNA and protein levels. Interestingly, ICAM-1 clusters aligned with newly-induced stress fibres. Physiological flow induced stress fibre alignment along the direction of flow, with monocytes preferentially migrating in the same direction. ICAM-1 has been reported to interact with ERMs and α-actinin in vitro, and Shp-2 in vivo. However, none of these proteins were detected in ICAM-1 immunoprecipitates, but instead a novel binding partner called Major Vault Protein (MVP) was identified. Both MVP and ERM proteins interacted with ICAM-1 cytosolic tails in vitro, and MVP coclustered with ICAM-1. Mutational analysis of the juxtamembrane lysine residue cluster (481kik484k) within the cytosolic tail revealed that different amino acids were important for MVP and ERM binding. This work demonstrates that ICAM-1 controls transmigration by both initiating signaling events and relocalizing to align with actin stress fibres at the apical membrane.

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