Unearthing the molecular mechanisms that govern L-selectin-dependent adhesion and migration

Abstract

L-selectin has been well characterised as a cell adhesion molecule, which plays a role in the recruitment of leukocytes to sites of inflammation and is responsible for the recirculation of lymphocytes to secondary lymphoid organs. Recent evidence has shown that L-selectin also acts as a signalling molecule to activate pathways and regulate the inflammatory response. The cytosolic tail of L-selectin plays a crucial role in regulating its activity through its interaction with binding partners, such as calmodulin (CaM) and the ERM protein family. However, little is known about how the interaction between L-selectin and its binding partners is regulated. The aim of this multidisciplinary PhD project is to use biophysical and cell biological methods to address the role of the interaction between L-selectin and its binding partners during leukocyte recruitment. To this end, the interaction between CaM and the L-selectin cytosolic tail was assessed using isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. Analysis revealed that phosphorylation of serine residues within the cytosolic tail of L-selectin did not affect CaM binding. To enable the observation of the interaction between L-selectin and CaM whilst leukocytes are undergoing transendothelial migration (TEM), the THP-1 monocytic cell line was engineered to stably express L-selectin-GFP and CaM-RFP so their interaction could be monitored at different stages of TEM. The data showed that phosphorylation of serine 364 in the L-selectin tail is important for regulating CaM interaction. Discrepancies were identified between the biophysical and cell biological results, implying the leukocyte plasma membrane may play a vital role in regulating the interaction between L-selectin and CaM. This highlights the importance of studying transmembrane proteins in the correct context.

Date of Award	2015
Original language	English
Awarding Institution	King's College London
Supervisor	Alex Ivetic (Supervisor) & Maria Conte (Supervisor)