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Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium
Published on: February 11, 2009
Serine Phosphorylation of L-Selectin Regulates ERM Binding, Clustering, and Monocyte Protrusion in Transendothelial
Abigail Newe1, Karolina Rzeniewicz1, Melanie König2
1BHF Centre of Research Excellence, James Black Centre, King's College London, London, United Kingdom.
Abstract:
The migration of circulating leukocytes toward damaged tissue is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first cellular barrier that is breached in this process. Human CD14+ inflammatory monocytes express L-selectin, bestowing a non-canonical role in invasion during TEM. In vivo evidence supports a role for L-selectin in regulating TEM and chemotaxis, but the intracellular mechanism is poorly understood. The ezrin-radixin-moesin (ERM) proteins anchor transmembrane proteins to the cortical actin-based cytoskeleton and additionally act as signaling adaptors. During TEM, the L-selectin tail within transmigrating pseudopods interacts first with ezrin to transduce signals for protrusion, followed by moesin to drive ectodomain shedding of L-selectin to limit protrusion. Collectively, interaction of L-selectin with ezrin and moesin fine-tunes monocyte protrusive behavior in TEM. Using FLIM/FRET approaches, we show that ERM binding is absolutely required for outside-in L-selectin clustering. The cytoplasmic tail of human L-selectin contains two serine (S) residues at positions 364 and 367, and here we show that they play divergent roles in regulating ERM binding. Phospho-S364 blocks direct interaction with ERM, whereas molecular modeling suggests phospho-S367 likely drives desorption of the L-selectin tail from the inner leaflet of the plasma membrane to potentiate ERM binding. Serine-to-alanine mutagenesis of S367, but not S364, significantly reduced monocyte protrusive behavior in TEM under flow conditions. Our data propose a model whereby L-selectin tail desorption from the inner leaflet of the plasma membrane and ERM binding are two separable steps that collectively regulate protrusive behavior in TEM.
Insights
L-selectin binding to ezrin-radixin-moesin (ERM) proteins regulates monocyte invasion during transendothelial migration (TEM). Phosphorylation of serine 367 on L-selectin is critical for ERM binding and monocyte protrusion during TEM.
Area of Science:
- Cellular Biology
- Immunology
- Biochemistry
Background:
- Leukocyte migration is crucial for inflammation, with transendothelial migration (TEM) being a key step.
- Human CD14+ inflammatory monocytes utilize L-selectin for invasion during TEM, but the intracellular mechanisms remain unclear.
- Ezrin-radixin-moesin (ERM) proteins link cell surface proteins to the actin cytoskeleton and act as signaling hubs.
Purpose of the Study:
- To elucidate the intracellular mechanism of L-selectin-mediated monocyte TEM.
- To investigate the role of ERM proteins in L-selectin signaling during monocyte extravasation.
- To determine the function of specific serine residues in the L-selectin cytoplasmic tail in regulating ERM binding and monocyte behavior.
Main Methods:
- Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster Resonance Energy Transfer (FRET) to assess ERM binding and L-selectin clustering.
- Molecular modeling to predict the effect of serine phosphorylation on L-selectin tail conformation.
- Site-directed mutagenesis (serine-to-alanine) to study the functional roles of L-selectin serine residues.
Main Results:
- ERM binding to L-selectin is essential for outside-in L-selectin clustering.
- Phosphorylation of serine 364 (S364) inhibits direct ERM interaction, while phosphorylation of serine 367 (S367) promotes L-selectin tail desorption from the plasma membrane, enhancing ERM binding.
- Mutating S367 to alanine significantly impaired monocyte protrusive behavior during TEM under flow conditions, unlike S364 mutations.
Conclusions:
- L-selectin interaction with ERM proteins fine-tunes monocyte protrusive behavior during TEM.
- L-selectin tail desorption from the plasma membrane and subsequent ERM binding are distinct, sequential steps regulating monocyte protrusion.
- This study reveals a novel mechanism controlling leukocyte migration essential for inflammatory responses.
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