A congenital activating mutant of WASp causes altered plasma membrane topography and adhesion under flow in lymphocytes.

Leukocytes rely on dynamic actin-dependent changes in cell shape to pass through blood vessels, which is fundamental to immune surveillance. Wiskott-Aldrich Syndrome protein (WASp) is a hematopoietic cell-restricted cytoskeletal regulator important for modulating cell shape through Arp2/3-mediated actin polymerization. A recently identified WASp(I294T) mutation was shown to render WASp constitutively active in vivo, causing increased filamentous (F)-actin polymerization, high podosome turnover in macrophages, and myelodysplasia. The aim of this study was to determine the effect of WASp(I294T) expression in lymphocytes. Here, we report that lymphocytes isolated from a patient with WASp(I294T), and in a cellular model of WASp(I294T), displayed abnormal microvillar architecture, associated with an increase in total cellular F-actin. Microvillus function was additionally altered as lymphocytes bearing the WASp(I294T) mutation failed to roll normally on L-selectin ligand under flow. This was not because of defects in L-selectin expression, shedding, cytoskeletal anchorage, or membranal positioning; however, under static conditions of adhesion, WASp(I294T)-expressing lymphocytes exhibited altered dynamic interaction with L-selectin ligand, with a significantly reduced rate of adhesion turnover. Together, our results demonstrate that WASp(I294T) significantly affects lymphocyte membrane topography and L-selectin-dependent adhesion, which may be linked to defective hematopoiesis and leukocyte function in affected patients.

The cytoplasmic domains of TNFalpha-converting enzyme (TACE/ADAM17) and L-selectin are regulated differently by p38 MAPK and PKC to promote ectodomain shedding.

L-selectin mediates the initial tethering and subsequent rolling of leucocytes along luminal walls of inflamed venules. TACE [TNFalpha (tumour necrosis factor alpha)-converting enzyme] is responsible for cleaving the membrane-proximal extracellular domain of L-selectin (also known as shedding), which reduces the efficiency of leucocyte recruitment to sites of inflammation. Many reports have highlighted roles for PKC (protein kinase C) and p38 MAPK (mitogen-activated protein kinase) in promoting L-selectin shedding with little insight into the mechanism involved. By using PMA and the phosphatase inhibitors cantharidin and calyculin A, we could selectively activate PKC or p38 MAPK respectively to promote TACE-dependent shedding of L-selectin. Interestingly, the intracellular mechanisms leading to the shedding event differed dramatically. For example, regulatory elements within the L-selectin cytoplasmic tail, such as ERM (ezrin/radixin/moesin)-binding and serine residues, were important for PKC- but not p38 MAPK-dependent shedding. Also, increased and sustained cell surface levels of TACE, and phosphorylation of its cytoplasmic tail (a hallmark of TACE activation), occurred in lymphocytes and monocytes following p38 MAPK activation. Finally, we showed that TNFalpha-induced shedding of L-selectin in monocytes was strikingly similar to cantharidin-induced shedding and suggest that this newly characterized mechanism could be physiologically relevant in inflammatory cells.

In Vitro and in Vivo Characterization of Molecular Interactions between Calmodulin, Ezrin/Radixin/Moesin, and L-selectin.

L-selectin is a cell adhesion molecule that tethers leukocytes to the luminal walls of venules during inflammation and enables them to roll under the force of blood flow. Clustering of L-selectin during rolling is thought to promote outside-in signals that lead to integrin activation and chemokine receptor expression, ultimately contributing to leukocyte arrest. Several studies have underscored the importance of the L-selectin cytoplasmic tail in functionally regulating adhesion and signaling. Interestingly, the L-selectin tail comprises only 17 amino acids, and yet it is thought to bind simultaneously to several proteins. For example, constitutive association of calmodulin (CaM) and ezrin/radixin/moesin (ERM) to L-selectin confers resistance to proteolysis and microvillar positioning, respectively. In this report we found that recombinant purified CaM and ERM bound non-competitively to the same tail of L-selectin. Furthermore, molecular modeling supported the possibility that CaM, L-selectin, and moesin could form a heterotrimeric complex. Finally, using fluorescence lifetime imaging microscopy to measure fluorescence resonance energy transfer, it was shown that CaM, L-selectin, and ERM could interact simultaneously in vivo. Moreover, L-selectin clustering promoted CaM/ERM interaction in cis (i.e. derived from neighboring L-selectin tails). These results highlight a novel intracellular event that occurs as a consequence of L-selectin clustering, which could participate in transducing signals that promote the transition from rolling to arrest.