Nucleofection of primary human peripheral blood T cells (PBTs) with a dominant negative GFP-tagged ezrin protein led to marked changes of both the surface microvilli features and the F-actin distribution.
PBTs transfected with point substitution mutant constructs of moesin, mimicking the phosphorylated form of the protein, maintained long, coarse microvilli. In addition, these cells showed delayed polarization after chemokine stimulation, revealing the importance of the phosphorylation state of ERM for these peripheral processes in PBTs.
Lymphocyte microvilli mediate initial rolling-adhesion along endothelium but are lost during transmigration from circulation to tissue. However, the mechanism for resorption of lymphocyte microvilli remains unexplored. We show that chemokine stimulation of human peripheral blood T (PBT) cells is sufficient to induce rapid resorption of microvilli. Microvilli in other cells are regulated by ezrin/radixin/moesin (ERM) proteins, which link the plasma membrane to the cortical F-actin cytoskeleton; maintenance of these linkages requires ERM activation, reflected by phosphorylation at a specific carboxy-terminal threonine residue. Carboxyphosphorylated-ERM (cpERM) proteins in resting PBT cells show a punctate peripheral distribution consistent with localization to microvilli. cpERM dephosphorylation begins within seconds of stimulation by chemokines (stromal derived factor 1 alpha [SDF-1 alpha] or secondary lymphoid tissue cytokine), and ERM proteins lose their punctate distribution with kinetics paralleling the loss of microvilli. The cpERM proteins are preferentially associated with the cytoskeleton at rest and this association is lost with chemokine-induced dephosphorylation. Transfection studies show that a dominant-negative ERM construct destroys microvilli, whereas a construct mimicking cpERM facilitates formation of microvilli, retards chemokine-induced loss of microvilli, and markedly impairs chemokine-induced polarization. Thus, chemokine induces rapid dephosphorylation and inactivation of cpERM, which may in turn facilitate 2 aspects of cytoskeletal reorganization involved in lymphocyte recruitment: loss of microvilli and polarization.