Cytoskeletal interactions regulate inducible L-selectin clustering

Abstract

selectin (CD62L) amplifies neutrophil capture within the microvasculature at sites of inflammation. Activation by G protein-coupled stimuli or through ligation of L-selectin promotes clustering of Lselectin and serves to increase its adhesiveness, signaling, and colocalization with ␤2-integrins. Currently, little is known about the molecular process regulating the lateral mobility of L-selectin. On neutrophil stimulation, a progressive change takes place in the organization of its plasma membrane, resulting in membrane domains that are characteristically enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins and exclude the transmembrane protein CD45. Clustering of L-selectin, facilitated by E-selectin engagement or antibody cross-linking, resulted in its colocalization with GPI-anchored CD55, but not with CD45 or CD11c. Disrupting microfilaments in neutrophils or removing a conserved cationic motif in the cytoplasmic domain of L-selectin increased its mobility and membrane domain localization in the plasma membrane. In addition, the conserved element was critical for L-selectin-dependent tethering under shear flow. Our data indicate that L-selectin's lateral mobility is regulated by interactions with the actin cytoskeleton that in turn fortifies leukocyte tethering. We hypothesize that both membrane mobility and stabilization augment L-selectin's effector functions and are regulated by dynamic associations with membrane domains and the actin cytoskeleton. membrane domains; adhesion; leukocyte; inflammation NEUTROPHIL RECRUITMENT from the bloodstream to sites of inflammation requires overcoming tremendous shear forces to initially tether to the vascular wall. Participation by the selectin adhesion protein family (E-, L-, and P-selectin) is critical for this process. The selectins are type 1 integral membrane proteins that contain a C-type lectin domain and recognize specific glycan moieties. L-selectin (CD62L) is expressed constitutively by leukocytes of myeloid and lymphoid origin; E-and P-selectin (CD62E and CD62P) are expressed by activated endothelial cells and by activated platelets (P-selectin). Neutrophils attach and roll along the vascular endothelium via the selectins, and then, if properly stimulated, integrins on the surface of neutrophils become activated and participate in adhesion strengthening and transmigration (18,40,41,78).Neutrophils that have accumulated within the microvasculature capture free-flowing leukocytes (13,69). This process of indirect leukocyte tethering accelerates neutrophil accumulation and is facilitated by L-selectin (2,4,74,85). Consistent with its role in amplifying the extent and rate of neutrophil accumulation, L-selectin is tightly regulated, which involves rapid and transient increases in its binding activity (26,70). C-type lectins achieve high-affinity binding through the presentation of multiple carbohydrate recognition domains in a single polypeptide or by clustering (reviewed in Ref. 86). L-selectin, which contains a single carbohydrate recognition domai...

Up‐regulation of leukocyte CXCR4 expression by sulfatide: An L‐selectin‐dependent pathway on CD4+ T cells

“…The binding of L-selectin with its ligands such as sulfatide or glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) or by antibody cross-linking has been demonstrated to trigger multiple signaling events [27,34]. L-Selectin signaling appears to involve physical clustering of L-selectin, including co-localization with integrins, which is regulated by cytoskeletal anchorage through the intracellular domain of L-selectin [23,35,36]. However, sulfatide can also signal through an L-selectin-independent pathway [14,21].…”

“…The human myeloid cell line K562 was cultured in RPMI 1640 medium supplemented with 10% FBS from Gibco (Grand Island, NY). K562 parent cells do not express endogenous L-selectin, whereas K562 WT cells are stably transfected with human WT L-selectin, and~16 cells with a mutant L-selectin (16 of 17 amino acid from the cytoplasmic region of L-selectin were truncated), as previously described [22,23].…”

“…To prove this directly, K562 cells were transfected with either WT L-selectin or a mutant form (~16) of L-selectin.~16 L-selectin lacks 16 of 17 intracellular amino acids, significantly impacting intracellular signaling upon ligand binding [22,23]. The parent cells (L-selectin negative) and~16 L-selectin-transfected cells did not show any significant upregulation of endogenous CXCR4 while WT L-selectintransfected cells displayed a prominent up-regulation of CXCR4 (Fig.…”

The Monoclonal Antibody CHO-131 Identifies a Subset of Cutaneous Lymphocyte-Associated Antigen T Cells Enriched in P-Selectin-Binding Cells

“…Isotype-matched negative control mAbs were used to evaluate levels of background staining. Analysis of fluorescence was performed on a Bio-Rad 1024 confocal laserscanning microscope, as described previously (32).…”

“…1A) was initially examined using an in vitro shear flow assay that allows for cellular analyses under hydrodynamic conditions simulating the microvasculature (29,30,32). CHO-131 ϩ and CHO-131 Ϫ CLA ϩ CD3 ϩ T cells were first isolated from the peripheral blood of normal individuals by sorting, and then the shear resistance of each subset was assessed over Nonspecific Ab labeling was determined using the appropriate isotype negative control Abs, as indicated.…”