| Abstract: | L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.The passage of leukocytes from the circulation toward the surrounding extravascular space is a critical event in the inflammatory response (1–3). The multistep adhesion cascade defines the increasingly adhesive steps leukocytes make with the endothelium to exit the circulation and enter the surrounding microenvironment (4). Each step of the cascade tethering, rolling, slow rolling, firm adhesion, and transendothelial migration (TEM)] critically depends on cell adhesion molecules expressed on both leukocytes and endothelial cells. Such cell adhesion molecules have been identified and characterized to act in a sequential and interdependent manner. The molecular mechanism driving leukocyte integrin function during the multistep adhesion cascade is relatively well defined (4–6). In contrast, mechanisms underpinning the regulation of nonintegrin receptors are only beginning to emerge. One such example, L-selectin, is known to promote the initial tethering and subsequent rolling of leukocytes along activated endothelial cells (7). Knocking out L-selectin in mice has provided compelling evidence for this cell adhesion molecule in directing neutrophils toward sites of inflammation (8). Intriguingly, knocking out L-selectin has a dramatic impact on neutrophil chemotaxis in vivo, but the molecular basis for this observation remains elusive (9). Following firm adhesion, L-selectin is broadly considered to be proteolytically cleaved (or shed) at a defined extracellular membrane/proximal domain by membrane-associated matrix metalloproteinases (10). The best-characterized “sheddase” is a disintegrin and metalloproteinase (ADAM17; or TNF-α concerting enzyme) (11). Shedding of L-selectin hinges on its interaction with calmodulin (CaM); binding of CaM to L-selectin protects from shedding, and leukocyte activation leads to CaM dissociation from the L-selectin tail to drive shedding (12). What promotes CaM dissociation is not known, but mutating the L-selectin tail can have a profound impact on the shedding response (13–15). L-selectin shedding is a rapid event, and the biological significance of its outcome may be numerous (10). In respect to leukocyte recruitment, L-selectin shedding would rapidly halt any further contribution of this molecule toward cell adhesion, signaling, or migration. The timing of L-selectin shedding is therefore critical in relation to input signals derived from other surface receptors contributing to the same cellular event (7). Our understanding of coordinated receptor signaling during TEM is extremely poor, mainly because insight into the molecular regulation of each individual cell adhesion molecule is still lacking.The contribution of L-selectin to the multistep adhesion cascade has been defined through two major experimental approaches: function-blocking studies (using soluble ligand or monoclonal antibody) and gene KO/knock-in mice. Such studies have built the foundation of our knowledge of the adhesion cascade. However, these approaches limit our understanding of where the targeted molecule’s first nonredundant step is required. Direct imaging of cell adhesion molecules in space and time, during recruitment under flow conditions, can provide additional clues beyond the first nonredundant point of execution. Here, we have used a series of advanced imaging techniques to pinpoint where and when L-selectin is cleaved during the multistep adhesion cascade. Using primary human monocytes, we reveal the shedding event was occurring specifically during TEM and not before. Through stable expression of WT and mutant forms of L-selectin tagged to GFP or red fluorescent protein (RFP), we could define the shedding event on a mechanistic level in THP-1 cells. Fluorescence lifetime imaging microscopy (FLIM) enabled quantitative measurement of the fluorescence resonance energy transfer (FRET) efficiency between L-selectin–GFP and CaM-RFP, allowing us to monitor the subcellular distribution of their interaction during TEM. We show, for the first time to our knowledge, that L-selectin shedding can regulate the invasive behavior of monocytes crossing activated endothelial monolayers under flow. Furthermore, we show that polarity in transmigrated monocytes is disrupted if shedding of L-selectin is blocked. Taken together, these results reveal previously unidentified roles for L-selectin that extend beyond tethering and rolling. |
