期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：12
页码：E1461-E1470
DOI：10.1073/pnas.1417100112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceDuring an inflammatory response, L-selectin, an immune cell-specific adhesion molecule, guides monocytes from the bloodstream toward the surrounding extravascular environment (termed "transmigration"). We show, under conditions that mimic blood flow, that L-selectin is proteolytically cleaved (or shed) exclusively within leading migratory fronts (pseudopods) of actively transmigrating monocytes. Calmodulin/L-selectin interaction, which acts to block shedding, is lost through Ser phosphorylation of the L-selectin cytoplasmic tail, occurring specifically within transmigrating pseudopods. Blocking L-selectin shedding specifically during transmigration increases pseudopod numbers, leading to defective front/back polarity that is essential for migration. These findings are the first to report, to our knowledge, an extended role for L-selectin in regulating morphological changes in leukocytes that are required for migration. 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.
