title: L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro.
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.
authors.affiliation: Membrane/Cytoskeleton Signalling Group, Cardiovascular Division, King's College London, British Heart Foundation Centre of Research Excellence, London SE5 9NU, United Kingdom;
authors.forename: Karolina
authors.initials: K
authors.lastname: Rzeniewicz
authors.affiliation: Membrane/Cytoskeleton Signalling Group, Cardiovascular Division, King's College London, British Heart Foundation Centre of Research Excellence, London SE5 9NU, United Kingdom;
authors.forename: Abigail
authors.initials: A
authors.lastname: Newe
authors.affiliation: Membrane/Cytoskeleton Signalling Group, Cardiovascular Division, King's College London, British Heart Foundation Centre of Research Excellence, London SE5 9NU, United Kingdom;
authors.forename: Angela
authors.initials: A
authors.lastname: Rey Gallardo
authors.affiliation: Membrane/Cytoskeleton Signalling Group, Cardiovascular Division, King's College London, British Heart Foundation Centre of Research Excellence, London SE5 9NU, United Kingdom;
authors.forename: Jessica
authors.initials: J
authors.lastname: Davies
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom;
authors.forename: Mark R
authors.initials: MR
authors.lastname: Holt
authors.affiliation: Cardiovascular Division, Academic Department of Surgery, King's College London, Biomedical Research Centre at Guy's & St. Thomas' National Health Service Foundation Trust and King's College London, London SE1 7EH, United Kingdom;
authors.forename: Ashish
authors.initials: A
authors.lastname: Patel
authors.affiliation: London Centre for Nanotechnology, London WC1H 0AH, United Kingdom; and.
authors.forename: Guillaume T
authors.initials: GT
authors.lastname: Charras
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom;
authors.forename: Brian
authors.initials: B
authors.lastname: Stramer
authors.affiliation: Membrane/Cytoskeleton Signalling Group, Cardiovascular Division, King's College London, British Heart Foundation Centre of Research Excellence, London SE5 9NU, United Kingdom;
authors.forename: Chris
authors.initials: C
authors.lastname: Molenaar
authors.affiliation: Department of Immunology, Duke University Medical Center, Durham, NC 27710.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Tedder
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, United Kingdom;
authors.forename: Maddy
authors.initials: M
authors.lastname: Parsons
authors.affiliation: Membrane/Cytoskeleton Signalling Group, Cardiovascular Division, King's College London, British Heart Foundation Centre of Research Excellence, London SE5 9NU, United Kingdom; aleksandar.ivetic@kcl.ac.uk.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Ivetic
mesh_terms: D000595:Amino Acid Sequence; D002448:Cell Adhesion; D002465:Cell Movement; D016764:Cell Polarity; D003593:Cytoplasm / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007249:Inflammation; D019041:L-Selectin / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D018715:Microscopy, Video; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry
chemical_list: D019041:L-Selectin; D049452:Green Fluorescent Proteins; D012694:Serine
keywords: biglycan; calmodulin; invasion; leukocyte; migration