Nanoscale Imaging Reveals a Tetraspanin-CD9 Coordinated Elevation of Endothelial ICAM-1 Clusters

Jonas Franz; Benjamin F Brinkmann; Michael König; Jana Hüve; Christian Stock; Klaus Ebnet; Christoph Riethmüller

doi:10.1371/journal.pone.0146598

Nanoscale Imaging Reveals a Tetraspanin-CD9 Coordinated Elevation of Endothelial ICAM-1 Clusters

PLoS One. 2016 Jan 5;11(1):e0146598. doi: 10.1371/journal.pone.0146598. eCollection 2016.

Authors

Jonas Franz¹, Benjamin F Brinkmann², Michael König¹, Jana Hüve³, Christian Stock⁴, Klaus Ebnet², Christoph Riethmüller⁵

Affiliations

¹ Medical faculty, University of Münster, Münster, Germany.
² Institute-associated Research Group "Cell adhesion and cell polarity", Institute of Medical Biochemistry, ZMBE, 48419 Münster, Germany.
³ Fluorescence Microscopy Facility Münster, Institute of Medical Physics and Biophysics, Center for Nanotechnology, University of Münster, Münster, Germany.
⁴ Centre for Internal Medicine, Hannover Medical School, Hannover, Germany.
⁵ Serend-ip GmbH, Centre for Nanotechnology, Münster, Germany.

Abstract

Endothelial barriers have a central role in inflammation as they allow or deny the passage of leukocytes from the vasculature into the tissue. To bind leukocytes, endothelial cells form adhesive clusters containing tetraspanins and ICAM-1, so-called endothelial adhesive platforms (EAPs). Upon leukocyte binding, EAPs evolve into docking structures that emanate from the endothelial surface while engulfing the leukocyte. Here, we show that TNF-α is sufficient to induce apical protrusions in the absence of leukocytes. Using advanced quantitation of atomic force microscopy (AFM) recordings, we found these structures to protrude by 160 ± 80 nm above endothelial surface level. Confocal immunofluorescence microscopy proved them positive for ICAM-1, JAM-A, tetraspanin CD9 and f-actin. Microvilli formation was inhibited in the absence of CD9. Our findings indicate that stimulation with TNF-α induces nanoscale changes in endothelial surface architecture and that--via a tetraspanin CD9 depending mechanism--the EAPs rise above the surface to facilitate leukocyte capture.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Adhesion / drug effects
Human Umbilical Vein Endothelial Cells / drug effects
Human Umbilical Vein Endothelial Cells / metabolism*
Humans
Intercellular Adhesion Molecule-1 / metabolism*
Leukocytes / drug effects
Leukocytes / metabolism
Microscopy, Atomic Force
Tetraspanin 29 / metabolism*
Tetraspanins / metabolism*
Tumor Necrosis Factor-alpha / pharmacology

Substances

Tetraspanin 29
Tetraspanins
Tumor Necrosis Factor-alpha
Intercellular Adhesion Molecule-1

Grants and funding

The work was financially supported by the German Ministry of Research, BMBF grant 01DJ13022A to CR, by grants from the Deutsche Forschungsgemeinschaft DFG (SFBs 629 and 944) to Jürgen Klingauf, and (EB 160/4-1 and -/4-2) and from the University Münster (IZKF Eb2/028/09 and Eb2/020/14) to KE. The authors acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publication Fund of University of Muenster. Serend-ip GmbH provided support in the form of salaries for authors CR, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.