Cell Adhesion Molecule Distribution Relative to Neutrophil Surface Topography Assessed by TIRFM

Hocdé, Sandrine; Hyrien, Ollivier; Waugh, Richard E.

doi:10.1016/j.bpj.2009.04.035

Cited by 30 publications

(37 citation statements)

References 32 publications

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“…Leukocyte membrane protrusions known as microvilli serve as active grouping sites for a variety of surface receptors including L-selectin (4), ␣4-integrins (5), P-selectin glycoprotein ligand 1 (PSGL-1) 3 (6,7), CD4, and the chemokine receptors CCR5 and CXCR4 (8). In contrast, the hyaluronan receptor CD44 (4) and the integrins ␣M␤2 (Mac-1) and ␣L␤2 (LFA-1) (9,10) show expression on the planar cell body excluding microvilli.…”

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confidence: 99%

The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow

Buscher

Riese

Shakibaei

et al. 2010

Journal of Biological Chemistry

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During inflammation and immune surveillance, initial contacts (tethering) between free-flowing leukocytes and the endothelium are vitally dependent on the presentation of the adhesion receptor L-selectin on leukocyte microvilli. Determinants that regulate receptor targeting to microvilli are, however, largely elusive. Therefore, we systematically swapped the extracellular (EC), transmembrane (TM), and intracellular (IC) domains of L-selectin and CD44, a hyaluronan receptor expressed on the cell body and excluded from microvilli. Electron microscopy of transfected human myeloid K562 cells showed that the highly conserved TM domains are responsible for surface positioning. The TM segment of L-selectin forced chimeric molecules to microvilli, and the CD44 TM domain evoked expression on the cell body, whereas the IC and EC domains hardly influenced surface localization. Transfectants with microvillus-based chimeras showed a significantly higher adhesion rate under flow but not under static conditions compared with cells with cell body-expressed receptors. Substitution of the IC domain of L-selectin caused diminished tethering but no change in surface distribution, indicating that both microvillus positioning and cytoskeletal anchoring contribute to leukocyte tethering. These findings demonstrate that TM domains of L-selectin and CD44 play a crucial role in cell adhesion under flow by targeting receptors to microvilli or the cell body, respectively.Neutrophil invasion at sites of inflammation as well as lymphocyte recirculation from blood into secondary lymphoid tissue require that free-flowing leukocytes become activated and subsequently exit the bloodstream. At the molecular level, the cells undergo a closely orchestrated cascade of interactions with the endothelium, each step consisting of a characteristic pattern of receptors, their ligands, cytokines, and chemoattractants (1, 2).Distinct cell surface receptor segregation is a widely observed phenomenon with effects on cell proliferation, migration, and tumorigenesis (3). Leukocyte membrane protrusions known as microvilli serve as active grouping sites for a variety of surface receptors including L-selectin (4), ␣4-integrins (5), P-selectin glycoprotein ligand 1 (PSGL-1) 3 (6, 7), CD4, and the chemokine receptors CCR5 and CXCR4 (8). In contrast, the hyaluronan receptor CD44 (4) and the integrins ␣M␤2 (Mac-1) and ␣L␤2 (LFA-1) (9, 10) show expression on the planar cell body excluding microvilli.Both L-selectin and CD44 are type I integral membrane glycoproteins expressed on most circulating leukocytes. L-selectin knock-out mice show a profound defect of lymphocyte accumulation in secondary lymphatic tissue and of neutrophils at sites of inflammation (11). CD44 is involved in many physiological processes, including lymphocyte activation, adhesion, and proliferation (12). There is also increasing evidence that CD44 and L-selectin contribute to tumor metastasis (13,14).Under many physiologic conditions, L-selectin initiates the first step of the adhesion cascade (15,1...

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confidence: 99%

The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow

Buscher

Riese

Shakibaei

et al. 2010

Journal of Biological Chemistry

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“…It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3 , that topography limits adhesive bond formation with other surfaces 9 , and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3,7 . We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6 .…”

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Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Marsh

Waugh

2013

JoVE

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Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3 , that topography limits adhesive bond formation with other surfaces 9 , and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3,7 . We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6 . It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4 . In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2 . The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5 . The thickness and structure of the glycocalyx have been studied using electron microscopy 8 , and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx. Video LinkThe video component of this article can be found at http://www.jove.com/video/50163/ Protocol 1. Methods Cell Flow ChamberA flow chamber, shown in Figure 1, was constructed so that cells could be grown under a shear of 1.0 Pa (10 dyn/cm 2 ) and then transferred directly to an Asylum MFP3D AFM (Santa Barbara, CA).1. The flow chamber was prepared for the experiment by first cleaning the glass slides in piranha solution (3:1 H 2 SO 4 :H 2 O 2 ) for 15 min and then washing them with distilled water. They were then baked to dry and coated with aminopropyl triethoxy silane (APTES) in a vacuum deposition chamber. 2. A silicone gasket was cut using a Silhouette SD cutting tool. This allowed us to finely control the flow chamber dimensions for control of the flow rate and shear stress during cell growth. Typically, a channel was cut 6.4 mm wide by 19 mm long from a sheet of 0.4 mm silicone. The flow rate necessary to generate a shear stress of 1.0 Pa (10 dyn/cm 2 ) is calculated assuming laminar flow in a rectangular channel with the equation:

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“…Rolling adhesion models [31][32][33] have assumed a uniformly stochastic distribution of microvilli and adhesion molecules based on fluorescence imaging of PSGL-1 antibodies on leukocytes 21 . Consequently, variations in rolling velocity were explained by the stochastic nature of adhesion bond rupture alone.…”

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Mapping Cell Surface Adhesion by Rotation Tracking and Adhesion Footprinting

Chemla

2017

Biophysical Journal

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Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell's surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.Rolling adhesion is a common process by which cells attach themselves to surfaces under shear flow, such as in the circulatory system. Leukocytes in the blood utilize this mechanism to locate inflammation sites throughout the body. During an inflammation response, endothelial cells lining the blood vessels surrounding an infection site express adhesion proteins called selectins that are specific to leukocyte surface receptors. As the first step of the leukocyte adhesion cascade, leukocytes captured via selectin-specific interactions passively roll on the blood vessel wall under blood flow toward the inflammation site in a process known as rolling adhesion [1][2][3] . Malfunction of any adhesion molecules involved in this process leads to severe immune disorders such as the leukocyte adhesion deficiencies (LAD) 4 . Rolling adhesion behavior is also exhibited by circulating tumor cells (CTCs) which is believed to enhance cancer metastasis [5][6][7][8] . Therefore, quantitative understanding of rolling adhesion is necessary to enable practical applications such as cancer screening and treatment [9][10][11] . At the molecular level, this adhesion is mediated by catch-bond-like interactions 12,13 between P-14 and E-selectins 15 expressed on endothelial cells lining blood vessels and P-selectin glycoprotein ligand-1 (PSGL-1) found at microvilli tips of leukocytes 16 . Despite our understanding of the individual components, how the molecular details of adhesion bonds scale to cell-surface adhesion and rolling behavior remains poorly understood 2,17,18 . Here, we developed a label-free method that maps the functional adhesion sites and strengths on a cell ...

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Cell Adhesion Molecule Distribution Relative to Neutrophil Surface Topography Assessed by TIRFM

Cited by 30 publications

References 32 publications

The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow

The Transmembrane Domains of L-selectin and CD44 Regulate Receptor Cell Surface Positioning and Leukocyte Adhesion under Flow

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Mapping Cell Surface Adhesion by Rotation Tracking and Adhesion Footprinting

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