Functional Mapping of Adhesiveness on Live Cells Reveals How Guidance Phenotypes Can Emerge From Complex Spatiotemporal Integrin Regulation.

Immune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially in vivo. Here, we developed a novel in vitro method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/µm2 in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance.

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] µm(2) for uninfected and 41 [34-51] µm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 µm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 µm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

Biophysical description of multiple events contributing blood leukocyte arrest on endothelium.

Blood leukocytes have a remarkable capacity to bind to and stop on specific blood vessel areas. Many studies have disclosed a key role of integrin structural changes following the interaction of rolling leukocytes with surface-bound chemoattractants. However, the functional significance of structural data and mechanisms of cell arrest are incompletely understood. Recent experiments revealed the unexpected complexity of several key steps of cell-surface interaction: (i) ligand-receptor binding requires a minimum amount of time to proceed and this is influenced by forces. (ii) Also, molecular interactions at interfaces are not fully accounted for by the interaction properties of soluble molecules. (iii) Cell arrest depends on nanoscale topography and mechanical properties of the cell membrane, and these properties are highly dynamic. Here, we summarize these results and we discuss their relevance to recent functional studies of integrin-receptor association in cells from a patient with type III leukocyte adhesion deficiency. It is concluded that an accurate understanding of all physical events listed in this review is needed to unravel the precise role of the multiple molecules and biochemical pathway involved in arrest triggering.

A new method for rapid detection of T lymphocyte decision to proliferate after encountering activating surfaces.

A critical step of the adaptive response is the detection of foreign peptides on antigen presenting cells by T lymphocytes. It is a major challenge for a T lymphocyte to detect the presence of a few tens of cognate ligands or less on the membrane of a cell exposing millions of protein molecules. Detection is followed by the cell decision to undergo full or partial activation or even to start an inhibitory program. While the measurement of cell proliferation or cytokine synthesis is accepted as a reliable means of monitoring T lymphocyte activation, this requires hours or days to complete, which is a significant drawback to relate decision to particular signaling events or to assess lymphocyte reactivity in patients. Here we show that the contact area formed between T lymphocytes and potentially activating surfaces is exquisitely correlated to the proliferative response measured with the standard CFSE technique. Correlation is even better than the Erk activation that was reported as a digital reporter of cell activation. The simple and accurate method of assessing lymphocyte-to-surface contact extension that we describe might be very useful both to monitor lymphocyte reactivity for clinical purposes and to identify early steps of lymphocyte activation.

How cells tiptoe on adhesive surfaces before sticking.

Cell membranes are studded with protrusions that were thoroughly analyzed with electron microscopy. However, the nanometer-scale three-dimensional motions generated by cell membranes to fit the topography of foreign surfaces and initiate adhesion remain poorly understood. Here, we describe the dynamics of surface deformations displayed by monocytic cells bumping against fibronectin-coated surfaces. We observed membrane undulations with typically 5 nm amplitude and 5-10 s lifetime. Cell membranes behaved as independent units of micrometer size. Cells detected the presence of foreign surfaces at 50 nm separation, resulting in time-dependent amplification of membrane undulations. Molecular contact then ensued with apparent cell-membrane separation of 30-40 nm, and this distance steadily decreased during the following tens of seconds. Contact maturation was associated with in-plane egress of bulky molecules and robust membrane fluctuations. Thus, membrane undulations may be the major determinant of cell sensitivity to substrate topography, outcome of interaction, and initial kinetics of contact extension.

Dissecting subsecond cadherin bound states reveals an efficient way for cells to achieve ultrafast probing of their environment.

Cells continuously probe their environment with membrane receptors, achieving subsecond adaptation of their behaviour [Diez, G., Gerisch, G., Anderson, K., Müller-Taubenberger, A. and Bretschneider, T. (2006) Subsecond reorganization of the actin network in cell motility and chemotaxis. Proc. Natl. Acad. Sci. USA 102, 7601-7606, Shamri, R., Grabovsky, V., Gauguet, J.M., Feigelson, S., Manevich, E., Kolanus, W., Robinson, M.K., Staunton, D.E., von Andrian, U.H. and Alon, R. (2005) Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat. Immunol. 6, 497-606, Jiang, G., Huang, A.H., Cai, Y., Tanase, M. and Sheetz, M.P. (2006) Rigidity sensing at the leading edge through alpha(V)beta(3) integrins and RPTPalpha. Biophys. J. 90, 1804-2006]. Recently, several receptors, including cadherins, were found to bind ligands with a lifetime of order of one second. Here we show at the single molecule level that homotypic C-cadherin association involves transient intermediates lasting less than a few tens of milliseconds. Further, these intermediates transitionned towards more stable states with a kinetic rate displaying exponential decrease with piconewton forces. These features enable cells to detect ligands or measure surrounding mechanical behaviour within a fraction of a second, much more rapidly than was previously thought.

Cell membrane alignment along adhesive surfaces: contribution of active and passive cell processes.

Cell adhesion requires nanometer scale membrane alignment to allow contact between adhesion receptors. Little quantitative information is presently available on this important biological process. Here we present an interference reflection microscopic study of the initial interaction between monocytic THP-1 cells and adhesive surfaces, with concomitant determination of cell deformability, using micropipette aspiration, and adhesiveness, using a laminar flow assay. We report that 1), during the first few minutes after contact, cells form irregular-shaped interaction zones reaching approximately 100 micro m(2) with a margin extension velocity of 0.01-0.02 micro m/s. This happens before the overall cell deformations usually defined as spreading. 2), These interference reflection microscopic-detected zones represent bona fide adhesion inasmuch as cells are not released by hydrodynamic forces. 3), Alignment is markedly decreased but not abolished by microfilament blockade with cytochalasin or even cell fixation with paraformaldehyde. 4), In contrast, exposing cells to hypotonic medium increased the rate of contact extension. 5), Contacts formed in presence of cytochalasin, after paraformaldehyde fixation or in hypotonic medium, were much more regular-shaped than controls and their extension matched cell deformability. 6), None of the aforementioned treatments altered adhesiveness to the surface. It is concluded that adhesive forces and passive membrane deformations are sufficient to generate initial cell alignment to adhesive surfaces, and this process is accelerated by spontaneous cytoskeletally-driven membrane motion.

Dissecting streptavidin-biotin interaction with a laminar flow chamber.

A laminar flow chamber was used to study single molecule interactions between biotinylated surfaces and streptavidin-coated spheres subjected to a hydrodynamic drag lower than a piconewton. Spheres were tracked with 20 ms and 40 nm resolution. They displayed multiple arrests lasting between a few tens of milliseconds and several minutes or more. Analysis of about 500,000 positions revealed that streptavidin-biotin interaction was multiphasic: transient bound states displayed a rupture frequency of 5.3 s(-1) and a rate of transition toward a more stable configuration of 1.3 s(-1). These parameters did not display any significant change when the force exerted on bonds varied between 3.5 and 11 pN. However, the apparent rate of streptavidin-biotin association exhibited about 10-fold decrease when the wall shear rate was increased from 7 to 22 s(-1), which supports the existence of an energy barrier opposing the formation of the transient binding state. It is concluded that a laminar flow chamber can yield new and useful information on the formation of molecular bonds, and especially on the structure of the external part of the energy landscape of ligand-receptor complexes.