T cell adhesion mechanisms revealed by receptor lateral mobility^†

INTRODUCTION

Cell surface adhesion receptors have two major functions. First, these molecules positively and negatively regulate the binding of a cell to other cells or to solid-phase supports (e.g., extracellular matrix), controlling the formation and dissolution of both stable (e.g., tight junction) and dynamic (e.g., focal adhesion) adhesive structures. Second, these receptors are critical signaling molecules in the plasma membrane, facilitating the exchange of information between cells (e.g., immune synapse). Unlike receptors that interact with freely diffusing small molecules, adhesion receptors typically function by interacting with a counter-receptor or ligand on an opposing cell or solid-phase support. Because both binding partners are generally confined to the plasma membrane, these interactions are acutely sensitive to the peculiarities of the membrane environment. The restriction of receptors to the two-dimensional, viscous, and heterogeneous plasma membrane slows their diffusion and therefore the rate of the biological events they mediate.1 Measurements of receptor diffusion in the plasma membrane have consistently shown that these molecules experience dramatically slowed diffusion, even when the viscosity of the environment is taken into account.2 A variety of mechanisms have been found to contribute to this phenomenon of anomalous diffusion within the plasma membrane (vide infra), and these mechanisms provide a means of specifically regulating the lateral mobility of cell surface molecules. As a central parameter in determining the rate of biological interactions, the two-dimensional diffusion, or lateral mobility, of these receptors is a critical determinant of their role in cellular biology.

Biological Function of Lateral Mobility

The range of two-dimensional diffusion coefficients that have been measured for membrane proteins and lipids spans several orders of magnitude. Lipids generally diffuse relatively rapidly in the plasma membrane, with diffusion coefficients close to 1 × 10⁻⁸ cm² s⁻¹ (1 μm² s⁻¹), whereas transmembrane adhesion proteins generally diffuse relatively slowly, with diffusion coefficients as low as 1 × 10⁻¹¹ cm² s⁻¹ (0.001 μm² s⁻¹).3, 4 But how do these values affect receptor function? A useful starting point is to consider the surface area coverage of a freely diffusing molecule in the plasma membrane. We approximate the surface area, SA, of a simple spherical cell, such as a human T cell (∼3 μm in radius, r), as

()

We further assume that the area of coverage, A, for a particle diffusing in two dimensions with a two-dimensional diffusion coefficient D is given by

()

Rearrangement of Eqs. (1) and (2) allows estimation of the time required for a particle experiencing free diffusion to explore an area equivalent to the cell's surface area

()

The measured diffusion coefficients of membrane proteins and lipids span three orders of magnitude, and the time required for a molecule to explore the surface area of a cell would also be expected to vary over this range—for our model cell, the range is from 2.8 × 10¹ to 2.8 × 10⁴ s (or about 30 s to 8 h). Similar considerations are expected to govern the rates of collision of two laterally mobile molecules on the cell surface.5

This oversimplified model of protein and lipid diffusion in the membrane provides a sense of the timescale over which these interactions take place. However, it is important to note that molecules in the membrane often experience anomalous diffusion because of complex mechanisms which are still not completely understood.3 A number of important molecular and cellular events require the lateral motion of membrane proteins and lipids, and therefore depend on receptor lateral mobility. These phenomena include receptor clustering, microdomain formation, cell adhesion, and receptor-ligand interactions among others.4, 6, 7 Therefore, accurate measurements of lateral mobility are required to understand these events at the molecular level.

REORGANIZATION

Probably the best known example of lateral organization in T cells is the complex of cell surface molecules that assemble with the T cell receptor (TCR). The TCR complex typically contains multiple TCR polypeptides (α/β or γ/δ) in a complex with CD3 chains (ε, γ, δ, and ζ). Fluorescence microscopy has shown that the TCR is rapidly clustered at contact sites displaying peptide-MHC protein (pMHC) binding sites49; this phenomenon is most dramatically seen in the ultimate formation of supramolecular activation complexes (SMACs).50 Recent work has shown that the TCR complex can include multiple TCR heterodimers (α/β) and, interestingly, these complexes can exist in a range of clustered states.51 Clustering may be a mechanism for controlling the avidity and sensitivity of the TCR complex for its cognate antigen, and cluster formation may depend on the lipid environment in which the complex resides. Furthermore, clustered TCR complexes may serve not only as a mechanism for signal attenuation but also as the initial site of signaling—whereas the subsequently formed SMACs appear to be sites for terminating the signal as the TCR is degraded.52

An important component of the TCR counter-receptor, the MHC protein, also experiences lateral reorganization in the membrane. Both FPR26, 28, 53 and SPT30, 54, 55 studies have shown that the MHC protein is a highly mobile cell surface receptor, and its mobility is in part determined by its cytoplasmic tail. FRET56-58 and single-molecule fluorescence imaging59 studies have shown that MHC proteins also reorganize into oligomeric clusters. The lateral mobility of the lymphocyte receptor CD2 has been examined by FPR and SPT.41, 60, 61 Using both confocal FM and SPT, Douglass and Vale observed that CD2 associates specifically with membrane microdomains.22 The receptor traverses these domains with reduced lateral mobility, and other signaling molecules are found within these domains along with CD2. Other receptors, such as L-selectin62 and LFA-1,63 are also known to reorganize into clusters. The binding of multivalent ligands may mimic adhesion systems by enforcing surface receptor clustering.64, 65 In some cases, the functional consequences of this clustering may include activation and proteolytic shedding of the receptor.66, 67

RECRUITMENT

The selectin family of cell surface receptors provides an excellent example of the importance of subcellular localization for receptor function. Selectins are found on a variety of leukocytes and are critical for the process of inflammation. The extracellular domain of the receptor contains a lectin binding site that is essential for receptor function. It has become clear, however, that the localization of the receptor on the cell surface is also critical for function.68 For example, L-selectin localizes to the microvilli of leukocytes, and experiments using selectin chimeras that contain the L-selectin binding domain and the transmembrane and intracellular domains of CD44—a cell surface molecule not generally found on microvilli—show that the localization of L-selectin to microvilli depends on the intracellular domain of the protein.69 Furthermore, the localization of L-selectin to microvilli depends on molecular interaction of the selectin intracellular domain with α-actinin, a cytoskeleton-associated protein.70 This molecular association is functionally important, as perturbations in the subcellular localization of L-selectin affect the ability of this receptor to mediate adhesion.70, 71 Other leukocyte receptors, such as the MHC protein on antigen presenting cells, are known to be associated with lipid raft domains, and the domains themselves appear to be actively recruited to the site of adhesion.72 In a similar fashion, both the SMACs and individual molecular components within the immune synapse—such as LFA-1, CD2, and the TCR—are directed via extracellular contacts and cytoskeletal mechanisms to the appropriate subcellular location, where an adhesive contact is formed.32, 50

DISPERSION

In the case of lymphocyte73 and related integrins,74 it appears that the resting state of the receptor has low lateral mobility. Both cellular stimulation and cytoskeletal disruption lead to a large increase in integrin lateral mobility and, thus, to dispersion of the receptor in the plasma membrane. One potential rationale for the effect of cellular stimulation on integrin mobility would be to “prime” the cell for adhesion by ensuring that the receptor binding sites are well distributed so as to foster binding. The lymphocyte receptor CD2 is also randomly distributed (i.e., disperse) on the surface of resting cells.38 Dispersion of adhesion receptors is often followed temporally by a localized recruitment and/or anchoring mechanism. For example, LFA-1 is ultimately recruited to the immune synapse as part of a SMAC50 and CD2 is selectively immobilized at sites of receptor ligation.41 One can therefore think of the dispersion mechanism as the counterpoint to receptor recruitment and anchoring.

ANCHORING

Adhesion receptor anchoring is an established mechanism of mobility regulation for several members of the integrin family. These heterodimeric adhesion receptors bind to a variety of counter-receptors, providing a physical link between the cellular cytoskeleton and the external environment. Measurements of integrin lateral mobility illustrate the molecular regulation of this process, and it is striking how many regulatory elements are conserved across different cell types and integrin subtypes. In fibroblasts, antibody-labeled β₁ integrins diffuse rapidly, but the same integrins labeled with a counter-receptor, fibronectin, show a dramatic reduction in lateral mobility.74 Notably, the effect of ligation on integrin diffusion is observed only in integrins with intact cytoplasmic domains. The lateral mobility of the lymphocyte integrin, LFA-1 (α_Lβ₂), has been studied using FM and SPT. The receptor is immobilized by cytoskeletal contacts in resting cells, and this anchored state can be disrupted by cell activation76 or cystoskeletal disruption.73 Several cytoskeleton-associated proteins appear to be involved in LFA-1 mobility regulation, including dynamitin77 and talin.78

Similar to the immobilizing effect of fibronectin ligation on β₁ integrin diffusion,74 the lateral mobility of the β₂ integrin LFA-1 is affected by binding to its counter-receptor, ICAM-1. Peters et al. observed that LFA-1 lateral diffusion is reduced when the receptor is labeled with ICAM-1, and hypothesized that the anchoring of bound LFA-1 would be expected to reinforce integrin adhesion.79 This observation was confirmed and extended by examining not only the diffusion of LFA-1 bound to ICAM-1, but also the mobility of different conformational epitopes of the receptor.15 Cairo et al. found that LFA-1 is indeed restricted in its lateral mobility on resting cells, but that activated conformations of the receptor are more mobile. These data fit a mass-transport model of LFA-1 diffusion, which may also be relevant for other adhesion receptor systems.48 A key observation from this study was the co-existence of multiple populations of receptors with different lateral mobilities. This heterogeneous diffusion profile suggests that the α_Lβ₂ integrin exists in a dynamic equilibrium between its cytoskeleton-associated and freely diffusing forms.

We suggest that the observation of multiple states of lateral mobility coexisting at the cell surface is not unique to this system, and could well be a ubiquitous feature of membrane adhesion receptors. The most direct statement of this model can be found in Cairo et al. for LFA-1,15 although SPT experiments on other integrins have also found evidence for multiple populations of molecules with different diffusion coefficients (personal communication from M. Cho and H.-F. Chen).80 Similar observations of heterogeneous diffusion have been made for the lymphocyte receptor CD2.22, 41 It is important to recognize that this hypothesis is directly supported by even the earliest studies of membrane protein lateral mobility, in which FPR data on live cells typically showed a mobile and an immobile fraction—indeed, based on this evidence alone, we would have predicted that SPT experiments would reveal a heterogeneous population for most, if not all, adhesion receptors.

INTEGRATION OF MECHANISMS

We have seen several individual examples of mechanisms that affect lateral mobility, but few of these mechanisms could be said to operate independently. Indeed, it is likely that many adhesion systems integrate multiple mechanisms of mobility regulation to accomplish their function. Several key examples from T cell biology demonstrate this interplay: the integrin LFA-1, L-selectin, and the TCR complex. Each of these systems has been studied to the extent that the true complexity of its regulation has begun to be revealed.

Using a variety of methods, studies of the lymphocyte integrin LFA-1 have elucidated a complex network of elements coordinating T cell adhesion during lymphocyte rolling, extravasation, and antigen presentation. Some models of LFA-1 function have been controversial, most likely because the receptor's behavior is complex and highly regulated. It has been established that conformational changes in integrin heterodimers alter their affinity for ligand and that this alteration in affinity is necessary for the function of many integrin receptors, including LFA-1.81, 82 A longstanding debate has focused on whether conformational changes or receptor clustering events are more central to integrin function.34, 83, 84 As discussed earlier, because lateral mobility is required for the formation of receptor clusters, considerations of integrin lateral mobility regulation are necessary to resolve the issue.

Single-molecule studies of the relationship between LFA-1 conformation and lateral mobility have provided an emerging model that orchestrates these two factors into a single mass transport model.15, 48 Current data suggest that an intermediate-affinity receptor conformation is anchored, that cellular activation causes dispersion of the receptor, and that receptor ligation induces recruitment and anchoring of a high-affinity receptor conformation at the contact site in order to strengthen the adhesion.73 This model provides for negative regulation by the first anchoring mechanism and for positive regulation by dispersion and by the second anchoring mechanism (most likely, involving alternative cytoskeletal regulators).78 Reorganization of LFA-1 into multivalent complexes is also likely to be key to receptor function during these steps.63 Importantly, models with this level of resolution begin to approach a quantitative understanding of the system. By separately considering each conformational state of the receptor and the molecular interactions that affect its lateral mobility, we can view the overall system as a set of dynamic equilibria.15 This model is satisfying in its simplicity, and suggests that the cell can efficiently regulate integrin-mediated adhesion through modification of the binding affinity of the integrin to the relevant cytoplasmic proteins.82

L-selectin represents a second intensively studied adhesion system that integrates multiple mechanisms of mobility regulation. L-selectin-mediated adhesion is regulated by both receptor recruitment and receptor anchoring. Several lines of investigation show that recruitment of L-selectin, resulting in its localization to the tips of microvilli, is essential for the initiation of contact between the leukocyte and the vascular endothelium.69, 71 In this system, recruitment appears to be mediated primarily by the transmembrane and intracellular domains of the receptor. In addition to recruitment, anchoring of the receptor to the cytoskeleton appears to be required for leukocyte rolling.70 Anchoring is mediated by the interaction of the cytoplasmic domain of L-selectin with α-actinin.

The group of proteins that form the TCR complex is perhaps the most complex cell surface receptor on the T cell. The TCR participates in the formation of an immune signaling complex that includes many other receptors and cytoplasmic components, ultimately forming microclusters and supramolecular activation complexes (SMACs) (Figure 5).50 These processes require exquisite regulation of TCR lateral mobility, depending on receptor reorganization, recruitment, and anchoring mechanisms. The TCR has been found to exist in a mixture of clustered states on the resting cell, and can be reorganized to form multiple complexes with different stoichiometries with other surface molecules.51 This reorganization is thought to assist in controlling the fidelity of TCR response as the functional affinity of the cluster is altered.85 TCR signaling also leads to the recruitment of membrane protein and lipid components as well as a bevy of cytoplasmic signaling molecules.52 Finally, the recruitment of TCR complexes to the appropriate subcellular locations is an active process mediated by anchoring to components of the cytoskeleton.86

CONCLUSIONS

The lateral mobility of cell surface receptors is an important regulator of cellular adhesion. We propose a general framework for understanding the role of lateral mobility in adhesion regulation. Our framework includes four interrelated mechanisms of regulation: reorganization, recruitment, dispersion, and anchoring. This framework allows for comparisons among different molecular adhesion systems and provides a useful classification of existing data. As more complete models of membrane receptor function and protein-protein interactions are elucidated, the measurement of lateral mobility will become increasingly important for understanding cellular adhesion mechanisms.