The dendritic cell cytoskeleton promotes T cell adhesion and activation by constraining ICAM-1 mobility

Integrity of the dendritic cell (DC) actin cytoskeleton is essential for T cell priming, but the underlying mechanisms are poorly understood. We show that the DC F-actin network regulates the lateral mobility of intracellular cell adhesion molecule 1 (ICAM-1), but not MHCII. ICAM-1 mobility and clustering are regulated by maturation-induced changes in the expression and activation of moesin and α-actinin-1, which associate with actin filaments and the ICAM-1 cytoplasmic domain. Constrained ICAM-1 mobility is important for DC function, as DCs expressing a high-mobility ICAM-1 mutant lacking the cytoplasmic domain exhibit diminished antigen-dependent conjugate formation and T cell priming. These defects are associated with inefficient induction of leukocyte functional antigen 1 (LFA-1) affinity maturation, which is consistent with a model in which constrained ICAM-1 mobility opposes forces on LFA-1 exerted by the T cell cytoskeleton, whereas ICAM-1 clustering enhances valency and further promotes ligand-dependent LFA-1 activation. Our results reveal an important new mechanism through which the DC cytoskeleton regulates receptor activation at the immunological synapse.     

Lineage diversion of T cell receptor transgenic thymocytes revealed by lineage fate mapping

Background

The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRαβ lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRαβ prematurely at the double negative stage and abnormal TCRαβ populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice.

Methodology and Principal Findings

To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRαβ diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORγt-positive CD4⁺8⁺ (double positive, DP) stage to accumulate either as CD4⁻8⁻ (double negative, DN) or as CD8α⁺ T cells in lymph nodes or gut epithelium. Likewise, DN TCRαβ cells in lymphoid tissue of female mice were not derived from DP thymocytes.

Conclusion

The results further support the hypothesis that the premature expression of the TCRαβ can divert DN thymocytes into gamma delta lineage cells.     

Influence of receptor lateral mobility on adhesion strengthening between membranes containing LFA-3 and CD2

We have used an in vitro model system of glass-supported planar membranes to study the effects of lateral mobility of membrane-bound receptors on cell adhesion. Egg phosphatidylcholine (PC) bilayers were reconstituted with two anchorage isoforms of the adhesion molecule lymphocyte function-associated antigen 3 (LFA-3). The diffusion coefficient of glycosyl phosphatidylinositol (GPI)-anchored LFA-3 approached that of phospholipids in the bilayers, whereas the transmembrane (TM)-anchored isoform of LFA-3 was immobile. Both static and laminar flow assays were used to quantify the strength of adherence to the lipid bilayers of the T lymphoma cell line Jurkat that expresses the counter-receptor CD2. Cell adhesion was dependent on LFA-3 density and was more efficient on membranes containing the GPI isoform than the TM isoform. Kinetic measurements demonstrated an influence of contact time on the strength of adhesion to the GPI isoform at lower site densities (25-50 sites/microns2), showing that the mobility of LFA-3 is important in adhesion strengthening. At higher site densities (1,500 sites/microns2) and longer contact times (20 min), Jurkat cell binding to the TM and GPI isoforms of LFA-3 showed equivalent adhesion strengths, although adhesion strength of the GPI isoform developed twofold more rapidly than the TM isoform. Reduction of CD2 mobility on Jurkat cells at 5 degrees C greatly decreased the rate of adhesion strengthening with the TM isoform of LFA-3, resulting in a 30-fold difference between the two LFA-3 isoforms. Our results demonstrate that the ability of a membrane receptor and its membrane-bound counter-receptor to diffuse laterally enhances cell adhesion both by allowing accumulation of ligands in the cell contact area and by increasing the rate of receptor-ligand bond formation.     

Analysis of cell surface interactions by measurements of lateral mobility

Interactions of cell surface components with one another and with structures inside and outside the cell may have important physiological functions in the transmission of signals and the assembly of specialized structures. These interactions may be detected and analyzed through their effects on the lateral mobility of cell surface molecules. Measurements by a fluorescence photobleaching method have shown that in general lipid-like molecules diffuse rapidly and freely through the plasma membrane, whereas proteins move much more slowly or appear to be immobile. This dichotomy has been supposed to result from forces beyond the viscosity of the lipid bilayer, which specifically retard the diffusion of membrane proteins. This general picture should be qualified, however, by noting that the lateral mobility of lipid-like molecules can be influenced in detail by changes in the state of the plasma membrane such as result from mitosis or fertilization. The interactions of cell surface proteins that limit their lateral mobility are unknown. The effects of binding concanavalin A to localized regions of cell surface show that these interactions can vary in subtle and complex ways. It may soon be useful to interpret mobility experiments in terms of simple reaction models that attempt to describe surface interactions in physicochemical terms. More experimental data are needed to carry out this program and to relate interactions that affect mobility to the structural connections between cell surface components and the cytoskeleton, which have been detected by biochemical methods and electron and immunofluorescence microscopy.     

Cooperation between lateral ligand mobility and accessibility for receptor recognition in selectin-induced cell rolling

Selectin-induced leukocyte rolling along the endothelial surface is an essential step in the immune response. Several in vitro studies showed that this cell rolling is a highly regulated adhesion phenomenon, controlled by the kinetics and forces of selectin-ligand interactions. In the flow chamber study presented here, we focused on the requirements on the ligand structure in this context. A series of neoglycolipids bearing the binding epitope Sialyl Lewis X was synthesized and used as artificial ligands. These lipids differed in their spacer structures between headgroup and membrane anchor, resulting in a gradual variation in accessibility and mobility of the binding epitope when immobilized in model membranes. Consequently, analysis of cell rolling along such membranes allowed correlation of ligand structures and functionality. All model membranes containing such ligands were further characterized by film balance measurements, epifluorescence, and atomic force microscopy. Generally, the glycolipids exhibited a high tendency for lateral aggregation, but the resulting clusters were of different morphology. This was also reflected by strong differences in the rolling experiments. Our results confirm that, in addition to a sufficient headgroup accessibility, the cell rolling process is governed by two further interdependent factors: (i) the headgroup flexibility caused by the intramolecular uncoupling between the headgroup and the hydrophobic moiety due to introduction of a spacer, and (ii) the stiffness of the molecules resulting from their supramolecular arrangement in clustered assemblies. Since both factors are influenced simultaneously by the spacer modification, we present for the first time a clear correlation between structural aspects of selectin ligands and their ability to mediate cell rolling. This might help to develop a better understanding for the function of the natural selectin ligands.     

T cell adhesion molecules

Cell adhesion or conjugate formation between T lymphocytes and other cells is an important early step in the generation of the immune response. Although the antigen-specific T cell receptor confers antigen recognition and specificity, a number of other molecules expressed on the T cell surface are involved in the regulation of lymphocyte adhesion. T cell molecules that function to strengthen adhesion include lymphocyte function-associated antigen (LFA)-1, CD2, CD4, and CD8. Their ligands have recently been identified. LFA-1 is a member of the integrin family of adhesion receptors and one of its ligands is intercellular adhesion molecule-1 (ICAM-1); a ligand for CD2 is LFA-3; and ligands for CD4 and CD8 appear to be major histocompatibility complex class II and class I molecules, respectively. In addition, T cells express a number of receptors thought to be involved in cell matrix adhesion. The function and significance of these T cell adhesion receptors and their ligands are reviewed.     

Redundant mechanisms for stable cell locomotion revealed by minimal models

Crawling of eukaryotic cells on flat surfaces is underlain by the protrusion of the actin network, the contractile activity of myosin II motors, and graded adhesion to the substrate regulated by complex biochemical networks. Some crawling cells, such as fish keratocytes, maintain a roughly constant shape and velocity. Here we use moving-boundary simulations to explore four different minimal mechanisms for cell locomotion: 1), a biophysical model for myosin contraction-driven motility; 2), a G-actin transport-limited motility model; 3), a simple model for Rac/Rho-regulated motility; and 4), a model that assumes that microtubule-based transport of vesicles to the leading edge limits the rate of protrusion. We show that all of these models, alone or in combination, are sufficient to produce half-moon steady shapes and movements that are characteristic of keratocytes, suggesting that these mechanisms may serve redundant and complementary roles in driving cell motility. Moving-boundary simulations demonstrate local and global stability of the motile cell shapes and make testable predictions regarding the dependence of shape and speed on mechanical and biochemical parameters. The models shed light on the roles of membrane-mediated area conservation and the coupling of mechanical and biochemical mechanisms in stabilizing motile cells.     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

Dynamic adhesion of T lymphocytes to endothelial cells revealed by atomic force microscopy

The recruitment of T lymphocytes to lymphoid organs or sites of inflammation is a crucial step in adaptive immunity. These processes require endothelial activation and expression of adhesion molecules, including E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). However, the complete characterization of the adhesion strength and dynamics between lymphocytes and endothelial cells has been hampered by the lack of sensitive quantitative techniques. Here we report on the application of atomic force microscopy to characterize the interaction between individual pairs of living T lymphocytes (i.e., Jurkat cells) and human umbilical vein endothelial cells (HUVECs). The detachment of individual cell-cell conjugates was a complex process involving several step-like rupture events and the viscoelastic deformation of cells on the scale of several microns. Adhesion between Jurkat cells and activated endothelial cells increased with compression force and contact time, with the most dramatic changes occurring within the first half second of contact. After 0.25 sec of contact, E-selectin, ICAM-1, and VCAM-1 contributed to 18%, 39%, and 41% of total adhesion strength, respectively, suggesting that ICAM-1 and VCAM-1 contributed more than the selectins in supporting cell attachment.     

Interdomain mobility in di-ubiquitin revealed by NMR

Domain orientation and dynamics can play an essential role in the function of multidomain proteins. Lys48-linked polyubiquitin chains, the principal signal for proteasomal protein degradation, adopt a closed conformation at physiological conditions, in which the functionally important residues Leu8, Ile44, and Val70 are sequestered at the interdomain interface. This interface must open in order for these groups to become available for interactions with various chain-recognition factors. Knowledge of the mechanism of domain motion leading to the opening of the interdomain interface in polyubiqutin is, therefore, essential for the understanding of the processes controlling molecular recognition events in polyubiquitin signaling. Here we use NMR to characterize the interdomain dynamics that open the interface in a di-ubiquitin chain. This process occurs via domain reorientations on a 10-ns time scale and with the amplitudes that are sufficient for making functionally important hydrophobic residues in polyubiquitin available for direct interactions with various ubiquitin-binding factors. The analysis revealed the structures of the interconverting conformational states of di-ubiquitin and the rates and amplitudes of this process at near-physiological and acidic pH. The proposed mechanism of domain reorientation is quite general and could serve as a paradigm of interdomain mobility in other multidomain systems.     

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor‐type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPρ is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto‐domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto‐domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane–proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain‐specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.     

Disparate modulation of plasma membrane protein lateral mobility by various cell permeabilizing agents

The mobility of a cell surface protein on cells osmotically swollen by treatment with several different cell permeabilizing agents retains specific restraints despite detachment of the plasma membrane from the cortical cytoskeleton. Fluorescence photobleaching recovery experiments indicate that the lateral diffusion constants of immunoglobulin E (IgE)-receptor complexes on the surface of rat basophilic leukemia cells increase 2–5 × following permeabilization with streptolysin O or digitonin, with little change in their mobile fractions. Swelling by hypo-osmotic treatment in water enhances lateral diffusion of IgE-receptor complexes and raises the mobile fractions to near 100%. In contrast, swelling by treatment with filipin arrests lateral diffusion, although rotational mobility remains unhindered. Lateral mobility of a fluorescent lipid analogue remains unchanged under these conditions. Crosslinking by anti-IgE antibodies redistributes the IgE-receptor complexes into large patches on untreated cells and on cells swollen by permeabilization with streptolysin O or digitonin, but rot on cells swollen by treatment with filipin. The results indicate a diversity of effects of the various permeabilizing agents on the mobility of membrane proteins. In particular, treatment with filipin appears to reorganize the plasma membrane into a network of fluid domains on a scale smaller than the bleaching spot size used (～1.5 μm). © 1994 Wiley-Liss, Inc.     

Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids

Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein- phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.     

Regulation of lateral mobility and cellular trafficking of the CCK receptor by a partial agonist

Partial agonists are effective tools for advancing developmentof highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of apartial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in nativepancreatic acinar cells and Chinese hamster ovary cells expressing CCKreceptor (CHO-CCKR). We developed and characterized a novel fluorescentpartial agonist,rhodamine-Gly-[(Nle^28,31)CCK-26-32]-phenethylester, that binds specifically and with high affinity to CCK receptors.Such analogs are fully efficacious pancreatic acinar cell secretagogueswithout supramaximal inhibition that mobilize intracellular calciumwith little or no increase in phospholipase C (PLC) activity. Despiteminimal phosphorylation of CCK receptors in response to this partialagonist, receptor trafficking was the same as that observed with fullagonist (CCK). This included normal internalization viaclathrin-dependent endocytosis in CHO-CCKR cells and insulation on thesurface of pancreatic acinar cells. Also, as with CCK-occupiedreceptor, fluorescence recovery after photobleaching of partialagonist-occupied receptor on the acinar cell surface demonstrated amarked temperature-dependent slowing of its rate of diffusion. This wassimilarly associated with resistance to acid-induced dissociation ofligand. Thus some key molecular regulatory mechanisms for CCK receptorinternalization and insulation may be initiated by cellular signalingcascades that are not dependent on PLC activation or receptor phosphorylation.     

Red cell membrane protein lateral mobility in diabetes mellitus.

In a group of 24 diabetics subdivided for type, we evaluated the red cell membrane protein lateral mobility marking intact red cells with pyrene-3-maleimide (3-PM) and calculating the dimer to monomer fluorescence intensity ratio (Iex/Im). The same fluorescent parameter was determined in a group of 13 normal controls. From the obtained data, it is evident that the red cell membrane protein lateral mobility clearly discriminates normals from diabetics of type 1 and 2. In normals and in diabetics of type 1 and 2 no relationship is present between this fluorescent determinant and the glycometabolic parameters (FBGL and HbA1c); considering all the diabetics, a negative relationship is evident between Iex/Im ratio and HbA1c only.