Polarizing effects of productive dendritic cell (DC)-T-cell interactions on DC cytoskeleton have been known in some detail, but the effects on DC membrane have been studied to a lesser extent. We found that T-cell incubation led to DC elongation and segregation of characteristic DC veils to the broader pole of the cell. On the opposite DC pole, we observed a novel membrane feature in the form of bundled microvilli. Each villus was approximately 100 nm in diameter and 600 to 1200 nm long. Microvilli exhibited high density of antigen-presenting molecules and costimulatory molecules and provided the physical basis for the multifocal immune synapse we observed during human DC and T-cell interactions. T cells preferentially bound to this site in clusters often contained both CD4 ؉ and CD8 ؉ T cells. (Blood. 2008;112:5037-5045) IntroductionDendritic cells (DCs) are sentinels of immunity as they respond to environmental cues resulting in antigen uptake and processing and stimulation of antigen-specific T cells. 1,2 In addition, DCs activate the cells of innate immunity, eg, natural killer (NK) cells, NK T cells, and neutrophils. 3 Langerhans cells, the DCs predominantly located in the skin, were originally discovered because of their unique morphology of large neuron-like dendrites. These cells were recognized for their myeloid origin, role in lymphocyte activation, 4-8 and characteristic translucent membrane veils. 5,9 Similar to Langerhans cells in situ, DCs matured from monocytes in vitro display analogous membrane veils. 5,9 T cells are activated through cell-cell contact and by soluble molecules. Early response to cell-cell contact includes the formation of a large macromolecular membrane assembly: the immunologic synapse 10,11 and the consequent cell polarization. 12,13 The initial idea that immunologic synapses mediate T-cell activation has been challenged by the finding that T-cell receptor (TCR)-mediated signaling precedes or is independent of synapse formation. [14][15][16][17] Consequently, the full function of the synapse has been modified to include directed cytokine secretion, enhanced secondary signals, or internalization of TCRs. [18][19][20] Most studies of the synapse have focused on those formed between T cells and B cells [21][22][23][24][25] ; consequently, the synapse between T cells and DCs has been studied to a much lesser extent. In contrast to the unifocal T-cell-B-cell synapse, the structure of the DC-T-cell interaction site, studied mostly in murine cells, appears "immature" or "multifocal," containing numerous segregated microdomains. 26 Microdomain segregation could be important for the physical separation of ligands and receptors in the membrane plane; such separation appears essential in preventing the cis-acting inhibition, such as that observed within the Notch family ligands and receptors during DC-T-cell interaction. 27 In addition, recent evidence suggests that mechanical TCR entrapment into segregated microdomains of the synapse is required for optimal T-cell activation. 28 Even th...

Calmodulin binds to amphiphilic, helical peptides of a variety of amino-acid sequences. These peptides are usually positively charged, although there is spectroscopic evidence that at least one neutral peptide binds. The complex between calmodulin and one of its natural target peptides, the binding site for calmodulin on smooth muscle myosin light-chain kinase (RS20), has been investigated by crystallography and NMR which have characterized the interactions between the ligand and the protein. From these data, it appears that the calmodulin-binding surface is sterically malleable and van der Waals forces probably dominate the binding. To explore further this apparently permissive binding, we investigated the chiral selectivity of calmodulin using synthesized analogues of melittin and RS20 that consisted of only D-amino acids. Fluorescence and NMR measurements show that D-melittin and D-RS20 both bind avidly to calmodulin, probably in the same general binding site as that for peptides having all L-amino acids. The calmodulin-peptide binding surface is therefore remarkably tolerant sterically. Our results suggest a potentially useful approach to the design of non-hydrolysable or slowly hydrolysable intracellular inhibitors of calmodulin.

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.

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Scanning electron microscopy (SEM) can produce striking three-dimensional images of biological cells and tissues with submicron resolution of surface morphology. Such cell surfaces are often complex blends of folds, extrusions, and pockets that may be necessary in the positioning of specific molecules within interaction range of each other. Thus, surface changes can have a spatial control over some molecular functions, and identification of select molecules at distinct morphological locations becomes critical to our understanding of total cell function.