Eilon Sherman

Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor

Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated localization microscopy to study complexes downstream of the T cell antigen receptor (TCR) in single-molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adaptor LAT in activated cells, thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells, LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adaptor SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

The LAT Story: A Tale of Cooperativity, Coordination, and Choreography

The adapter molecule LAT is a nucleating site for multiprotein signaling complexes that are vital for the function and differentiation of T cells. Extensive investigation of LAT in multiple experimental systems has led to an integrated understanding of the formation, composition, regulation, dynamic movement, and function of LAT-nucleated signaling complexes. This review discusses interactions of signaling molecules that bind directly or indirectly to LAT and the role of cooperativity in stabilizing LAT-nucleated signaling complexes. In addition, it focuses on how imaging studies visualize signaling assemblies as signaling clusters and demonstrate their dynamic nature and cellular fate. Finally, this review explores the function of LAT based on the interpretation of mouse models using various LAT mutants.

Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions

Studies of protein-protein interactions, carried out in polymer solutions, are designed to mimic the crowded environment inside living cells. It was shown that crowding enhances oligomerization and polymerization of macromolecules. Conversely, we have shown that crowding has only a small effect on the rate of association of protein complexes. Here, we investigated the equilibrium effects of crowding on protein heterodimerization of TEM1-beta-lactamase with beta-lactamase inhibitor protein (BLIP) and barnase with barstar. We also contrasted these with the effect of crowding on the weak binding pair CyPet-YPet. We measured the association and dissociation rates as well as the affinities and thermodynamic parameters of these interactions in polyethylene glycol and dextran solutions. For TEM1-BLIP and for barnase-barstar, only a minor reduction in association rate constants compared to that expected based on solution viscosity was found. Dissociation rate constants showed similar levels of reduction. Overall, this resulted in a binding affinity that is quite similar to that in aqueous solutions. On the other hand, for the CyPet-YPet pair, aggregation, and not enhanced dimerization, was detected in polyethylene glycol solutions. The results suggest that typical crowding agents have only a small effect on specific protein-protein dimerization reactions. Although crowding in the cell results from proteins and other macromolecules, one may still speculate that binding in vivo is not very different from that measured in dilute solutions.

Using Fluorescence Correlation Spectroscopy to Study Conformational Changes in Denatured Proteins

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.

Imaging techniques for assaying lymphocyte activation in action

Imaging techniques have greatly improved our understanding of lymphocyte activation. Technical advances in spatial and temporal resolution and new labelling tools have enabled researchers to directly observe the activation process. Consequently, research using imaging approaches to study lymphocyte activation has expanded, providing an unprecedented level of cellular and molecular detail in the field. As a result, certain models of lymphocyte activation have been verified, others have been revised and yet others have been replaced with new concepts. In this article, we review the current imaging techniques that are used to assess lymphocyte activation in different contexts, from whole animals to single molecules, and discuss the advantages and potential limitations of these methods.

Non-random-coil Behavior as a Consequence of Extensive PPII Structure in the Denatured State

Unfolded proteins may contain a native or nonnative residual structure, which has important implications for the thermodynamics and kinetics of folding, as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique--fluorescence correlation spectroscopy--to probe the denatured state of a set of repeat proteins containing an increasing number of identical domains, from 2 to 20. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random coil statistics. The origin of this unexpected behavior is traced to the presence of an extensive nonnative polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show that the experimentally observed effects of polyproline II on the size scaling of the denatured state can be well-described by simple polymer models. Our findings suggest a hitherto unforeseen potential of nonnative structure to induce significant compaction of denatured proteins, significantly affecting folding pathways and kinetics.

Biophysical Characterization of the Unstructured Cytoplasmic Domain of the Human Neuronal Adhesion Protein Neuroligin 3

Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila protein, gliotactin, showed that it is intrinsically unstructured in vitro. Bioinformatic analysis suggested that the cytoplasmic domains of other CLAMs are also intrinsically unstructured, even though they bear no sequence homology to each other or to any known protein. In this study, we overexpress and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the Escherichia coli endogenous proteases that cause its rapid degradation. Using bioinformatic analysis, sensitivity to proteases, size exclusion chromatography, fluorescence correlation spectroscopy, analytical ultracentrifugation, small angle x-ray scattering, circular dichroism, electron spin resonance, and nuclear magnetic resonance, we show that the cytoplasmic domain of human neuroligin 3 is intrinsically unstructured. However, several of these techniques indicate that it is not fully extended, but becomes significantly more extended under denaturing conditions.

Super-resolution characterization of TCR-dependent signaling clusters

Multi‐molecular signaling complexes drive the earliest events of immune cell activation via immunoreceptors with unexplained specificity and speed. Fluorescence microscopy has shown that these complexes form microclusters at the plasma membrane of activated T cells upon engagement of their antigen receptors (TCRs). Although crucial for cell function, much remains to be learned about the molecular content, fine structure, formation mechanisms, and function of these microclusters. Recent advancements in super‐resolution microscopy have enabled the study of signaling microclusters at the single molecule level with resolution down to approximately 20 nm. These techniques have now helped to characterize the size distributions of signaling clusters at the plasma membrane of intact cells and to shed light on the formation mechanisms that govern their assembly. Surprisingly, dynamic and functional nanostructures have been identified within the signaling clusters. We expect that these novel methodologies, combined with older techniques, will shed new light on the nature of signaling clusters and their critical role in T‐cell activation.

Fluorescence Correlation Spectroscopy of Fast Chain Dynamics within Denatured Protein L

Intramolecular dynamics in the denatured state of a protein are of importance for protein folding. Native-like contact formation within the ensemble of denatured conformations of a protein may guide its transformation towards the native conformation. The efficiency of folding is thus dependent on the diffusion of chain fragments, which facilitates contact formation. Herein we investigate intramolecular diffusion of denatured molecules of the small two-state-folding protein L with fluorescence correlation spectroscopy (FCS). We utilize the specific quenching of the fluorescence of the oxazine dye Atto655 labeling a cysteine at position 64 (the C-terminus of the protein) by the side chain of a tryptophan at position 47. FCS measurements allow us to probe processes ranging in timescales from tens of nanoseconds to seconds. Two fast photophysical processes can be distinguished in the fluorescence correlation curves. The slower of the two is found to be due to triplet dynamics, while the faster process is attributed to the quenching of the Atto655 by the tryptophan upon transient ground-state complex formation. We study the dependence of the intrachain dynamics of the denatured protein on the concentration of the denaturant guanidinium chloride (GdmCl), and extract complex association and dissociation rates. While the dissociation rate does not depend on the denaturant, the association rate decreases as denaturant concentration is increased from 3 to 7 M GdmCl. This decrease in contact formation rate tracks the expansion of denatured protein L, measured in our previous work. Thus, the intramolecular diffusion coefficient calculated from the results is found to be essentially independent of the denaturant concentration over this range, even as the protein expands by more than 20%.

Resolving multi-molecular protein interactions by photoactivated localization microscopy

Multi-molecular protein complexes are critical to many cellular functions, including signaling, DNA transcription and enzymatic reactions. In spite of their importance, current research techniques such as biochemistry and diffraction-limited microscopy cannot resolve the heterogeneity and nanoscale organization of protein complexes in intact cells. Here we describe a technique that enables the study of multi-molecular protein complexes at the single molecule level in intact cells. The technique uses photoactivated localization microscopy (PALM) to resolve individual proteins with a resolution down to 20nm in intact cells, and second-order statistics to study the spatial interactions of the proteins. We demonstrate the feasibility of this technique by studying signaling complexes that form in activated T cells. We first use single color PALM imaging and univariate second-order statistics to resolve the clustering of Linker for Activation of T cells (LAT) at the plasma membrane (PM) of the cells. We then use two color PALM and bivariate second-order statistics to resolve the interaction of LAT with key interacting proteins. We discuss potential caveats in studying molecular clustering and the robustness of the technique to study bimolecular interactions. Our proposed technique, combined with older techniques, could help shed new light on the nature of multimolecular protein complexes and their significance to cell function.