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Lymphocyte T cells are responsible for cell-mediated adaptive immune responses, involving transient interactions of the T-cell receptor (TCR) with peptides presented by MHC proteins. A productive interaction triggers the T-cell signaling forming the immunological synapse. Initially, the TCR is phosphorylated by the Lck, a membrane-anchored tyrosine kinase, producing membrane microclusters. Understanding the T-cell pre-synaptic triggering implies comparing resting vs. activated states. Super-resolution imaging techniques (i.e. dSTORM and PALM) suggested protein pre-clustering into “nano-domains” in resting cells, contradicting the classical view of cluster formation upon activation. However, observations with cells touching non-activating surfaces probably don’t resemble a true resting state. Instead, avoiding contacts would allow better understanding the resting and early stages of activation. We studied live T-cells in suspension employing a hydrogel in a density gradient and used STED nanoscopy to unravel the plasma membrane distribution and dynamics of Lck in T-cells on glass and in suspension. T-cells suspended in the hydrogel do not triggered calcium, indicating absence of activation; while classically considered resting states triggered calcium in a similar fashion as when cells were deposited onto a surface functionalized by antibody (antiCD3 and antiCD28) coating. In contrast to surface-contacting, suspended T-cells showed mostly a uniform distribution of Lck. Nevertheless, Lck still displayed heterogeneity in mobility in the true resting state. We found several components of mobility rather than a single one. These different diffusion coefficients were remarkably slower when T-cells were in contact with any of the studied surfaces. Our results suggest that pre-clustering of signaling receptors and cell-surface proteins in resting T-cells needs reconsideration and that understanding the T cell activation requires a true resting state, which we can be obtained by means of hydrogels.

Summary Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the “glycosylation problem” can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

The natural variation of Mg and Ca stable isotopes of carbonates has been determined in carbonate skeletons of perforate foraminifera and reef coral together with Mg/Ca ratios to assess the influence of biomineralisation processes. The results for coral aragonite suggest its formation, in terms of stable isotope behaviour, approximates to inorganic precipitation from a seawater reservoir. In contrast, results for foraminifera calcite suggest a marked biological control on Mg isotope ratios presumably related to its low Mg content compared with seawater. The bearing of these observations on the use of Mg and Ca isotopes as proxies in paleoceanography is considered.

Among the most contentious questions in immunology is: how is the T cell receptor (TCR) triggered? using a combination of different single-molecule tracking, novel calcium signalling assays, crystallography and super-resolution imaging techniques we have demonstrated evidence to support the kinetic segregation model as the molecular mechanism behind the adaptive immune system.The phosphorylation of the T cell receptor is required for T cell activation. The kinetic segregation model predicts that this is achieved by the spatial partitioning of important components such as the TCR and Lck kinase from the CD45 phosphatase according to the difference in size of the extracellular domain of the proteins on the cell membrane. We test this prediction by imaging the formation of contacts between T cells with a model antigen presenting cells in early stage contacts at endogenous expression levels. The approach allows the interrogation of the kinetic segregation with other models based upon aggregation or conformation change of the TCR. We present the results of this study.

In this presentation I show how hydrodynamic forces can be used to locally trap and move membrane-associated molecules in lipid bilayers. We use the liquid flow through a ∼1 μm pipette to create a localized force field that acts on molecules protruding from the lipid bilayer (see Fig. 1). In addition to introducing the hydrodynamic trap and some of the possibilities and challenges of using this technique on living cells, I will also present examples of using this technique to: (i) vary the concentration of molecules in lipid bilayers, (ii) study intermolecular interactions between different membrane-bound proteins as a function of surface coverage and (iii) induce and study pore formation in lipid bilayers. In particular, I show how the hydrodynamic trap can be used to obtain information about the orientation and mechanical properties of the extracellular domain of the tyrosine phosphatase CD45 involved in the early stages of T-cell immune response.Fig. 1Liquid flow through a conical pipette is used to locally trap molecules in lipid bilayers.View Large Image | View Hi-Res Image | Download PowerPoint Slide