Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions.
PNAS 113(20):5682 (2016)
The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a so...
Initiation of T cell signaling by CD45 segregation at 'close contacts'.
Nature Immunology 17(5):574 (2016)
It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracell...
Super-Resolved Traction Force Microscopy (STFM).
Nano Letters 16(4):2633 (2016)
Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greate...
Phagocytes Get Close to Their Enemies.
Developmental Cell 36(2):131 (2016)
Phagocytosis is key for many organismal functions. In a recent issue of Cell, Freeman et al. (2016) demonstrate a feed-forward signaling mechanism wherein F-actin and integrin receptors drive contact formation between phagocytes and antibody-coated solid particles, signaling their engulfment. Th...
Referenced Single-Molecule Measurements Differentiate between GPCR Oligomerization States.
Biophysical Journal 109(9):1798 (2015)
The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at...
Membrane nanoclusters-tails of the unexpected.
Cell 161(3):433 (2015)
The existence, nature, and role of highly ordered membrane domains, often referred to as lipid rafts, have been highly debated by cell biologists for many years. In this issue, Raghupathy et al. describe molecular mechanisms leading to the formation of ordered lipid-protein clusters.
CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses.
Scientific reports 5:16487 (2015)
Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell leve...
Glycan Remodeling with Processing Inhibitors and Lectin-Resistant Eukaryotic Cells.
Methods in Molecular Biology 1321:307 (2015)
Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, t...
Type-3 BRET, an improved competition-based bioluminescence resonance energy transfer assay.
Biophysical Journal 106(12):L41 (2014)
We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membr...
TCR signaling: the barrier within.
Nature Immunology 15(2):136 (2014)
Structure and interactions of the human programmed cell death 1 receptor.
Journal of Biological Chemistry 288(17):11771 (2013)
PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based struc...
PD-1 promotes immune exhaustion by inducing antiviral T cell motility paralysis.
Journal of Experimental Medicine 210(4):757 (2013)
Immune responses to persistent viral infections and cancer often fail because of intense regulation of antigen-specific T cells-a process referred to as immune exhaustion. The mechanisms that underlie the induction of exhaustion are not completely understood. To gain novel insights into this pro...
Imaging the cell surface and its organization down to the level of single molecules.
Philosophical Transactions of the Royal Society... 368(1611):20120027 (2013)
Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we re...
A streamlined implementation of the glutamine synthetase-based protein expression system.
BMC Biotechnology 13:74 (2013)
The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultu...
A quantitative comparison of single-dye tracking analysis tools using Monte Carlo simulations.
PLoS ONE 8(5):e64287 (2013)
Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of shor...
IL-2 and IL-15 signaling complexes: different but the same.
Nature Immunology 13(12):1141 (2012)
TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis.
Nature 488(7412):508 (2012)
Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these varia...
T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements.
Journal of Biological Chemistry 287(16):13324 (2012)
Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found...
On the Control of TCR Phosphorylation.
Frontiers in Immunology 3:92 (2012)
Unraveling receptor stoichiometry using bret.
Frontiers in Endocrinology 3:86 (2012)