13 research outputs found
Dynamics of Natural Killer cell receptor revealed by quantitative analysis of photoswitchable protein
Natural Killer (NK) cell activation is dynamically regulated by numerous
activating and inhibitory surface receptors that accumulate at the immune
synapse. Quantitative analysis of receptor dynamics has been limited by
methodologies which rely on indirect measurements such as fluorescence recovery
after photobleaching. Here, we report a novel approach to study how proteins
traffic to and from the immune synapse using NK cell receptors tagged with the
photoswitchable fluorescent protein tdEosFP, which can be irreversibly
photoswitched from a green to red fluorescent state by ultraviolet light. Thus,
following a localized switching event, the movement of the photoswitched
molecules can be temporally and spatially resolved by monitoring fluorescence
in two regions of interest. By comparing images with mathematical models, we
evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06
micron^2/s) and assessed how synapse formation affects receptor dynamics. Our
data conclude that the inhibitory NK cell receptor KIR2DL1 is continually
trafficked into the synapse and remains surprisingly stable there. Unexpectedly
however, in NK cells forming synapses with multiple target cells
simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus,
our results reveal a previously undetected inter-synaptic exchange of protein.Comment: 25 pages, 5 figure
Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination
Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination
New Insights into How Trafficking Regulates T Cell Receptor Signaling
There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions.publishe
Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data
Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins
Superresolution Microscopy Reveals Nanometer-Scale Reorganization of Inhibitory Natural Killer Cell Receptors upon Activation of NKG2D
An intermolecular FRET sensor detects the dynamics of T cell receptor clustering
Clustering of the T-cell receptor (TCR) is thought to initiate downstream signalling. However, the detection of protein clustering with high spatial and temporal resolution remains challenging. Here we establish a Förster resonance energy transfer (FRET) sensor, named CliF, which reports intermolecular associations of neighbouring proteins in live cells. A key advantage of the single-chain FRET sensor is that it can be combined with image correlation spectroscopy (ICS), single-particle tracking (SPT) and fluorescence lifetime imaging microscopy (FLIM). We test the sensor with a light-sensitive actuator that induces protein aggregation upon radiation with blue light. When applied to T cells, the sensor reveals that TCR triggering increases the number of dense TCR-CD3 clusters. Further, we find a correlation between cluster movement within the immunological synapse and cluster density. In conclusion, we develop a sensor that allows us to map the dynamics of protein clustering in live T cells.publishe
Distinct Mechanisms Regulate Lck Spatial Organization in Activated T Cells
Phosphorylation of the T cell receptor (TCR) by the kinase Lck is the first detectable signaling event upon antigen engagement. The distribution of Lck within the plasma membrane, its conformational state, kinase activity, and protein-protein interactions all contribute to determine how efficiently Lck phosphorylates the engaged TCR. Here, we used cross-correlation raster image correlation spectroscopy and photoactivated localization microscopy to identify two mechanisms of Lck clustering: an intrinsic mechanism of Lck clustering induced by locking Lck in its open conformation and an extrinsic mechanism of clustering controlled by the phosphorylation of tyrosine 192, which regulates the affinity of Lck SH2 domain. Both mechanisms of clustering were differently affected by the absence of the kinase Zap70 or the adaptor Lat. We further observed that the adaptor TSAd bound to and promoted the diffusion of Lck when it is phosphorylated on tyrosine 192. Our data suggest that while Lck open conformation drives aggregation and clustering, the spatial organization of Lck is further controlled by signaling events downstream of TCR phosphorylation.publishe