Imaging the Single Cell Dynamics of CD4+ T Cell Activation by Dendritic Cells in Lymph Nodes

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+ T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+ T cells and in vivo–labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11–12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events

STIM1, an essential and conserved component of store-operated Ca2+ channel function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels

Orai3 TM3 point mutation G158C alters kinetics of 2-APB–induced gating by disulfide bridge formation with TM2 C101

After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident STIM proteins to form the Ca(2+)-selective Ca(2+) release–activated Ca(2+) (CRAC) channel. However, in the absence of Ca(2+) store depletion and STIM interaction, the mammalian homologue Orai3 can be activated by 2-aminoethyl diphenylborinate (2-APB), resulting in a nonselective cation conductance characterized by biphasic inward and outward rectification. Here, we use site-directed mutagenesis and patch-clamp analysis to better understand the mechanism by which 2-APB activates Orai3. We find that point mutation of glycine 158 in the third transmembrane (TM) segment to cysteine, but not alanine, slows the kinetics of 2-APB activation and prevents complete channel closure upon 2-APB washout. The “slow” phenotype exhibited by Orai3 mutant G158C reveals distinct open states, characterized by variable reversal potentials. The slow phenotype can be reversed by application of the reducing reagent bis(2-mercaptoethylsulfone) (BMS), but in a state-dependent manner, only during 2-APB activation. Moreover, the double mutant C101G/G158C, in which an endogenous TM2 cysteine is changed to glycine, does not exhibit altered kinetics of 2-APB activation. We suggest that a disulfide bridge, formed between the introduced cysteine at TM3 position 158 and the endogenous cysteine at TM2 position 101, hinders transitions between Orai3 open and closed states. Our data provide functional confirmation of the proximity of these two residues and suggest a location within the Orai3 protein that is sensitive to the actions of 2-APB

State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation.

After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident stromal interacting molecule (STIM) proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. Of the three human Orai channel homologues, only Orai3 can be activated by high concentrations (>50 µM) of 2-aminoethyl diphenylborinate (2-APB). 2-APB activation of Orai3 occurs without STIM1-Orai3 interaction or store depletion, and results in a cationic, nonselective current characterized by biphasic inward and outward rectification. Here we use cysteine scanning mutagenesis, thiol-reactive reagents, and patch-clamp analysis to define the residues that assist in formation of the 2-APB-activated Orai3 pore. Mutating transmembrane (TM) 1 residues Q83, V77, and L70 to cysteine results in potentiated block by cadmium ions (Cd(2+)). TM1 mutants E81C, G73A, G73C, and R66C form channels that are not sensitive to 2-APB activation. We also find that Orai3 mutant V77C is sensitive to block by 2-aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). Block induced by reaction with MTSEA is state dependent, as it occurs only when Orai3-V77C channels are opened by either 2-APB or by cotransfection with STIM1 and concurrent passive store depletion. We also analyzed TM3 residue E165. Mutation E165A in Orai3 results in diminished 2-APB-activated currents. However, it has little effect on store-operated current density. Furthermore, mutation E165C results in Cd(2+)-induced block that is state dependent: Cd(2+) only blocks 2-APB-activated, not store-operated, mutant channels. Our data suggest that the dilated pore of 2-APB-activated Orai3 is lined by TM1 residues, but also allows for TM3 E165 to approach the central axis of the channel that forms the conducting pathway, or pore

Orai3 TM3 point mutation G158C alters kinetics of 2-APB-induced gating by disulfide bridge formation with TM2 C101.

After endoplasmic reticulum (ER) Ca(2+) store depletion, Orai channels in the plasma membrane (PM) are activated directly by ER-resident STIM proteins to form the Ca(2+)-selective Ca(2+) release-activated Ca(2+) (CRAC) channel. However, in the absence of Ca(2+) store depletion and STIM interaction, the mammalian homologue Orai3 can be activated by 2-aminoethyl diphenylborinate (2-APB), resulting in a nonselective cation conductance characterized by biphasic inward and outward rectification. Here, we use site-directed mutagenesis and patch-clamp analysis to better understand the mechanism by which 2-APB activates Orai3. We find that point mutation of glycine 158 in the third transmembrane (TM) segment to cysteine, but not alanine, slows the kinetics of 2-APB activation and prevents complete channel closure upon 2-APB washout. The "slow" phenotype exhibited by Orai3 mutant G158C reveals distinct open states, characterized by variable reversal potentials. The slow phenotype can be reversed by application of the reducing reagent bis(2-mercaptoethylsulfone) (BMS), but in a state-dependent manner, only during 2-APB activation. Moreover, the double mutant C101G/G158C, in which an endogenous TM2 cysteine is changed to glycine, does not exhibit altered kinetics of 2-APB activation. We suggest that a disulfide bridge, formed between the introduced cysteine at TM3 position 158 and the endogenous cysteine at TM2 position 101, hinders transitions between Orai3 open and closed states. Our data provide functional confirmation of the proximity of these two residues and suggest a location within the Orai3 protein that is sensitive to the actions of 2-APB

Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes.

The adaptive immune response is initiated in secondary lymphoid organs by contact between antigen-bearing dendritic cells (DCs) and antigen-specific CD4+ T cells. However, there is scant information regarding the single cell dynamics of this process in vivo. Using two-photon microscopy, we imaged the real-time behavior of naive CD4+ T cells and in vivo-labeled DCs in lymph nodes during a robust T cell response. In the first 2 h after entry into lymph nodes, T cells made short-lived contacts with antigen-bearing DCs, each contact lasting an average of 11-12 min and occurring mainly on dendrites. Altered patterns of T cell motility during this early stage of antigen recognition promoted serial engagement with several adjacent DCs. Subsequently, T cell behavior progressed through additional distinct stages, including long-lived clusters, dynamic swarms, and finally autonomous migration punctuated by cell division. These observations suggest that the immunological synapse in native tissues is remarkably fluid, and that stable synapses form only at specific stages of antigen presentation to T cells. Furthermore, the serial nature of these interactions implies that T cells activate by way of multiple antigen recognition events

The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers

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