Development of nanoscale structure in LAT-based signaling complexes

Barr, Valarie A.; Sherman, Eilon; Yi, Jason; Akpan, Itoro; Rouquette-Jazdanian, Alexandre K.; Samelson, Lawrence E.

doi:10.1242/jcs.194886

Cited by 15 publications

(12 citation statements)

References 74 publications

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“…To date, SMLM studies have revealed patterns of molecular assembly within microclusters, the nanoscale clustering of molecular components, and the 3D distribution of concentrated receptors in resting T cells 13,14,25 . However, in addition to the limitations of SMLM mentioned above, we and others have reported artifactual clustering of photoactivatable fluorescent proteins used in SMLM 26,27 and poor resolution in antibody-based SMLM methods due to inadequate labeling of targeted structures 16,28,29 . Given these issues, we sought to use TIRF-SIM, which yields ~100 nm resolution, and is compatible with well characterized, monomeric fluorescent proteins to perform live imaging of microcluster formation in T cells 30 .…”

Section: Resultsmentioning

confidence: 92%

TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps

Balagopalan

Nguyen

et al. 2019

Nat Commun

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Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.

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Section: Resultsmentioning

confidence: 92%

TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps

Balagopalan

Nguyen

et al. 2019

Nat Commun

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“…dSTORM images were acquired by a commercially available Nikon system based on an inverted microscope (Nikon T i -E) equipped with an oil-immersion TIRF objective (Apochromat TIRF100X, 1.49 NA; Nikon) as well as four lasers and a filter set suitable for multiple color imaging. L-function was used for examining the bivariate pair correlation between IgG-BCRs and PI(4,5)P2 in the two-color superresolution imaging experiments (Wiegand and Moloney, 2004; Barr et al, 2016). In detail, L-function is a transformation of K-function.…”

Section: Methodsmentioning

confidence: 99%

PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

Wan

Chen

et al. 2018

Journal of Cell Biology

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B lymphocytes use B cell receptors (BCRs) to sense the chemical and physical features of antigens. The activation of isotype-switched IgG-BCR by mechanical force exhibits a distinct sensitivity and threshold in comparison with IgM-BCR. However, molecular mechanisms governing these differences remain to be identified. In this study, we report that the low threshold of IgG-BCR activation by mechanical force is highly dependent on tethering of the cytoplasmic tail of the IgG-BCR heavy chain (IgG-tail) to the plasma membrane. Mechanistically, we show that the positively charged residues in the IgG-tail play a crucial role by highly enriching phosphatidylinositol (4,5)-biphosphate (PI(4,5)P2) into the membrane microdomains of IgG-BCRs. Indeed, manipulating the amounts of PI(4,5)P2 within IgG-BCR membrane microdomains significantly altered the threshold and sensitivity of IgG-BCR activation. Our results reveal a lipid-dependent mechanism for determining the threshold of IgG-BCR activation by mechanical force.

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“…Interestingly, LAT and SLP-76 were reported to form nanostructures with LAT tending to be in the center and SLP-76 distributed on the outside (Sherman et al, 2011 ). Further investigation showed that this LAT-SLP nanostructure develops during the spreading process (Barr et al, 2016 ), suggesting that the nanostructure of signaling complexes is dynamic and changes with time. This research has also revealed the difficulty in analyzing the patterns of multiple proteins.…”

Section: Spatial Organization Of Microclustersmentioning

confidence: 99%

Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

Balagopalan

Raychaudhuri

Samelson

2021

Front. Cell Dev. Biol.

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When T cell receptors (TCRs) engage with stimulatory ligands, one of the first microscopically visible events is the formation of microclusters at the site of T cell activation. Since the discovery of these structures almost 20 years ago, they have been studied extensively in live cells using confocal and total internal reflection fluorescence (TIRF) microscopy. However, due to limits in image resolution and acquisition speed, the spatial relationships of signaling components within microclusters, the kinetics of their assembly and disassembly, and the role of vesicular trafficking in microcluster formation and maintenance were not finely characterized. In this review, we will summarize how new microscopy techniques have revealed novel insights into the assembly of these structures. The sub-diffraction organization of microclusters as well as the finely dissected kinetics of recruitment and disassociation of molecules from microclusters will be discussed. The role of cell surface molecules in microcluster formation and the kinetics of molecular recruitment via intracellular vesicular trafficking to microclusters is described. Finally, the role of post-translational modifications such as ubiquitination in the downregulation of cell surface signaling molecules is also discussed. These results will be related to the role of these structures and processes in T cell activation.

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Development of nanoscale structure in LAT-based signaling complexes

Cited by 15 publications

References 74 publications

TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps

TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps

PI(4,5)P2 determines the threshold of mechanical force–induced B cell activation

Microclusters as T Cell Signaling Hubs: Structure, Kinetics, and Regulation

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