Distribution of lipid raft markers in live cells

2004 ◽  
Vol 32 (5) ◽  
pp. 673-675 ◽  
Author(s):  
O.O. Glebov ◽  
B.J. Nichols
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Receptor ◽  
Specific Protein ◽  
Live Cells ◽  
Jurkat T Cells

GPI (glycosylphosphatidylinositol)-anchored proteins are characteristic components of biochemically defined lipid rafts. Rafts may be involved in T-cell stimulation, but it is not clear whether molecules involved in TCR (T-cell receptor) signalling are partitioned to T-cell synapses through raft microdomains or through specific protein–protein interactions. We have used FRET (fluorescence resonance energy transfer) analysis to study the distribution of GPI-anchored fluorescent proteins in the plasma membrane of live cells. Multiple criteria suggested that FRET between different GPI-anchored fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, unclustered distribution. Stimulation of TCR signalling in Jurkat T-cells by beads coated with antibodies against TCR subunits resulted in localized increases in fluorescence of raft markers. However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of raft markers in these regions.

scholarly journals ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+microdomains upon T cell activation

2018 ◽  
Vol 11 (561) ◽  
pp. eaat0358 ◽  
Author(s):  
Björn-Philipp Diercks ◽  
René Werner ◽  
Paula Weidemüller ◽  
Frederik Czarniak ◽  
Lola Hernandez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ryanodine Receptors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Receptor ◽  
Intracellular Signals

The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+microdomains. Here, we identified a Ca2+entry component involved in Ca2+microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+release through RYR1, in coordination with Ca2+entry through ORAI1 and STIM, rapidly increases the number of Ca2+microdomains, thereby initiating spread of Ca2+signals deeper into the cytoplasm to promote full T cell activation.

scholarly journals Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
Author(s):  
Michael Winkler ◽  
Florian Wrensch ◽  
Pascale Bosch ◽  
Maike Knoth ◽  
Michael Schindler ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Interactions ◽  
Viral Entry ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

scholarly journals Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3096-3103 ◽  
Author(s):  
Kenrick Semple ◽  
Antony Nguyen ◽  
Yu Yu ◽  
Honglin Wang ◽  
Claudio Anasetti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Protein Tyrosine Kinase ◽  
Cell Receptor ◽  
Specific Protein ◽  
Cell Immunity ◽  
Cd28 Costimulation

Abstract CD28 costimulation is required for the generation of naturally derived regulatory T cells (nTregs) in the thymus through lymphocyte-specific protein tyrosine kinase (Lck) signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naive CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naive CD4 T cells (CD25−Foxp3−) by T-cell receptor stimulation with additional transforming growth factorβ (TGFβ) in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. Using a series of transgenic mice on a CD28-deficient background that bears wild-type or mutated CD28 in its cytosolic tail that is incapable of binding to Lck, phosphoinositide 3-kinase (PI3K), or IL-2–inducible T-cell kinase (Itk), we found that CD28-mediated Lck signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide a rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28 signaling.

scholarly journals Ras Activation in Jurkat T cells following Low-Grade Stimulation of the T-Cell Receptor Is Specific to N-Ras and Occurs Only on the Golgi Apparatus

2004 ◽  
Vol 24 (8) ◽  
pp. 3485-3496 ◽  
Author(s):  
Ignacio Perez de Castro ◽  
Trever G. Bivona ◽  
Mark R. Philips ◽  
Angel Pellicer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Golgi Apparatus ◽  
Cell Receptor ◽  
Ras Signaling ◽  
Low Grade ◽  
Jurkat T Cells ◽  
Ras Activation ◽  
Ras Isoforms ◽  
Stimulation Of

ABSTRACT Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cγ- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.

QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING

2012 ◽  
Vol 05 (03) ◽  
pp. 1250015 ◽  
Author(s):  
XIAO-PING WANG ◽  
HUAI-NA YU ◽  
TONG-SHENG CHEN
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Region Of Interest ◽  
Living Cells ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Microscopy Imaging ◽  
Acceptor Photobleaching ◽  
Induced Apoptosis

Fluorescence resonance energy transfer (FRET) technology had been widely used to study protein–protein interactions in living cells. In this study, we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest (ROI) function of confocal microscope and partial acceptor photobleaching. We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3, and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine (STS)-induced apoptosis. Our results for the first demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.

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