Single-molecule microscopy reveals heterogeneous dynamics of lipid raft components upon TCR engagement

Drbal, Karel; Moertelmaier, Manuel; Holzhauser, Christa; Muhammad, Arshad; Fuertbauer, Elke; Howorka, Stefan; Hinterberger, Maria; Stockinger, Hannes; Schütz, Gerhard J.

doi:10.1093/intimm/dxm032

Cited by 41 publications

(34 citation statements)

References 49 publications

(70 reference statements)

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“…Previous work has examined the diffusion of CD45 by FPR (15), and experiments with activated cells are in agreement with our observations of reduced lateral mobility (17). CD45 receptors lacking a cytoplasmic domain have been reported to have much higher diffusion coefficients than observed in our experiments, implicating the cytoplasmic domain in the regulation of mobility (16).…”

Section: Discussionsupporting

confidence: 92%

“…Single-particle tracking (SPT) experiments on CD45 with a cytoplasmic domain deletion showed rapid diffusion that was unaffected by cellular activation (16). SPT measurements on CD45 with an intact cytoplasmic domain revealed a significant decrease in mobility upon cellular activation, suggesting increased cytoskeletal interactions (17). Thus, the lymphocyte cytoskeleton plays an active role in CD45 regulation and lateral mobility.…”

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confidence: 92%

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Dynamic Regulation of CD45 Lateral Mobility by the Spectrin-Ankyrin Cytoskeleton of T Cells

Cairo

Das

Albohy

et al. 2010

Journal of Biological Chemistry

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The leukocyte common antigen, CD45, is a critical immune regulator whose activity is modulated by cytoskeletal interactions. Components of the spectrin-ankyrin cytoskeleton have been implicated in the trafficking and signaling of CD45. We have examined the lateral mobility of CD45 in resting and activated T lymphocytes using single-particle tracking and found that the receptor has decreased mobility caused by increased cytoskeletal contacts in activated cells. Experiments with cells that have disrupted ␤⌱ spectrin interactions show decreased cytoskeletal contacts in resting cells and attenuation of receptor immobilization in activated cells. Applying two types of population analyses to single-particle tracking trajectories, we find good agreement between the diffusion coefficients obtained using either a mean squared displacement analysis or a hidden Markov model analysis. Hidden Markov model analysis also reveals the rate of association and dissociation of CD45-cytoskeleton contacts, demonstrating the importance of this analysis for measuring cytoskeleton binding events in live cells. Our findings are consistent with a model in which multiple cytoskeletal contacts, including those with spectrin and ankyrin, participate in the regulation of CD45 lateral mobility. These interactions are a major factor in CD45 immobilization in activated cells. Furthermore, cellular activation leads to CD45 immobilization by reduction of the CD45-cytoskeleton dissociation rate. Short peptides that mimic spectrin repeat domains alter the association rate of CD45 to the cytoskeleton and cause an apparent decrease in dissociation rates. We propose a model for CD45-cytoskeleton interactions and conclude that the spectrin-ankyrin-actin network is an essential determinant of immunoreceptor mobility.The lymphocyte receptor-like protein tyrosine phosphatase, CD45, 2 is a transmembrane protein that acts as a critical component of T cell activation (1). The phosphatase activity of the CD45 cytoplasmic domain contributes to T cell activation through the removal of an inhibitory phosphate group on a membrane-associated Src family kinase, such as Tyr 505 of p56 lck (Lck) (2). Other CD45 substrates have been established in addition to Lck, including Fyn, SKAP-55, JAK, ZAP-70, and CK2 (1). The involvement of CD45 as a regulator of critical signaling pathways and autoimmune diseases highlights the importance of understanding its function.In addition to phosphoprotein substrates, CD45 interacts with a variety of molecular partners that modulate its activity. Its heavily glycosylated extracellular domain interacts with Thy1 (3) and galectins (4), and it self-associates to form dimers. Dimer formation is a process linked to both regulation of its phosphatase activity and sialylation of its ectodomain (1, 5, 6). The cytoplasmic domain of CD45 contains one catalytically active protein-tyrosine phosphatase domain (Domain 1) and one inactive domain (Domain 2) and binds to key cellular regulators of immune function such as CD45AP (7), CD2 (8), CD11a (9),...

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Section: Discussionsupporting

confidence: 92%

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confidence: 92%

Dynamic Regulation of CD45 Lateral Mobility by the Spectrin-Ankyrin Cytoskeleton of T Cells

Cairo

Das

Albohy

et al. 2010

Journal of Biological Chemistry

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“…We found that T cell stimulation on planar CD3 mAb coated surfaces induced the immobilization of CD48 in the center of the T cell interface whereas the second GPI protein, CD59, remained highly mobile (14). Heterogeneity in the behavior of lipid raft molecules and different lipid composition of microdomains has been already demonstrated by others (15)(16)(17), however, it is still not clear what molecular interactions determine this diversity.…”

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confidence: 69%

Sequential Cooperation of CD2 and CD48 in the Buildup of the Early TCR Signalosome

Muhammad

Schiller

Förster

et al. 2009

The Journal of Immunology

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The buildup of TCR signaling microclusters containing adaptor proteins and kinases is prerequisite for T cell activation. One hallmark in this process is association of the TCR with lipid raft microdomains enriched in GPI-proteins that have potential to act as accessory molecules for TCR signaling. In this study, we show that GPI-anchored CD48 but not CD59 was recruited to the immobilized TCR/CD3 complex upon activation of T cells. CD48 reorganization was vital for T cell IL-2 production by mediating lateral association of the early signaling component linker for activated T cells (LAT) to the TCR/CD3 complex. Furthermore, we identified CD2 as an adaptor linking the Src protein tyrosine kinase Lck and the CD48/LAT complex to TCR/CD3: CD2 associated with TCR/CD3 upon T cell activation irrespective of CD48 expression, while association of CD48 and LAT with the TCR/CD3 complex depended on CD2. Consequently, our data indicate that CD2 and CD48 cooperate hierarchically in the buildup of the early TCR signalosome; CD2 functions as the master switch recruiting CD48 and Lck. CD48 in turn shuttles the transmembrane adapter molecule LAT.

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“…To elucidate further the significance of CD48 colocalization with 2B4, we performed solid-phase signalosome precipitation for the receptor complex. Using a procedure that does not solubilize the raft, it was possible to visualize components in the detergent-insoluble raft complex (16). As shown in Fig.…”

Section: Coclustering Of Cd48 With 2b4mentioning

confidence: 99%

Mechanism of Induction of NK Activation by 2B4 (CD244) via Its Cognate Ligand

Sinha

Gao

Guo

et al. 2010

The Journal of Immunology

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We have previously shown that coincubation of purified B cells with IL-2–propagated NK cells can result in the induction of IL-13 mRNA and that the induction requires the presence of CD48 on B cells and 2B4 on NK cells. Because both of these molecules are expressed on NK cells, it is surprising that very little IL-13 mRNA can be detected in the absence of B cells. We have now found that incubation of NK cells on plates containing immobilized anti-CD48 Abs results in the clustering of CD48 and colocalization with 2B4 on the same cell. This colocalization, together with the requirement for SAP, the signal transducer for 2B4, is necessary for the induction of IL-13 mRNA expression. Activation of NK cell via CD48 on another cell may require a similar ability to alter the configuration of 2B4 to activate downstream signaling. By the use of double CD2/2B4 knockout mice, we have also shown that the induction of NK cell activation by anti-CD48 or by B cells is not due to the release of inhibitory effects of 2B4.

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Single-molecule microscopy reveals heterogeneous dynamics of lipid raft components upon TCR engagement

Cited by 41 publications

References 49 publications

Dynamic Regulation of CD45 Lateral Mobility by the Spectrin-Ankyrin Cytoskeleton of T Cells

Dynamic Regulation of CD45 Lateral Mobility by the Spectrin-Ankyrin Cytoskeleton of T Cells

Sequential Cooperation of CD2 and CD48 in the Buildup of the Early TCR Signalosome

Mechanism of Induction of NK Activation by 2B4 (CD244) via Its Cognate Ligand

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