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The role of PSD-95 in the rearrangement of Kv1.3 channels to the immunological synapse

  • Signaling and Cell Physiology
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Abstract

Establishment of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells is a key step in the adaptive immune response. Several proteins accumulate in the IS, such as the Kv1.3 potassium channel; however, the mechanism of this translocation is unknown. PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3. Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells. These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.

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Acknowledgments

The authors are thankful to Prof. Hannes Stockinger (University of Vienna, Vienna, Austria) for help in the optimization of retroviral transduction of Jurkat cells and to Prof. Jim Trimmer (University of California, Davis, USA) for the PSD-95 and SAP97 plasmids. The technical assistance of Cecilia Nagy is greatly appreciated. This project was funded by the following grants: The Hungarian Social Renewal Operational Program (TAMOP-4.2.2-A-11/1/KONV-2012-0025; TÁMOP-4.2.2/B-10/1-2010-0024; TÁMOP-4.2.2-08/1/2008-0019; TÁMOP-4.2.1/B-09/1/KONV-2010-007) and the Hungarian Scientific Research Fund (Grants K75904, CK 78179, and NK 101337). P.H. is supported by Janos Bolyai and Lajos Szodoray Fellowships.

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  1. Department of Biophysics and Cell Biology, University of Debrecen, Nagyerdei krt. 98, 4032, Debrecen, Hungary

    Orsolya Szilágyi, György Panyi & Péter Hajdu

  2. Department of Medical Chemistry, University of Debrecen, Nagyerdei krt. 98, 4032, Debrecen, Hungary

    Anita Boratkó

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  1. Orsolya Szilágyi
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Correspondence to Péter Hajdu.

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Supplementary Fig. 1

shRNA knockdown of PSD-95 and SAP97. Jurkat and Jurkat-mGFP-Kv1.3-WT cells were transduced with two specific shRNA constructs each to down-regulate the expression level of PSD-95 (clone 27 and 28) or SAP97 (clone 01 and 02). Scrambled shRNA was used as transduction control. In case of PSD-95 construct 27 has been chosen for further experiments. Western blot of Jurkat cell lysates using a) anti-PSD-95 (top set: WB PSD-95): 1) Jurkat, 2) Jurkat-PSD-95 knockdown clone 27 (KD-PSD-95-27), 3) Jurkat PSD-95 knockdown clone 28 (KD-PSD-95-28), 4) Jurkat scrambled shRNA control (SCR-control), 5) Jurkat stable transfected with mGFP-Kv1.3-WT, 6) Jurkat stable transfected with mGFP-Kv1.3-WT and PSD-95 knocked down, clone 27 (Jurkat- mGFP-Kv1.3-WT -KD-PSD-95-27), 7) Jurkat stable transfected with mGFP-Kv1.3-WT and PSD-95 knocked down, clone 28 (Jurkat-mGFP-Kv1.3-WT-KD-PSD-95-28), 8) Jurkat stable transfected with mGFP-Kv1.3-WT, scrambled shRNA control (Jurkat-mGFP-Kv1.3-WT-SCR-control), b) anti-SAP97 (top set: WB SAP97): 1) Jurkat, 2) Jurkat-SAP97 knockdown clone 01 (KD-SAP97-01), 3) Jurkat SAP97 knockdown clone 02 (KD-SAP97-02), 4) Jurkat scrambled shRNA control (SCR-control), 5) Jurkat stable transfected with mGFP-Kv1.3-WT, 6) Jurkat stable transfected with mGFP-Kv1.3-WT and SAP97 knocked down, clone 01 (Jurkat- mGFP-Kv1.3-WT -KD-SAP97-01), 7) Jurkat stable transfected with mGFP-Kv1.3-WT and SAP97 knocked down, clone 02 (Jurkat-mGFP-Kv1.3-WT-KD-SAP97-02), 8) Jurkat stable transfected with mGFP-Kv1.3-WT, scrambled shRNA control (Jurkat-mGFP-Kv1.3-WT-SCR-control). The actin blot (bottom set: WB actin) shows the protein quantity loaded of each total cell lysate for both figure parts (a-b). (PDF 69 kb)

Supplementary Fig. 2

Recruitment of Kv1.3 and CD3 into the immune synapse in SAP97 knocked down or scramble control Jurkat cells. Representative examples of immune synapse formation between SEE-pulsed Raji and a) SAP97 knocked-down Jurkat expressing mGFP-tagged Kv1.3 (Jurkat-mGFP-Kv1.3-WT-KD-SAP97) or b) Jurkat transduced with scramble control shRNA. The 1st column shows the Kv1.3 signal, mGFP-tagged channels are shown in green. Jurkat cells were also labeled with anti-CD3 primary and Alexa Fluor 647 GAMIG secondary antibodies to identify the synapses (2nd column, magenta). The 3rd column shows the overlay images of the Kv1.3 and anti-CD3 fluorescence signal whereas phase contrast images of the cells forming the IS are displayed in the 4th column. (For details concerning synapse formation and fluorescent labeling see Materials and Methods.) Scale bar is 10 μm. (PDF 111 kb)

Supplementary Fig. 3

Accumulation of Kv1.3 channels in the IS is not influenced by SAP97 or non-specific shRNA knockdown. The percentage of cells showing polarized Kv1.3 expression during IS formation for Jurkat expressing the wild type (Jurkat-mGFP-Kv1.3-WT, empty bars), SAP97 knocked-down (Jurkat-mGFP-Kv1.3-WT-KD-SAP-97, for 1 min: n = 33, for 5 min: n = 41) or scramble shRNA transfected Jurkat cells (Jurkat-mGFP-Kv1.3-SCR, for 1 min: n = 29, for 5 min: n = 85) transduced the same, mGPF-tagged Kv1.3 construct. (PDF 325 kb)

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Szilágyi, O., Boratkó, A., Panyi, G. et al. The role of PSD-95 in the rearrangement of Kv1.3 channels to the immunological synapse. Pflugers Arch - Eur J Physiol 465, 1341–1353 (2013). https://doi.org/10.1007/s00424-013-1256-6

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