Journal: Journal of Cell Science
Article Title: Lysosomal agents inhibit store-operated Ca 2+ entry
doi: 10.1242/jcs.248658
Figure Lengend Snippet: Lysosomal agents and Stim1 signalling. (A-D) Cells transfected with EYFP-tagged Stim1 (or mutants) were treated with 50 µM CPA for 15-20 min to initiate SOCE, and then lysosomal agents were added acutely (200 µM GPN, 5 mM LLOMe and 20 µM nigericin). Binary threshold masking determined the length of the Stim1 structures (length before GPN=13.7±3.0 µm). (A) Wild-type EYFP-Stim1 before and 5 min after agents were added (GPN, n =21, N =16; LLOMe, n =7, N =5; and nigericin, n =5, N =3). (B) The cell-average length of Stim1 structures was normalized to the basal value. (C) Quantification of the effect of 200 µM GPN on the length of structures formed by Stim1 variants, wild type (WT, n =21, N =16), Stim1-ΔK ( n =9, N =3) and Stim1-D76A ( n =13 cells, N =3). (D) Morphology of Stim1 mutants (Stim1-ΔK and Stim1-D76A) treated with 50 µM CPA then 200 µM GPN. (E-G) Cells were co-transfected with (or without) EYFP-tagged Stim1 (or mutants) and the ratiometric Ca 2+ reporter GEM-GECO1. SOCE was initiated with 50 µM CPA, and then 200 µM GPN was acutely applied. (E) Representative single-cell Ca 2+ traces. (F) Collated data expressed as percentage of the CPA peak ratio. (G) Plateau phase as a percentage of the pre-GPN value. Data are mean±s.e.m. of 67-86 cells ( N =3-9). (H-M) Effect of lysosomal agents on SOCE evoked optogenetically by hBACCS2 (see inset cartoon) activated with 488 nm light (indicated by the bar, hν), as measured with the red Ca 2+ reporter JRGECO1a. (H) Cells expressing the EGFP transfection marker alone. (I-K) Cells co-expressing EGFP plus hBACCS2. Ca 2+ influx was inhibited by 3 mM EGTA, 200 µM GPN or 5 mM LLOMe. (L) Ca 2+ amplitudes before and after agent addition. (M) Rate of fall in JRGECO1a signal upon removal of light (hν off) or addition of agents. Data are mean±s.e.m. of 22-101 cells ( N =3-4). ** P <0.01, *** P <0.001 (ANOVA, Tukey-Kramer post-test). Scale bars: 10 μm.
Article Snippet: The following plasmids were obtained as gifts from these authors via Addgene: GCaMP6s (Douglas Kim and the GENIE Project, 40753) ( ); R-CEPIA1er (Masamitsu Iino, 58216) ( ); the following from Tobias Meyer , SP-YFP-STIM1(23-685) (18857), SP-YFP-STIM1(D76A) (18859) and YFP-STIM1-deltaK (18861); GEM-GECO1 (Robert Campbell, 32442) ( ); hBACCS2-IRES-GFP (Takao Nakata, 72891) ( ); mTagRFP-Membrane-1 (Michael Davidson, 57992); Orai1-YFP (Anjana Rao, 19756) ( ); K44A HA-dynamin 2 (Sandra Schmid, 34685); tdTomato-BicD2-FKBP12 (64205) or KIF5C-tdTomato-FKBP12 (64211), both from Gary Banker ( ); GPI-EGFP was a generous gift from Sergio Grinstein (Hospital for Sick Children, Toronto, ON, Canada); LAMP1-ECFP-FRB* was a generous gift from Takanari Inoue (Johns Hopkins School of Medicine, Baltimore, MD, USA).
Techniques: Transfection, Expressing, Marker
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