stim1 deltak fragment  (Addgene inc)

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

Journal: Journal of cell science

Article Title: STIM1L traps and gates Orai1 channels without remodeling the cortical ER.

doi: 10.1242/jcs.164228

Figure Lengend Snippet: Fig. 5. Cluster expansion requires the STIM1 lysine-rich tail but not cytosolic Ca2+ elevations. (A) Left, TIRF images of cells expressing YFP–STIM1 and YFP–STIM1L taken before (left) and 10 min after thapsigargin (Tg) addition (right) in conditions preventing cytosolic Ca2+ elevations (20 mM BAPTA-AM and 50 mM La3+). Insets, a 2.6-fold magnification to show the morphology of fluorescent clusters. Scale bars: 10 mm. Right, quantitative analysis of TIRF images showing the effects of thapsigargin on the absolute increase in cluster size. (n533/5/3, 38/5/3, 36/5/3 and 44/5/3 cells/recordings/transfections). (B) Left, TIRF images of cells expressing YFP–STIM1DK and YFP–STIM1LDK taken before (left) and 10 min after thapsigargin addition (right). Right, quantitative analysis of TIRF images showing the effects of thapsigargin on the absolute increase in cluster size. (n541/7/6, 49/6/5, 51/7/5 and 42/6/5 cells/recordings/transfections). Quantitative data show the mean6s.e.m.; *P,0.05, **P,0.01; ****P,0.0001; ns, not significant (unpaired Student’s t-test).

Article Snippet: YFP– STIM1DK was obtained from Addgene (Plasmid 18861; Cambridge, MA) and YFP–STIM1LDK was obtained by mutagenesis (GeneCust; Dudelange, Luxembourg), Orai1 was obtained from Addgene (Plasmid 12199; Cambridge, MA) and pCMV/myc/ER/GFP (KDEL–GFP) was purchased from Life Technologies (Plasmid V823-20).

Techniques: Expressing, Transfection