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: JCI Insight

Article Title: ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome

doi: 10.1172/jci.insight.174866

Figure Lengend Snippet: ( A ) Between 1 and 4 months of age, body weight measurements showed a higher growth curve for Stim1 R304W/+ Orai1 R93W/+ mice compared with Stim1 R304W/+ littermates ( n = 11–14, 2-way ANOVA and Tukey’s post hoc test). ( B ) 3D reconstruction of the femur microarchitecture illustrated a similar trabecular density in Stim1 R304W/+ Orai1 R93W/+ bones and healthy WT and Orai1 R93W/+ controls at 4 months (representative images, n = 7–8). ( C – E ) At 4 months of age, spleen weight, megakaryocyte numbers, and spleen histology (H&E staining) were similar in Stim1 R304W/+ Orai1 R93W/+ mice and healthy controls and markedly differed from Stim1 R304W/+ littermates (spleen, n = 6–10, 1-way ANOVA and Tukey’s post hoc test; megakaryocytes, n = 7–8, Kruskal-Wallis and Dunn’s multiple comparison test). Black arrows indicate megakaryocytes. Scale bar: 250 μm. ( F ) Platelet numbers were doubled in Stim1 R304W/+ Orai1 R93W/+ mice compared with Stim1 R304W/+ littermates at 4 months without reaching WT levels ( n = 9–14, Kruskal-Wallis and Dunn’s multiple comparison test). Data are shown as the mean ± SEM. Significant differences are indicated as * ,α,$ P < 0.05, ** ,αα,$$ P < 0.01, ααα P < 0.001, and **** ,αααα P < 0.0001, with * reflecting comparison of Stim1 R304W/+ with the WT group, α comparison with the Orai1 R93W/+ group, and $ comparison with the Stim1 R304W/+ Orai1 R93W/+ group.

Article Snippet: Stim1 R304W/+ and Orai1 +/– mice (a gift from Paul F. Worley, Johns Hopkins University, Baltimore, Maryland, USA) and Ora1 R93W/+ mice (a gift from Stefan Feske, New York University School of Medicine, New York, New York, USA) have been described previously ( , , ).

Techniques: Staining, Comparison

Journal: JCI Insight

Article Title: ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome

doi: 10.1172/jci.insight.174866

Figure Lengend Snippet: ( A and B ) Stim1 R304W/+ Orai1 R93W/+ mice outperformed Stim1 R304W/+ littermates in the hanging test throughout the first 4 months ( n = 11–14, 2-way ANOVA and Tukey’s post hoc test). ( B ) Premature muscle contraction of Stim1 R304W/+ mice at low stimulation frequencies was normalized in Stim1 R304W/+ Orai1 R93W/+ mice at 4 months ( n = 5–7, 2-way ANOVA and Tukey’s post hoc test). ( C – F ) Following single and tetanic stimulations, the relaxation time was significantly delayed in Stim1 R304W/+ tibialis anterior and almost normalized in Stim1 R304W/+ Orai1 R93W/+ mice at 4 months (mean traces shown, n = 5–7, 2-way ANOVA and Tukey’s post hoc test). ( G and H ) Resting cytosolic Ca 2+ levels and SOCE amplitude were strongly increased in Stim1 R304W/+ myotubes and shifted toward WT levels in Stim1 R304W/+ Orai1 R93W/+ myotubes (resting Ca 2+ , n = 53–89 cells; SOCE amplitude, n = 18–55 cells; Kruskal-Wallis and Dunn’s multiple comparison test). Data are shown as the mean ± SEM. Significant differences are indicated as * ,α,$ P < 0.05, ** ,αα,$$ P < 0.01, *** ,ααα P < 0.001, and **** ,αααα,$$$$ P < 0.0001, with * reflecting comparison of Stim1 R304W/+ with the WT group, α comparison with the Orai1 R93W/+ group, and $ comparison with the Stim1 R304W/+ Orai1 R93W/+ group.

Article Snippet: Stim1 R304W/+ and Orai1 +/– mice (a gift from Paul F. Worley, Johns Hopkins University, Baltimore, Maryland, USA) and Ora1 R93W/+ mice (a gift from Stefan Feske, New York University School of Medicine, New York, New York, USA) have been described previously ( , , ).

Techniques: Comparison

Journal: JCI Insight

Article Title: ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome

doi: 10.1172/jci.insight.174866

Figure Lengend Snippet: ( A ) H&E staining of tibialis anterior sections from 4-month-old Stim1 R304W/+ mice revealed internalized nuclei (blue arrow), regenerating fibers (green arrow), and immune cell infiltrations (black arrow), while the Stim1 R304W/+ Orai1 R93W/+ histology was indistinguishable from that of the WT and Orai1 R93W/+ controls (representative images, n = 5–7). Immunofluorescence detected prominent embryonic myosin (eMHC) signals, indicating regenerating fibers in Stim1 R304W/+ muscle sections and, to a much lesser extent, in WT, Orai1 R93W/+ , and Stim1 R304W/+ Orai1 R93W/+ myofibers. Scale bar: 50 μm (representative images, n = 5–7). ( B ) Quantification of myofibers with internal nuclei showed an increased ratio in Stim1 R304W/+ tibialis anterior compared with WT, Orai1 R93W/+ , and Stim1 R304W/+ Orai1 R93W/+ muscle sections ( n = 5–8, 1-way ANOVA and Tukey’s post hoc test). ( C ) Serum creatine kinase levels were significantly reduced in 4-month-old Stim1 R304W/+ Orai1 R93W/+ blood compared with Stim1 R304W/+ samples ( n = 4–6, 1-way ANOVA and Tukey’s post hoc test). ( D ) Quantification of eMHC signals disclosed an enhanced proportion of regenerating myofibers in Stim1 R304W/+ mice and a complete normalization in Stim1 R304W/+ Orai1 R93W/+ mice ( n = 6–8, Kruskal-Wallis and Dunn’s multiple comparison test). ( E ) The expression of the UPR markers Hspa5 and Hsp90b1 and the ratio of spliced/unspliced Xbp1 were comparable in muscle extracts from Stim1 R304W/+ Orai1 R93W/+ mice and healthy WT and Orai1 R93W/+ controls ( n = 5–7, 1-way ANOVA and Tukey’s post hoc test). Data are shown as the mean ± SEM. Significant differences are indicated as * ,α,$ P < 0.05, ** ,αα,$$ P < 0.01, ααα,$$$ P < 0.001, and **** ,αααα,$$$$ P < 0.0001, with * reflecting comparison of Stim1 R304W/+ with the WT group, α comparison with the Orai1 R93W/+ group, and $ the comparison with Stim1 R304W/+ Orai1 R93W/+ group.

Article Snippet: Stim1 R304W/+ and Orai1 +/– mice (a gift from Paul F. Worley, Johns Hopkins University, Baltimore, Maryland, USA) and Ora1 R93W/+ mice (a gift from Stefan Feske, New York University School of Medicine, New York, New York, USA) have been described previously ( , , ).

Techniques: Staining, Immunofluorescence, Comparison, Expressing

Journal: JCI Insight

Article Title: ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome

doi: 10.1172/jci.insight.174866

Figure Lengend Snippet: ( A ) ORAI1 inhibition ( Stim1 R304W/+ Orai1 R93W/+ mice) provided a higher overall rescue level of bone and spleen morphology, platelet numbers, muscle histology, contractility, and cytosolic Ca 2+ content in TAM/STRMK mice compared with Orai1 downregulation ( Stim1 R304W/+ Orai1 +/– mice). The WT phenotype was set at 100% and the Stim1 R304W/+ phenotype at 0%. ( B ) Hierarchical clustering of tibialis anterior RNA-Seq data revealed sample grouping of WT, Orai1 R93W/+ , and Stim1 R304W/+ Orai1 R93W/+ mice on one side and of Stim1 R304W/+ and Stim1 R304W/+ Orai1 +/– mice on the other side, confirming the higher therapeutic potential of ORAI1 inhibition at the transcriptomic level ( n = 4). ( C ) The percentage of genes with improved or rescued expression was substantially higher in Stim1 R304W/+ Orai1 R93W/+ mice (ORAI1 inhibition) compared with that in Stim1 R304W/+ Orai1 +/– mice ( Orai1 downregulation). ( D ) Venn diagram illustrating that Stim1 R304W/+ Orai1 +/– ( Orai1 downregulation) and Stim1 R304W/+ Orai1 R93W/+ (ORAI1 inhibition) muscle samples shared 113 genes with partially or completely normalized expression. ( E ) Normalized expression of Atp2a1 and Sln in Stim1 R304W/+ Orai1 R93W/+ compared with Stim1 R304W and Stim1 R304W/+ Orai1 +/– muscle samples ( n = 4–9, 1-way ANOVA and Tukey’s post hoc test). Data are shown as the mean ± SEM. Significant differences are indicated as ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Stim1 R304W/+ and Orai1 +/– mice (a gift from Paul F. Worley, Johns Hopkins University, Baltimore, Maryland, USA) and Ora1 R93W/+ mice (a gift from Stefan Feske, New York University School of Medicine, New York, New York, USA) have been described previously ( , , ).

Techniques: Inhibition, RNA Sequencing, Expressing

Journal: JCI Insight

Article Title: ORAI1 inhibition as an efficient preclinical therapy for tubular aggregate myopathy and Stormorken syndrome

doi: 10.1172/jci.insight.174866

Figure Lengend Snippet: ( A ) Circulating myostatin levels were significantly lower in 4-month-old Stim1 R304W/+ mice compared with healthy WT and Orai1 R93/W+ controls, and they were fully rescued in Stim1 R304W/+ Orai1 R93/W+ mice ( n = 4–7, 1-way ANOVA and Tukey’s post hoc test). ( B ) Analogously to mice, myostatin levels were decreased in patients with TAM/STRMK harboring different STIM1 mutations compared with healthy controls ( n = 3–4, t test). Data are shown as the mean ± SEM. Significant differences are indicated as ** P < 0.01 and **** ,αααα,$$$$ P < 0.0001, with * reflecting comparison of Stim1 R304W/+ /TAM/STRMK mice and patients with the WT/control group, α the comparison with the Orai1 R93W/+ group, and $ the comparison with the Stim1 R304W/+ Orai1 R93W/+ group.

Article Snippet: Stim1 R304W/+ and Orai1 +/– mice (a gift from Paul F. Worley, Johns Hopkins University, Baltimore, Maryland, USA) and Ora1 R93W/+ mice (a gift from Stefan Feske, New York University School of Medicine, New York, New York, USA) have been described previously ( , , ).

Techniques: Comparison, Control