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. 2017 Feb 27;7:43490.
doi: 10.1038/srep43490.

Atlastin Regulates Store-Operated Calcium Entry for Nerve Growth Factor-Induced Neurite Outgrowth

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Free PMC article

Atlastin Regulates Store-Operated Calcium Entry for Nerve Growth Factor-Induced Neurite Outgrowth

Jing Li et al. Sci Rep. .
Free PMC article

Abstract

Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by a class of dynamin-like GTPases known as atlastin (ATL). Depletion of or mutations in ATL cause an unbranched ER morphology and hereditary spastic paraplegia (HSP), a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity of the lower limbs. How ER shaping is linked to neuronal defects is poorly understood. Here, we show that dominant-negative mutants of ATL1 in PC-12 cells inhibit nerve growth factor (NGF)-induced neurite outgrowth. Overexpression of wild-type or mutant ATL1 or depletion of ATLs alters ER morphology and affects store-operated calcium entry (SOCE) by decreasing STIM1 puncta formation near the plasma membrane upon calcium depletion of the ER. In addition, blockage of the STIM1-Orai pathway effectively abolishes neurite outgrowth of PC-12 cells stimulated by NGF. These results suggest that SOCE plays an important role in neuronal regeneration, and mutations in ATL1 may cause HSP, partly by undermining SOCE.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Overexpression of ATL1 impaired neurite outgrowth in PC-12 cells.
(a and b) Representative images for neurite outgrowth in GFP-transfected PC-12 cells with (b) or without (a) 100 ng/ml NGF treatment for 48 hours. (c–h) Images of NGF-treated PC-12 cells transfected with Sec61β-GFP (c) or co-transfected with GFP and Myc-ATL1-wt (d) Myc-ATL1-K80A (e) Myc-ATL1-Y196C (f) Myc-ATL1-R217Q (g) or Myc-ATL1-P342S (h). (i) Western blot for Myc-vector, Myc-ATL1-wt, Myc-ATL1-K80A, Myc-ATL1-Y196C, Myc-ATL1-R217Q, and Myc-ATL1-P342S in PC-12 cells. GAPDH was used as a loading control. Full length blot are presented in Supplementary Figure S6a. (j) Quantification of the cells with neurites longer than 15 μm. The percentage was determined from three independent assays. (Vector without NGF, n = 62; vector, n = 230; Sec61β-GFP, n = 134; ATL1-wt, n = 134; ATL1-K80A, n = 170; ATL1-Y196C, n = 112; ATL1-217Q, n = 141; ATL1-P342S, n = 165). Scale bar = 20 μm. *P < 0.05; **P < 0.01.
Figure 2
Figure 2. Overexpression of ATL1 reduced TG-evoked SOCE.
(a) Representative [Ca2+]i data illustrating SOCE in HEK293A cells overexpressing GFP + Myc-vector (left), GFP + Myc-ATL1-wt (middle), or GFP + Myc-ATL1-K80A (right) after treatment with 2 μM TG for 5 minutes. (b) Representative intracellular free calcium ([Ca2+]i) recordings showing TG-triggered SOCE in COS-7 cells transfected with GFP + Myc-vector (left), GFP + Myc-ATL1-wt (middle), or GFP + Myc-ATL1-K80A (right). (c) Averaged peak values of [Ca2+]i were collected from corresponding HEK293A cells (Vector, n = 112; ATL1-wt, n = 43; ATL1-K80A, n = 54). (d) Averaged peak values of [Ca2+]i were collected from corresponding COS-7 cells (Vector, n = 24; ATL1-wt, n = 29; ATL1-K80A, n = 15). (e) Western blot of the endogenous expression levels of STIM1 and Orai1 in HEK293A or COS-7 cells. GAPDH was used as a loading control. Full length blot are presented in Supplementary Figure S6b. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. Depletion of ATLs reduced SOCE.
(a) Representative intracellular free calcium ([Ca2+]i) recordings showing TG-triggered SOCE in COS-7 cells transfected with control non-targeting siRNA (left) or siRNAs target ATL2 and ATL3 (right) for 72 h. (b) Averaged peak values of [Ca2+]i were collected from corresponding COS-7 cells (siCtrl, n = 70; siATL2/3, n = 71). (c) ATL2 and ATL3 expression was detected by immunoblotting. An asterisk (*) indicates a non-specific band. Full length blot are presented in Supplementary Figure S6c. (d) The ER morphology was visualized by calreticulin antibody staining. Enlarged areas are marked by the small squares, showing normal (left) and long, unbranched ER tubular networks (right). Scale bar = 15 μm. ***P < 0.001.
Figure 4
Figure 4. Overexpression of ATL1 inhibited STIM1 puncta formation.
(a) Endogenous STIM1 puncta (green) and overexpressed Myc-vector, Myc-ATL1-wt, or Myc-ATL1-K80A (red) were stained in COS-7 cells after TG treatment. (b) Quantification of STIM1 puncta per cell. (c) Quantification of the number of pericellular STIM1 puncta in 10 μm perimeter per cell (Vector, n = 42; ATL1-wt, n = 46; ATL1-K80A, n = 42). Scale bar = 15 μm. ***P < 0.001.
Figure 5
Figure 5. Extracellular Ca2+ was essential for NGF-induced neurite outgrowth.
(a) Representative images for neurite outgrowth in PC-12 cells treated with 2 mM Ca2+, NGF + 2 mM Ca2+, or NGF only and cultured in calcium-deficient DMEM (cdDMEM). (b) The percentage of neurited cells with neurites longer than 15 μm (No NGF, n = 342, NGF + Ca2+, n = 374; NGF only, n = 587). *P < 0.05.
Figure 6
Figure 6. NGF-induced Ca2+ mobilization was suppressed by CRAC channel inhibition.
(a) Representative [Ca2+]i response to 2 μg/ml NGF treatment in PC-12 cells (left); representative [Ca2+]i recording showing NGF-induced [Ca2+]i mobilization from PC-12 cells treated with 10 μM BTP2 (middle); and representative [Ca2+]i data illustrating [Ca2+]i mobilization after NGF treatment in PC-12 cells overexpressing GFP-Orai1-E106A (right). (b) The percentage of cells responsive to NGF was calculated from three independent assays (Ctrl, n = 188; + BTP2, n = 70; E106A, n = 36). *P < 0.05.
Figure 7
Figure 7. NGF-induced neurite outgrowth was sensitive to CRAC channel inhibition.
(a) Representative images for neurite outgrowth in PC-12 cells with DMSO, NGF, NGF and 10 μM BTP2, or 50 μM 2-APB treatment for 48 hours. (b) Representative images for neurite outgrowth in PC-12 cells transfected with GFP, GFP-Orai1-E106A, or GFP-Orai1-R91W with or without NGF treatment for 48 hours. (c) Representative images for neurite outgrowth in PC-12 cells transfected with control siRNA, siOrai1, or siSTIM1 with or without NGF treatment. (d) Quantification data for the cells with long neurites in panel (a) (Ctrl without NGF, n = 422; Ctrl, n = 721;+BTP2, n = 982;+2-APB, n = 1031). (e) Quantification data for panel (b) (GFP without NGF, n = 40; GFP, n = 106; E106A, n = 298; R91W, n = 334). (f) Quantification data for panel (c) (siCtrl without NGF, n = 480; siCtrl, n = 323; siOrai1, n = 287; siSTIM1, n = 401). (g) PC-12 cells were treated with NGF for 48 hours, followed by DMSO, 2-APB, or BTP2 treatment for 24 hours. (h) Neurite length was quantified 72 hours later (Ctrl, n = 360; +BTP2, n = 375;+2-APB, n = 342). *P < 0.05; **P < 0.01; ***P < 0.001; ns, P ≥ 0.05 compared to control. Scale bar = 20 μm.

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