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, 26 (2), 322-329.e3

Reduced SERCA Function Preferentially Affects Wnt Signaling by Retaining E-Cadherin in the Endoplasmic Reticulum

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Reduced SERCA Function Preferentially Affects Wnt Signaling by Retaining E-Cadherin in the Endoplasmic Reticulum

Annabelle Suisse et al. Cell Rep.

Abstract

Calcium homeostasis in the lumen of the endoplasmic reticulum is required for correct processing and trafficking of transmembrane proteins, and defects in protein trafficking can impinge on cell signaling pathways. We show here that mutations in the endoplasmic reticulum calcium pump SERCA disrupt Wingless signaling by sequestering Armadillo/β-catenin away from the signaling pool. Armadillo remains bound to E-cadherin, which is retained in the endoplasmic reticulum when calcium levels there are reduced. Using hypomorphic and null SERCA alleles in combination with the loss of the plasma membrane calcium channel Orai allowed us to define three distinct thresholds of endoplasmic reticulum calcium. Wingless signaling is sensitive to even a small reduction, while Notch and Hippo signaling are disrupted at intermediate levels, and elimination of SERCA function results in apoptosis. These differential and opposing effects on three oncogenic signaling pathways may complicate the use of SERCA inhibitors as cancer therapeutics.

Keywords: Drosophila; E-cadherin; Hippo; Notch; SERCA; Wnt; calcium; endoplasmic reticulum; wing disc; β-catenin.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. SERCA Mutations Affect Wg Signaling
(A) Diagram of the SERCA gene, showing the coding region in blue and the positions of the introns. Below is the encoded protein, showing the transmembrane domains (gray), actuator domain (red), phosphorylation domain (blue), and nucleotide-binding domain (green). Asterisks indicate the positions of the dsm, S5, and Kum170 mutations. (B-G) Third instar wing imaginal discs containing SERCAdsm (B), SERCAS5 (C and G), arr63D (D), arr63D, SERCAS5 (E), or cul1EX (F) clones marked by the absence of RFP (green). Anterior is to the left and dorsal up in this and all subsequent figures. Discs are stained with anti-Al (B’-E’, red in B-E), anti-Arm (B”-E”, F’, and G’; blue in B-E, red in F and G), or anti-phospho-Arm (F” and G”; blue in F and G). Al levels decrease and unphosphorylated Arm accumulates in SERCA mutant clones. Arm also accumulates in arr63D, SERCAS5 double mutant clones, but not in arr63D clones. Scale bar, 50 μm. n ≥ 10 discs for all stainings shown in this and all subsequent figures. See also Figure S1.
Figure 2.
Figure 2.. E-Cad Is Retained in the Endoplasmic Reticulum and Traps Arm There in the Absence of SERCA
(Aand B) Wing disc containing SERCAS5 clones marked bythe absence of RFP (green) stained with anti-Arm (A’ and A”’; red in A) and anti-E-Cad (A” and A””; blue in A). Subapical sections are shown in A-A”, and apical sections in A”’ and A””. Scale bar, 50 μm. (B) z projection through the disc at the position shown by the line in (A), with apical up. In mutant cells, E-Cad colocalizes with Arm below the AJs. (C-F) S2 cells co-transfected with Act-GAL4 and UAS-E-Cad and treated with DMSO (C and D)or10 μM thapsigargin (Tg) (E and F) were stained with anti-E-Cad (C’-F’; green in C-F), phalloidin (C” and E”; red in C-F), anti-Cnx99A (C”’ and E”’; blue in C and E), or anti-Arm (D” and F”; blue in D and F). Scale bar, 5 μM. (G) Quantification of the percentage of cells (means ± SEMs) in which E-Cad is localized at the plasma membrane in Tg- or DMSO-treated cells. ****p < 0.0001 by unpaired t test on three independent experiments. InTg-treated cells, E-Cad does not reach the plasma membrane and colocalizes with Cnx99A. Arm colocalizes with E-Cad in control and Tg-treated cells. (H-K) Wing discs with wild-type (H) or SERCAS5 clones (I) expressing E-Cad RNAi, E-GadΔS-αCat clones (J) or SERCAS5, E-GadΔS-αCat clones (K). Clones are positively marked with GFP (green) and stained with anti-Al (H’-K’; red in H-K) and anti-Arm (H”-K”; blue in H-K). E-CadΔS-αCat rescuesArm accumulation and al expression in SERCAS5 clones. See also Figure S2.
Figure 3.
Figure 3.. Three Levels of ER Stress Induced by SERCA and orai Mutations
(A-F) Wing discs with oraik11505 (A), SERCAS5, oraik11505 (B), SERCAdsm, orat11505 (Cand E), SERCAdsm (D), or SERCAS5 (F)clones labeled bytheabsence ofRFP (green). (A-C) Discs are stained with anti-Al (A’-C’; red in A-C) and anti-Arm (A”-C”; blue in A-C). Scale bar, 50 μm. Disrupting Orai channel function does not rescue SERCA mutants. (D-F) The endoplasmic reticulum (ER) stress reporter Xbp-1-GFP (D’-F’; red in D-F) is driven in the posterior compartment with hh-Gal4. Discs are also stained with anti-activated caspase 3 (basal sections shown in D”-F”; blue in D-F). Loss of orai increases ER stress in SERCAdsm cells, but not to the level seen in SERCAS5 cells, which induces apoptosis. (G) Quantification ofXbp1-GFP intensity in SERCAdsm; SERCAdsm, oraik11505; and SERCAS5 clones. Box-and-whisker plot shows median bounded by minimum, first quartile, third quartile, and maximum. SERCAdsm, n = 22 clones in 5 wing discs; SERCAdsm, oraik1505 n = 22 clones in 6 discs; SERCAS5 n = 29 clones in 7 discs; ****p < 0.0001 by Welch’s ANOVA. See also Figure S3.
Figure 4.
Figure 4.. Hippo and Notch Signaling Pathways Are Affected by Moderate ER Stress
(A-L) Wing imaginal discs with SERCAdsm (A, D, G, and J), SERCAdsm, oraik11505 (B, E, H, and K), or SERCAS5 clones (C, F, I, and L), marked by the absence of RFP (green) and stained with antibodies to Fat (A’-C’; magenta in A-C), Dachs (D’-F’; magenta in D-F), Notch (G’-I’; magenta in G-I), or Cut (J’-L’; magenta in J-L). Scale bar, 50 mm. Subapical sections are shown, except in (D)-(F), which show the apical membrane. Although there is a weak subapical accumulation of Fat and Notch in SERCAdsm clones, Dachs and Cut are not affected. Decreasing levels of ER Ca2+ in SERCAdsm, oraik11505 and SERCAS5 clones cause an increasing accumulation of Fat and Notch, leading to the membrane localization of Dachs and loss of Cut. See also Figure S4.

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