transgene

imaginary discs

in vivo

Xenopus laevis oocytes

Fig. 1. RECS1 induces cell death through the mitochondrial pathway of apoptosis. (A) Left: MEFs expressing doxycycline-inducible Flag-RECS1 were stimulated with doxycycline (DOX), and the levels of RECS1 were determined by Western blot. Right: Cell death was determined by PI and SYTO16 costaining followed by automated microscopy (n = 7). Mw, weight-average molecular weight; NT, not treated. (B) MEFs were transiently transfected with the indicated concentrations of MYC-RECS1 followed by Western blot analysis. (C) Left: Bright-field (BF) and PI fluorescent images of MEFs transfected with 0.4 μg of MYC-RECS1 for 24 hours. Right: Cell death was determined by PI staining followed by FACS (n = 3). (D) Left: Images of MEF Flag-RECS1 cells cultured with doxycycline in the presence or absence of the caspase inhibitor QvD-OPh (QvD; 40 μM) for 24 hours (red, PI staining). Right: Corresponding cell death kinetics (n = 3). (E) PI staining and FACS of cells treated as in (D) (n = 4). (F) MEF Flag-RECS1 wild-type (WT) or BAX and BAK (BB) double-knockout (DKO) cells were treated with doxycycline, and indicated proteins were assessed by Western blot. (G) Indicated cells were cultured with doxycycline for 38 hours, and cell death was determined by FACS (n = 3). (H) Images (left) and quantification (right) of RECS1 (Flag) colocalization with indicated organelle markers in Flag-RECS1 MEFs treated with doxycycline. GA, Golgi apparatus. Data represent means ± SD (A, C, and D) or means ± SEM. Statistically significant differences were determined comparing the best individual fit for each curve using the extra-sum-of-squares F test (A and D) or two-way analysis of variance (ANOVA) followed by Holm-Sidak’s multiple comparisons test (C, E, and G). *P < 0.05; **P < 0.01; ****P < 0.0001).

Apoptosis

transmembrane domains

Fig. 9. RECS1-like/CG9722 modulates viability in response to intracellular stress in D. melanogaster. (A) One copy of dRECS1 was overexpressed in the wing imaginal discs of animals fed with control food or food supplemented with CQ (100 μg/ml) or Tm (10 μg/ml). The phenotype of adult male (right) and female (left) wings was quantified (n = 3 to 7, >80 wings per condition). (B) Control (yw) and mutant (dRECS1KO) male and female flies were treated with Tm (10 μg/ml), and the animal death rate was quantified daily (n = 2 to 3). (C) WT, dRECS1KO, or dRECS1IR pupae were treated with Tm, and the percentage of eclosion was quantified (n = 2 to 4). (D) WT and dRECS1KO female flies were fed a normal diet or starved, and the death rate was daily quantified (n = 3). (E) WT and dRECS1KO knockout male flies were fed a normal diet or starved, and the death rate was daily quantified (n = 2 to 3). Data represent means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. ****P < 0.0001.

Fig. 7. RECS1 is a pH-dependent calcium and sodium channel. (A) Alignment showing the conservation of the critical Arg60 residue from BsYetJ. Right: Comparison of the conserved di-aspartyl sensor. (B) Tridimensional structural model of the human RECS1 in closed and open conformations. Zoom: Key conserved C-terminal residues involved in pH sensing and channel opening. (C) X. laevis oocytes were injected with mRNA for Flag-RECS1 WT or D295Q, and ionic single-channel currents were recorded in the cell-attached mode using patch clamp at pH 7 or 6.5. Inset: Amplified plots depicting three opening states for RECS1 (O1, O2, and O3). (D) Quantification of the probability to find the channel in the open state (NPo) for RECS1 WT and D295Q (left). The NPo for open states O1 (middle) and O2 (right) is also shown [n = 4 (RECS1 WT) or n = 3 (RECS1 D295Q)]. (E) Images of X. laevis oocytes injected with different concentrations of Flag-RECS1 (top) and oocyte survival (bottom). Arrowhead indicates morphological alterations (n = 3). NI, not injected. (F) Recordings of macroscopic currents using the cut-open technique. (G) Permeability ratios with respect to K+ for indicated cations (n = 3 to 7). (H) Top: Indicated MEF Flag-RECS1 cells were treated with doxycycline, and protein levels were monitored by Western blot. Dashed lines indicate Western blot splicing. Bottom: Cells were treated with doxycycline and 25 μM CQ for 24 hours. Cell death was determined by FACS and normalized to WT (100%) (n = 3). Data represent means ± SEM. Statistical differences were determined by two-way (D, E, and G) or one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Fig. 5. Inhibition of the endogenous RECS1 protects cells against ER stress. (A) Knockdown of the endogenous mouse RECS1 using two different siRNA pools was confirmed by quantitative polymerase chain reaction (qPCR). (B) Left: Cells transfected with RECS1 siRNAs were treated with Tm (50 ng/ml), and cell death was determined by SYTOX green staining. A representative experiment from four independent experiments is shown. Right: Quantification of cell death at 24 hours (n = 4). (C) Cell death kinetics of cells transfected as in (B) and treated with 25 nM Tg. Left: Representative kinetics. Right: Quantification at 24 hours (n = 3). Data are shown as means ± SD. Statistical differences were determined by one-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. RECS1 sensitizes cells to ER stress. (A) Annexin V versus PI FACS density plots from MEF Flag-RECS1 cells cultured with doxycycline for 16 hours and then treated with Tm (100 ng/ml) for 16 hours followed by FACS (n = 9). (B) Experimental design for cell death kinetic experiments. (C) Cell death kinetic analyses of MEFs treated as in (A) (n = 6). (D) Cell death of MEF Flag-RECS1 cells incubated with doxycycline and then treated with 100 nM Tg for 24 hours (n = 3). (E) Corresponding cell death kinetic analysis (n = 3). (F) Clonal assay of MEF Flag-RECS1 cells treated with doxycycline alone or in the presence of Tm (50 ng/ml) or 25 nM Tg. (G) Cell death kinetics of MEF Flag-RECS1 cells cultured with doxycycline and then treated with Tm (100 ng/ml) in the presence or absence of 40 μM QvD (n = 3). (H) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and treated with Tm (100 ng/ml) or 50 nM Tg for 24 hours. Cell death was determined by FACS (n = 2 to 3). Data are shown as means ± SD (C, E, and G) or means ± SEM. Statistical differences were determined by the extra-sum-of-squares F test (C, E, and G) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3. RECS1 causes LMP in cells undergoing stress. (A) HeLa Flag-RECS1 cells were cultured with doxycycline and then treated with indicated concentrations of CQ, QvD, or both for 16 hours. Immunofluorescence against galectin-1 was performed. (B) Quantification of galectin-1 puncta per field (n = 2; 6 to 27 fields in total). (C) MEF Flag-RECS1 cells were cultured with doxycycline and treated with 50 μM CQ for indicated time points. The enzymatic activities of cathepsin D, acid phosphatase, and hexosaminidase were assessed from cytosolic, lysosomal-free fractions (n = 3). (D) MEF Flag-RECS1 WT and BAX and BAK DKO cells were cultured with doxycycline and then treated with indicated concentrations of CQ for 24 hours. Cell death was determined by PI staining followed by FACS (n = 3). (E) MEF Flag-RECS1 cells were cultured with doxycycline and treated with CQ for 16 hours. Immunofluorescence against LAMP-2 (green), BAX N-terminal domain (red, BAX 6A7 antibody), and Flag-RECS1 (blue, anti-Flag antibody) was performed. The triple colocalization of Flag-RECS1, LAMP-2, and active BAX is visualized as white color. (F) Representative immunofluorescence images of Flag-RECS1 MEF cells treated as in (E). Bottom panels correspond to zoomed white boxes (marked as z) from the top panels. (G) Intensity profiles for LAMP-2, BAX, and Flag-RECS1 signals along the line shown with an L1 (left) or an L2 (right) in (F). Data are shown as means ± SEM. Statistically significant differences were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2. RECS1 triggers cell death upon lysosomal stress. (A) Flag-RECS1 MEFs were stimulated with doxycycline for 16 hours and treated with a library of 77 cytotoxic compounds for 24 hours. Cell death was determined by PI staining using an ImageXpress Micro XL automated microscope. ETO, etoposide; NVB, vinorelbine tartrate; DTX, docetaxel. (B) Heatmap of the top 10 sensitizing compounds in (A). For each compound, heatmap colors depict doxycycline-treated PI values after the subtraction of cell death induced by doxycycline alone and by the compound in the absence of doxycycline. *CQ and HCQ were used at 50 μM. **Cisplatin (CDDP) was used at 150 μM. (C) Left: Representative images of MEF Flag-RECS1 cells pretreated with doxycycline and treated with 50 μM CQ for 24 hours (red, PI staining). Right: Cell death kinetic analyses of cells treated as in (B) (n = 3). (D) Cells were cultured as in (C) and then treated with indicated concentrations of bafilomycin A1 for 24 hours. Cell death was determined by FACS (n = 4). (E) Left: Representative images of HeLa Flag-RECS1 cells treated as in (C). Right: Corresponding cell death kinetic analyses (n = 3). (F) HeLa Flag-RECS1 cells were treated as in (E), and cell death was assessed by FACS (n = 3). Data are shown as means ± SD (C and E) or means ± SEM (A, D, and F). Statistically significant differences were determined using the extra-sum-of-squares F test (C and E) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ****P < 0.0001.

anterior-posterior shortening

We found that dRECS1KO cells exhibited increased formation of autophagosomes and autolysosomes together with higher overall autophagy flux (fig. S8, D to F). Together, these results suggest that CG9722 has a proapoptotic activity in vivo and sensitizes flies to lysosomal- and ER stress–induced cell death.

autophagy

In addition, dRECS1KO female animals were more resistant to death induced by nutrient starvation, an effect that was still observed but not as prominent in male flies or larvae (Fig. 9, D and E).

Moreover, dRECS1 deficiency conferred resistance to Tm in male and female flies, increasing life span under ER stress (Fig. 9, B and C).

wing phenotype

transposon

Last, to define the relationship between the channel activity of RECS1 and the regulation of cell death induced under lysosomal stress, we generated a doxycycline-inducible MEF cell line expressing a single-point mutant version of RECS1 (Fig. 7H). When compared to WT RECS1, the overexpression of the D295Q mutant diminished cell death, behaving similarly to the mock conditions (Fig. 7H)

Mutation of the di-aspartyl sensor results in a marked decrease in the channel’s permeability to Ca2+ and Na+ but not to Ba2+ or Cs+ (Fig. 7G). Together, these results suggest that RECS1 is a pH-dependent cationic channel, with a main preference for calcium.

ltage clamp technique with potassium as the reference ion. Analyses of permeability ratios indicated that RECS1 allows the flux of Ca2+ and, to a lesser extent, Na+ (Fig. 7F).

Notably, overexpression of RECS1 triggered cell death and structural abnormalities in the oocytes in a dose-dependent manner, consistent with its role as a cell death regulator (Fig. 7E)

Mutation of the key Asp295 from the putative di-aspartyl sensor significantly reduced RECS1 opening probability at pH 7, while it only slightly affected the ability to respond to pH changes (Fig. 7D).

Lowering the pH to 6.5 caused a marked decrease in the number of current spikes in both channels (Fig. 7C), consistent with the hypothesis of RECS1 as a pH-regulated channel. Quantification of the total open probability (NPo) in addition to the probability for each independent opening state (NPo Ox) showed that the channel activity of RECS1 is heavily dependent on pH (Fig. 7D).

current spikes

single-channel currents in the cell-attached mod

electrophysiological properties

MODELLER platform

interaction between Arg158 with the Asp268-Asp295 di-aspartyl sensor in the closed conformation (Fig. 7B, green)

In the open state, this latch becomes dislodged, and the second transmembrane is displaced out, leading to the opening of the channel (Fig. 7B, magenta). Our model suggests that RECS1, as demonstrated for other proteins of the LFG and BI-1 families (11, 12, 25), could operate as an ion channel. Our bioinformatic analysis also suggests that Asp295 (D295) is a strong candidate to regulate the channel activity of RECS1 and was therefore selected for mutagenesis and functional studies.

homology models of the three-dimensional (3D)

morphology

Fig. 6. RECS1 increases lysosomal acidification and calcium content. (A) Lysosomal pH of HeLa Flag-RECS1 loaded with FuraDx and OGDx and cultured in the absence or presence of doxycycline for 24 hours (n = 5, 15 to 16 total coverslips). (B) Corresponding lysosomal calcium concentration. (C) Left: Cytosolic calcium levels were determined in HeLa Flag-RECS1 cells treated with doxycycline. Cells were treated with 5 μM Tg to stimulate ER calcium release. Right: Maximum ER calcium peak (n = 4, 10 to 11 coverslips). ns, not significant. (D) MEF WT and BAX and BAK DKO Flag-RECS1 cells were loaded as in (A), and lysosomal pH was determined after 24 hours of doxycycline treatment (n = 6, 12 to 17 coverslips per condition). (E) Lysosomal calcium measurements (n = 6). (F) Knockdown of the endogenous mouse RECS1 by siRNA was confirmed by qPCR after 48 hours of transient transfection. (G) Cells transfected as described in (F) were loaded as described in (A), and lysosomal pH was determined after 48 hours of transfection (n = 5, 14 to 15 coverslips). (H) Lysosomal calcium measurements (n = 5, 14 to 15 coverslips). (I) MEF Flag-RECS1 WT and BAX and BAK DKO cells were treated with doxycycline for 16 hours and analyzed by TEM. Image magnification, ×4000 (n = 13 to 17 cells per condition). (J) Diameter of primary and secondary lysosomes (n = 8 to 13 cells per condition). Data represent means ± SEM. Statistically significant differences were detected using ratio-paired two-tailed t test (A, B, D, E, G, and H) or two-way ANOVA followed by Holm-Sidak’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

ependent on BAX and BAK expression (Fig. 6D)

RECS1 overexpression resulted in lysosomal acidification in HeLa cells, with lysosomal pH (pHL) decreasing from 4.77 to 4.38 (Fig. 6A). The acidification of lysosomes correlated with an increase in lysosomal intraluminal calcium concentration ([Ca2+]lys) from an average of 149 to 286 μM (1.9-fold increase) (Fig. 6B),

pH (OGDx)

atiometric calcium (Fura-2Dx)

calcium-binding affinities

short hairpin RNA (shRNA)

Silencing

had no effects in basal cell viability (fig. S5, O and P), but it conferred protection against ER stress induced by Tm and Tg (Fig. 5, B and C).

(siRecs1#1 and siRecs1#2)

siRNAs

endogenous RECS1

(Fig. 4A). Since ER stress affects protein trafficking through the secretory pathway and the expression of RECS1 alone does not trigger ER stress, we allowed RECS1 to accumulate for 16 hours before challenging cells with Tm or other ER stressors (Fig. 4B). Using this protocol, we found that RECS1 overexpression hastened cell death to ER stress, as evidenced in cell death kinetic experiments (Fig. 4C). Moreover, we confirmed these observations in HeLa cells in dose-response experiments (fig. S5, M and N). We further validated these results in cells stimulated with Tg, followed by cell death measurements using both FACS and live microscopy (Fig. 4, D and E). Long-term survival assessment by clonogenic assays indicated that RECS1 expression reduced the growth of cells under ER stress (Fig. 4F). Last, we treated MEFs expressing Flag-RECS1 with QvD together with Tm and determined cell death by live microscopy. As expected, QvD treatment reduced cell death under ER stress (Fig. 4G). Similarly, BAX and BAK double deficiency fully inhibited cell death triggered by RECS1 overexpression under ER stress (Fig. 4H).

These results indicate that RECS1 overexpression alone does not cause ER stress. We also determined whether CQ treatment could induce ER stress. Treatment of Flag-RECS1–overexpressing MEFs with CQ failed to induce Xbp1 mRNA splicing (fig. S5, K and L), Bip/Grp78, or Chop/Gadd153 (fig. S5L). These results suggest that cell death induced by RECS1 under lysosomal stress conditions is not associated with the activation of the UPR.

mobility shift

ip/Grp78 and Chop/Gadd153 (fig. S5H), two sensitive markers of ER stress (13, 15).

UPR

XBP1s

Several proteins of the TMBIM family, such as BI-1 and GRINA, inhibit apoptosis triggered by ER stress, probably through the modulation of ER calcium (10, 13, 15, 17, 38). To gain further insights into the role of RECS1 in the control of cell death under cellular stress, we measured its expression in cells undergoing ER stress induced by the treatment with the N-glycosylation inhibitor tunicamycin (Tm). We found that Recs1 mRNA levels were up-regulated under ER stress, whereas Bi-1 mRNA levels remained unaltered (fig. S5A). This expression profile parallels that of certain BCL-2 proteins known to be induced by ER stress (i.e., Puma), while antiapoptotic components were unchanged (i.e., Bcl-2) (fig. S5B). Recs1 mRNA was also up-regulated in cells undergoing ER stress induced by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) calcium pump inhibitor thapsigargin (Tg) (fig. S5C). The kinetics of the up-regulation of Recs1 mRNA under ER stress paralleled the up-regulation of the ER chaperone Bip/Grp78 and the proapoptotic factor Chop/Gadd153 (fig. S5D)

As expected, BAX and BAK DKO cells were almost completely resistant to cell death induced by treatment with 25 μM CQ (Fig. 3D). However, we observed 30% of cell death in BAX and BAK DKO Flag-RECS1 cells treated with the higher CQ dose (50 μM), an effect that could not be reversed by inhibition of mitochondrial transition permeability pore (mPTP) (cyclosporin A) or necroptosis (necrostatin-1s) (Fig. 3D and fig. S4G).

In addition, we measured the enzymatic activities of three lysosomal enzymes—cathepsin D, acid phosphatase, and hexosaminidase—in cytosolic lysosomal-free fractions. In line with previous results, we found that the activity of these three enzymes is increased in the cytosol of MEFs expressing Flag-RECS1 after CQ treatment but not in control cells (Fig. 3C)

downstream

, treatment with CQ resulted in a dose-dependent increase in the number of LGALS1/galectin-1 puncta (Fig. 3, A and B

LMP

During LMP, lysosomal intraluminal cathepsins and hydrolases leak into the cytosol, where they act as cell death proteases

pathophysiological processes

We then explored the effects of lysosomal stress on RECS1 localization. Analysis of the distribution of endogenous mouse RECS1 indicated that, under basal conditions, RECS1 colocalizes mainly in lysosomes and late endosomes (around ~20%) (fig. S3, C to E). However, in cells undergoing starvation or starvation and treated with CQ, RECS1 was further recruited to lysosomes but not to late endosomes, suggesting that RECS1 distribution is highly dynamic (fig. S3, C to E). Notably, the levels of Recs1 mRNA were up-regulated in cells undergoing nutrient starvation (fig. S3F). In addition, RECS1 overexpression induced autophagosome formation, an effect that was increased by CQ treatment (fig. S3G). Overall, our results indicate that RECS1 is located at lysosomes and regulates the susceptibility of cells to lysosomal stress.

endogenous

HeLa

colocalization analysis

confocal microscopy

immunofluorescence

These results indicate that RECS1 potentiates cell death triggered by lysosomal stress.

bafilomycin A1

V-ATPase (vacuolar H+-ATPase)

pH alkalization

lysosomotropic agents

nexpectedly, RECS1 overexpression sensitized cells to the death triggered by ~14 compounds, whereas it repressed the activity of 12 compounds (fig. S2A). The top sensitizing drugs were chloroquine (CQ) and hydroxychloroquine (HCQ), which are known to induce lysosomal stress by inhibiting their acidification (Fig. 2, A and B).

BAX- and BAK-dependent manne

depolarize

Specific structural and functional criteria are required to validate a BH3 motif, since definitive bioinformatic procedures that rely purely on sequence analyses are currently lacking (28). To this end, we synthesized two peptides: RECS1280–299 and RECS1296–311, based on the N- and C-terminal BH3-like motifs, respectively.

deletion mutants: RECS1Δ910, lacking the C-terminal BH3 motif, and RECS1Δ868, lacking both

RECS1 has been traditionally classified within the TMBIM superfamily (8). However, detailed sequence and phylogenetic analyses suggest that RECS1, together with GRINA, LFG, GAAP, and Tmbim1b, forms a distinct and evolutionarily conserved protein family, termed the “LFG family,” characterized by a shared common ancestry and the lack of the signature BI-1 motif (fig. S1A) (7, 26). Previous sequence comparison using the BH3 motif consensus from Prosite (PS01259) identified one putative BH3 motif on the C-terminal domain of the human RECS1 and LFG proteins (22). The BH3-like motif in the C-terminal region of RECS1 contains the characteristic minimal Leu-X3-Gly-Asp/Glu sequence, but no additional BH3-like motifs of this kind were found in the other members of the LFG and BI-1 protein families (fig. S1B). However, an additional conserved BH3-like sequence consisting of Leu-X4-Asp is also present in an adjacent sequence of RECS1, in addition to GRINA and LFG (fig. S1B). These putative “BH3 motifs” are conserved in humans and in several species of non-human primates (fig. S1, C and D). However, only one such motif is present in rodents, suggesting that they represent a late evolutionary event.

BAX and BAK double-knockout (DKO) background (Fig. 1F)

caspases

o determine the requirement of caspases, we treated RECS1-overexpressing cells with the pan-caspase inhibitor QvD-OPh (QvD), followed by cell death kinetics and fluorescence-activated cell sorting (FACS) analysis. Caspase inhibition completely abrogated cell death induced by RECS1

intracellular signals

These observations suggest that RECS1 overexpression is sufficient to induce cell death.

transient transfection

propidium iodide (PI)

FLAG

doxycycline-inducible

embryonic fibroblast (MEF)

(D295Q)

proapoptotic

mutation

LMP,

ECS1 expression highly sensitizes cells to lysosomotropic agents and microtubule-destabilizing drugs.

cytotoxic agents

TMBIM family, RECS1 expression results in cell death via the mitochondrial pathway of apoptosis.

gain- and loss-of-function approaches

intracellular

Additional sequence analyses revealed the presence of a conserved di-aspartyl sensor and an arginine latch, shown to be critical for the regulation of the calcium channel function of the bacterial (Bacillus subtilis) homolog BsYetJ and human GAAP (11, 12, 25).

Golgi apparatus

BH3-like motif

However, no proapoptotic molecules have been identified in the mammalian TMBIM family.

ortholog

putative

rheostat

RECS1 and LFG are regarded as inhibitors of the extrinsic pathway of apoptosis as they block cell death induced by Fas ligand (18, 19), while BI-1, GRINA, and GAAP repress apoptosis induced by intracellular cytotoxic stimuli (10, 17).

ion channe

calcium homeostasis

In addition to BI-1, the TMBIM superfamily includes the following members: responsive to centrifugal force and shear stress 1 (RECS1/TMBIM1), lifeguard (LFG/TMBIM2), glutamate receptor ionotropic N-methyl-D-aspartate receptor 1 (GRINA/TMBIM3), Golgi anti-apoptotic protein (GAAP/TMBIM4), and growth hormone–inducible protein (GHITM/TMBIM5) (8).

cytotoxic stimuli

homologs

transmembrane BAX inhibitor motif containing (TMBIM) superfamily

pathophysiological

lysosomal membrane permeabilization (LMP)

caspase cascade

cytochrome c

homo-oligomerization

conformational activation

proapoptotic proteins

to ER stress. RECS1 overexpression resulted in reduced survival when zebrafish were exposed to Tg in the water, while overexpression of the mouse GRINA conferred protection (Fig. 10, H and I) as reported (17). W

Dysmorphic embryos

Next, we determined the importance of RECS1 channel activity to cell death control during zebrafish development. We injected single-cell embryos with RECS1 WT or the D295Q mutant and assessed animal viability 24 hpf. In line with our in vitro results, overexpression of the RECS1 D295Q mutant failed to induce cell death in these embryos (Fig. 10E). Moreover, while the injection of RECS1 WT resulted in a high proportion of morphological-altered embryos, expression of the mutant protein did not have any adverse effect (Fig. 10, F and G).

As expected, RECS1 overexpression induced a high number of AO spots, distributed along the head and, to a lesser extent, the body of the embryo (Fig. 10, C and D). Deletion of the C-terminal domain of RECS1 completely abrogated its cytotoxic effects (Fig. 10, C and D). T

cridine orange (

RECS1 overexpression significantly decreased animal survival, a phenotype that was independent of its C-terminal domain (Fig. 10A). As a control, we overexpressed the TMBIM protein GRINA, which, under the same conditions, did not result in any developmental defects (Fig. 10A).

Together, these results suggest that the C-terminal region of RECS1 is essential to induce cell death, but this sequence does not bear functional bona fide BH3 motifs.

itochondrial potential (ΔΨm)–sensitive dye JC-1

o investigate the role of RECS1 on the control of cell death in vivo in a vertebrate model, we overexpressed the full-length human RECS1 in zebrafish by injecting in vitro synthetized mRNA to one-cell stage embryos, followed by the analysis of animal viability and morphology 24 hours post-fertilization (hpf)

There is a high degree of functional conservation in the mechanisms regulating cell death between vertebrates, with almost all the members of the BCL-2 and the TMBIM families conserved in zebrafish

verexpression of dRECS1 strongly sensitizes to lysosomal perturbations and ER stress, resulting in exacerbated wing abnormalities (Fig. 9A).

The di-aspartyl sensor is conserved in all six TMBIM proteins, while the arginine latch is only present in the LFG subfamily (LFG1 to LFG4) but not in BI-1, where it has been replaced by His78 (Fig. 7A) (11).

BsYetJ has 27% identity with GAAP and 36% with RECS1 (fig. S7). In the bacterial BsYetJ, the closed channel conformation is maintained through the interaction of the Arg60 with a conserved di-aspartyl group formed by Asp171-Asp195, which are hydrogen-bonded to one another through their carboxylate groups

intraluminal cathepsins

annexin V

BAX has been shown to translocate to lysosomes where it is suggested to trigger LMP and cell death under different pathophysiological conditions, including Parkinson’s disease, oxidative stress, and autophagic cell death (35–37). To assess BAX translocation to the lysosomal membrane and its colocalization with RECS1, we detected active BAX using the 6A7 conformational antibody. Analysis of RECS1 (using the Flag antibody) and the distribution of the lysosomal protein LAMP-2 in MEFs indicated that under basal conditions (no RECS1), BAX remained mainly cytosolic (fig. S4H). However, BAX colocalized with RECS1 in LAMP-2–positive vesicles following RECS1 induction (Fig. 3, E to G). After CQ treatment, BAX was redistributed into large LAMP-2–positive vesicles, an effect that was dependent on RECS1 expression (Fig. 3E). Active BAX was present preferentially in Flag-RECS1–positive lysosomes in cells treated with CQ (Fig. 3, F and G). Together, these results suggest that RECS1 induces cell death through LMP in response to lysosomal stress, correlating with the translocation of BAX to lysosomes

cytotoxic

abrogate

Ablation

BAX and BAK

SYTO16 staining

lysosomotropic agents