The x2-versus-t relationship was linear for at least an hour (fig. S4C), consistent with random walk diffusion. Thus, over this time scale and this distance scale, we found no evidence for a trigger wave

Every egg that showed a wave had increased caspase activity (Fig. 4, E and F, red symbols). We also collected eggs during the same time period that had not displayed a wave. None of these eggs had increased caspase activity

The TMRE wave propagated at the same speed as the caspase trigger wave reported by Z-DEVD-R110 in the same tube (38 μm/min)

As shown in movie S1 and Fig. 1, B and C, apoptosis progressed up the thin tube at a constant speed of 27 μm/min over a distance of several millimeters. In five independent experiments, apoptosis always propagated linearly, without showing any signs of slowing down or diminishing, and the average speed was 29 ± 2 μm/min (mean ± SD). In contrast, the 10-kDa dye spread only a few hundred micrometers (Fig. 1B), implying that neither simple diffusion nor any unintended mixing could account for the spread of apoptosis.

These experiments implicate caspase-3 and/or -7 as well as Bcl-2 in the regulation of the apoptotic trigger waves. The experiments also show that the trigger waves are relatively robust; they are still present, though with reduced speeds, in extracts depleted of mitochondria or treated with maximal doses of GST–Bcl-2 or Ac-DEVD-CHO

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.

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).

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)

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),

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).

(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.

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)

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

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.

colocalization analysis

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

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).

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

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

(D295Q)

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

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

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

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

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).

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

Adding GST–Bcl-2 decreased the wave speed (Fig. 3, A and B, and movie S5). The maximum effect [at 400 nM added GST–Bcl-2, which compares to the estimated endogenous Bcl-2 concentration of approximately 140 nM (14)] was a reduction of the speed to about 13 μm/min, the speed seen in purified cytosol. Added GST–Bcl-2 had no effect on the trigger wave speed in cytosolic extracts (Fig. 3, C and D), which emphasizes that the waves seen in purified cytosol are probably not caused by contaminating mitochondria. GST–Bcl-2 decreased the trigger wave speed in reconstituted (cytosol plus mitochondria) extracts (Fig. 3, E and F), just as it did in cytoplasmic extracts

During this same time period, a surface wave often appeared, originating near the vegetal pole and propagating toward the animal pole, with a typical apparent speed of ~30 μm/min

These findings support the idea that spontaneous apoptosis typically initiates near the vegetal pole of the egg and propagates outward and upward from there as a ~30-μm/min trigger wave

The surface waves propagated at an apparent speed of ~30 μm/min, similar to the wave speeds seen in apoptotic extracts. Control oocytes injected with Texas Red–dextran in water did not exhibit these surface waves. These findings indicate that apoptotic trigger waves can be produced in oocytes

Both reporters showed that the wave speed decreased as the inhibitor concentration increased

However, in experiments with either cytoplasmic extracts or reconstituted extracts, more than half of the time (in 12 of 22 or 21 of 37 tubes, respectively) a second spontaneous apoptotic wave emerged elsewhere in the tube

we determined the trigger wave speed to be half maximal at a mitochondrial concentration of 1.3 ± 0.6% (mean ± SE), which is estimated to be ~40% of the physiological mitochondrial concentration in Xenopus eggs. Thus, an average concentration of mitochondria is sufficient to generate apoptotic trigger waves of near-maximal speed, and the wave speed would be expected to drop in mitochondrion-poor regions of the cytoplasm

As was the case in Fig. 2B, the speed of the wave front fell during the first ~120 min, consistent with diffusive propagation, but once the speed reached ~14 μm/min it remained constant for many hours (Fig. 2E). This suggests that purified cytosol is capable of generating apoptotic trigger waves, albeit with a substantially lower speed than that seen in cytoplasm or in cytosol supplemented with mitochondria

Moreover, the reconstitution restored the trigger waves (movie S3 and Fig. 2D). The propagation distance increased linearly with time (Fig. 2D), as it did in crude cytoplasmic extracts (Fig. 1), and propagation occurred at a constant speed of 39 μm/min, somewhat faster than that observed in cytoplasmic extracts. The signal propagated over a long distance (6000 μm) with little loss of amplitude and no loss of speed (Fig. 2D)

In agreement with previous reports (3, 13), the caspases were briskly activated (Fig. 2A). Thus, mitochondria are not essential for cytochrome c–induced activation of executioner caspases in Xenopus extracts

Fluorescence spread up the tube at a constant speed (in this experiment, 33 μm/min). In eight experiments, the average speed was 30 ± 3 μm/min (mean ± SD)

The apoptotic activity propagated from the induction terminus to the distal terminus at a constant speed of 32 μm/min, consistent with a self-sustaining process

trends noted here as being independent of the observational and analysis techniques used

large increase was seen in the number and proportion of hurricanes reaching categories 4 and 5

global data indicate a 30-year trend toward more frequent and intense hurricanes

These changes occur in all of the ocean basins.

has decreased monotonically as a percentage of the total number of hurricanes throughout the 35-year period

a simple attribution of the increase in numbers of storms to a warming SST environment is not supported

decreasing by 40% from 1995 to 2003

Decadal variability is particularly evident in the eastern Pacific

North Atlantic Ocean, which possesses an increasing trend in frequency and duration

a substantial decadal-scale oscillation

None of these time series shows a trend that is statistically different from zero over the period

trends in each of the ocean basins are significantly different from zero at the 95% confidence level or higher, except for the southwest Pacific Ocean

Significance

(H2) Flower and leaf bud break of high Arctic plants should be more sensitive to temperature increases than those of low Arctic and alpine plants.

Flower and leaf bud break are occurring earlier as sites become warmer.

temporal trends of heat sums (βTDD_x_YEAR) showed significantly increased heat sums over time for greening but a tendency for lower heat sums over time for flowering and senescence.

Trends in the timing of events as represented by βDOY_x_YEAR showed significantly later greening (positive slope) but tendencies for earlier flowering and earlier senescence (negative slopes) over the study.

Plant phenological responses to these increases in season length and temperature are uncertain, but understanding changes as they occur is essential for predicting future changes in tundra vegetation processes

First, anti-ISIS agencies can thwart development of large aggregates that are potentially far more potent (21) by breaking up smaller ones.