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Mol Biol Cell
Mol Biol Cell
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Molecular Biology of the Cell
1059-1524
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The American Society for Cell Biology

38696256
E24-02-0058
10.1091/mbc.E24-02-0058
Brief Report
new_hypothesis
Drosophila Nhe2 overexpression induces autophagic cell death
Peralta Jobelle a † ‡
DuPriest Blake a
Orozco Daniel a
Pacheco Juan Reyna a
Martins Laura a
Soriano Rachel Ann a
Wong Alan a
Wong Ramy a
Grillo-Hill Bree a *
a Department of Biological Sciences, San José State University, San José, CA 95112
Grinstein Sergio Monitoring Editor
Hospital for Sick Children
Author contributions: B.G. conceived and designed the experiments; B.G., J.P., B.D., D.O., J.R., L.M., R.S., and A.W. performed the experiments; B.G., J.P., B.D., J.R., L.M., R.S., and R.W. analyzed the data; B.G., J.P., and D.O. drafted the article; B.G., J.P., B.D., J.R., and L.M. prepared the digital images.

Present addresses: †Fred Hutchinson Cancer Center, Translational Science and Therapeutics Division and Molecular and Cellular Biology Program, Seattle, WA;

‡University of Washington, Seattle, WA 98195.

Conflicts of interests: The authors declare no financial conflict of interest.

ORCID ID: Bree Grillo-Hill, 0000-0001-6471-7025

*Address correspondence to: Bree Grillo-Hill (bree.grillo-hill@sjsu.edu).
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© 2024 Peralta et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
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https://creativecommons.org/licenses/by/4.0/ This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution 4.0 International Creative Commons CC-BY 4.0 License.

Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via overexpression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.

Cancer cells have constitutively increased intracellular pH (pHi), which is thought to facilitate cancer cell behaviors like increased proliferation and resistance to apoptotic cell death.

The authors show that overexpression of Drosophila Nhe2 is sufficient to decrease cell number through autophagic cell death.

This suggests that increased pHi may induce autophagic cell death.
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pmcINTRODUCTION

Regulation of complex processes like tissue growth control can be mediated by environmental cues, including pH. Intracellular pH (pHi) is regulated by ion transport proteins including the plasma membrane resident sodium-proton exchanger NHE1 (DNhe2 in Drosophila melanogaster, Grillo-Hill et al., 2015). NHE1 exchanges an extracellular Na+ for an intracellular H+ to maintain pHi near neutral at 7.2. Constitutively increased pHi is a common characteristic of most cancers and is sufficient to induce cancer cell behaviors, including dysplasia, increased proliferation, invasive cell migration, and impeded differentiation (Grillo-Hill et al., 2015; Ulmschneider et al., 2016). Increased pHi is caused by higher expression or activity of ion transport proteins like the ubiquitously expressed sodium-proton exchanger NHE1 (Reshkin et al., 2014). The adult Drosophila compound eye is composed of approximately 750 unit eyes, termed ommatidia, that are precisely arranged in a hexagonal lattice (Ready et al., 1976). Drosophila eye development is an excellent system to probe how cell signaling pathways coordinate complex biological processes like cell growth and regulated cell death. In the developing Drosophila eye, overexpression of DNhe2 is sufficient to increase pHi, disrupt tissue architectures, increase proliferation, and induce planar cell polarity defects.

Cell number is precisely regulated in the developing Drosophila eye in distinct temporal phases for proliferation (larval) and apoptosis (pupal). The eye imaginal disc, which is the precursor to the adult eye, begins as a sheet of undifferentiated cells. During the first and second larval instars, the Drosophila eye imaginal disc proliferates and remains largely undifferentiated. During the third larval instar, two bursts of regulated proliferation, termed the first and second mitotic wave, flank the morphogenetic furrow. Posterior to the second mitotic wave, stochastic cell division occurs (Ready et al., 1976; Wolff and Ready, 1991a). At the end of the larval phase of development, proliferation is complete.

Next, apoptosis plays an important role in refining the pattern during pupal eye development in two distinct phases that are regulated by Wnt, Notch, and EGFR signaling (Cagan and Ready, 1989; Yu et al., 2002; Cordero et al., 2004). This process removes exactly seven interommatidial lattice cells from each ommatidium (Wolff and Ready, 1991b). At the end of patterning in midpupal development, this leaves exactly six secondary pigment cells, three tertiary pigment cells, and three bristle complexes surrounding each ommatidium. Regulated cytosolic pH dynamics are observed during apoptosis, with alkalization of mitochondrial matrix pH and subsequent cytosolic acidification. Caspase activation requires decreased pHi, and the highest caspase activity was detected at pH 6.3–6.8 (Matsuyama et al., 2000). Further, one study has shown that constitutively increased pHi blocks apoptotic signaling as measured by downstream effects on DNA degradation (Pérez-Sala et al., 1995). These data led to a model where increased pHi protects cancer cells from apoptotic cell death (White et al., 2017).

Much less is understood about the regulation of necrotic and autophagic cell death in the developing Drosophila eye. Different modes of cell death are differentiated by distinct cell morphologies and often initiated through distinct signaling pathways (Ouyang et al., 2012). Necrosis is a form of cell death that is often brought on by injury, which results in the loss of cellular membrane structure and uncontrolled release of cellular contents (Proskuryakov et al., 2003). This initiates an inflammatory response that attracts phagocytes to remove dead cells and leukocytes, which contain microbicidal products to help clear invading microorganisms (Rock and Kono, 2008).

Autophagy is a conserved catabolic process where cellular components are packaged into a double-membrane vesicle known as an autophagosome that fuses with a lysosome to degrade macromolecules. Autophagosome contents are broken down into cellular building blocks (nucleotides, amino acids, and fatty acids) and recycled for homeostasis or growth in a cell autonomous manner (Khandia et al., 2019). In Drosophila eye imaginal discs, autophagy is required for tissue differentiation and the survival of precursor cells. Atg5-positive and Atg8a-positive focal labeling was localized within and posterior to the morphogenetic furrow (Billes et al., 2018). Knockdown of Atg3, Atg14, or Atg101 via RNA interference (RNAi) results in significantly reduced adult eyes (Billes et al., 2018). Furthermore, when Atg8a, Atg7, or Atg9 are knocked down with coexpression of oncogenic RasV12, adult eyes show overgrowth or enhanced roughness (Manent et al., 2017).

There are a few examples in development showing that autophagy can culminate in cell death, termed autosis (Das et al., 2012; Liu and Levine, 2015). During metamorphosis in Drosophila, larval tissues undergo histolysis in response to the hormone ecdysone. The most studied example is salivary glands; however, their destruction is not solely due to autophagy, as apoptotic caspases are also required for their destruction (Das et al., 2012). However, the amnioserosa cells in the Drosophila embryo and midgut cells in Drosophila larvae undergo cell death that requires autophagy proteins but does not require caspase activity (Mohseni et al., 2009). In cancer cells, autophagy is often up-regulated and is thought to play a protective, prosurvival role for tumor cells (Debnath et al., 2023). Previous studies have suggested that acidic extracellular pH may regulate autophagy (Wojtkowiak and Gillies, 2012; Zhao et al., 2016), but the mechanism remains unresolved.

We became interested in how dysregulated pHi modulates mechanisms of growth control. We overexpressed DNhe2 using the eye-specific Glass Multimerized Reporter Gal4 driver (GMRGAL4) and found that GMR>DNhe2 expressing flies showed increased pHi from ∼7.3 to ∼7.7 and induced patterning errors visible as a rough eye phenotype in adult flies (Grillo-Hill et al., 2015). We showed that the rough eye phenotype was due to ion exchange rather than another function of DNhe2 by expressing a transport dead DNhe2E358I transgene, which showed no change in pHi and also showed no retinal phenotypes. GMR>DNhe2 expressing flies showed an increase in stochastic proliferation in the posterior region of the eye imaginal disk (Grillo-Hill et al., 2015). With increased proliferation, we expected to see an increase in the size of the adult eye; however, external examination of adult fly eyes revealed an overall size decrease. Here, we investigate the cellular basis of this reduced eye size, and report that DNhe2 overexpression induces a 20% decrease in the number of interommatidial cells in the developing eye. In addition, we found that mutations in essential apoptotic genes including the viral caspase inhibitor p35 show no effect on cell loss. However, decreasing activity of autophagy genes suppressed the reduced eye phenotype and rescued missing cells. Further, molecular markers support increased autophagy. Collectively, our data suggest that regulated autophagy occurs in response to increased pHi, and suggests a model for regulation of autophagy in tumors where autophagic cell death may promote tumor growth.

RESULTS AND DISCUSSION

Overexpression of DNhe2 causes a smaller adult eye and loss of interommatidial lattice cells

To determine how increased DNhe2 expression impacts tissue size in the Drosophila eye, quantitative analysis of the area of adult eyes was performed. High-resolution images of adult Drosophila eyes were collected (Figure 1A), and the area of each eye was measured. We found that GMR>DNhe2-expressing flies have significantly smaller eyes (mean 74.28 µm2) compared with the genetic background control w1118 flies (mean 125.83 µm2) or the driver control GMRGAL4 heterozygous flies (mean 142.49 µm2), respectively 41% and 48% reduction in area (Figure 1B). This reduction in eye size, despite increased proliferation, suggests that cell death may be increased.

FIGURE 1: Overexpression of DNhe2 induces a rough, small eye and reduces interommatidial cell number. (A) High-resolution images show adult eyes for these genotypes: w1118 (parental strain control); GMRGAL4 heterozygotes (driver control); GMR>DNhe2. (B) Mean measured adult eye sizes were: w1118 (125.83 µm2, N = 7); GMRGAL4 (142.49 µm2, N = 9), and GMR>DNhe2 (74.28 µm2, N = 6). One eye per adult fly was imaged and quantified. Data are graphed as median values with interquartile ranges. Statistical significance was determined by unpaired t tests with Welch’s correction. (C) Confocal micrographs show w1118 and GMR>DNhe2 wandering third larval instar eye imaginal discs labeled with an antibody against Armadillo (Arm, Drosophila beta-catenin) to highlight cell-cell junctions. (D) Mean cell counts were: w1118(38.6 cells, N = 25 counting areas from 5 discs/animals); GMR>DNhe2 (30.10 cells, N = 15 counting areas from 3 discs/animals). Data are graphed as median values with interquartile ranges. Statistical significance was determined by unpaired t tests with Welch’s correction. (E) Confocal micrographs show w1118 and GMR>DNhe2 32H apf pupal eyes labeled with an antibody against Armadillo (Arm) to highlight cell-cell junctions. (F) Mean cell counts were: w1118 (15 cells, N = 25 counting areas from 5 eyes/animals); GMR>DNhe2 (11.42, N = 25 from 5 eyes/animals). Data are graphed as median values with interquartile ranges. Statistical significance was determined by unpaired t tests with Welch’s correction.

To determine when in development this decrease occurs, cell counts of interommatidial lattice cells were performed in wandering third larval eye imaginal discs and in pupal eyes at the end of patterning. Cell junctions were labeled and cells were counted through identification of a central ommatidium and its six nearest neighbors. Counting areas defined by lines connecting through centers of neighboring ommatidium. Cells completely contained within this hexagon count as one cell, and partially contained cells count as one-half cell. In w1118 wandering third larval eye imaginal discs, we found an average of 38.6 cells per counting area, compared with 30.1 cells in GMR>DNhe2 (Figure 1, C and D), a reduction of ∼22%. In pupal eyes, we counted interommatidial lattice cells and bristles at the end of pattern formation, and found a decrease from exactly 15 cells per counting area in control to 11.4 cells per counting area in GMR>DNhe2 expressing pupal eyes (Figure 1, E and F), a reduction of ∼24%. These data suggest that cell loss is apparent during larval development, and that these missing cells are not restored by the end of pattern formation in pupae.

As previously reported, ommatidial organization in GMR>DNhe2 is significantly disrupted compared with control w1118, specifically cells show aberrant cell-cell contacts, and aberrant cell numbers when cell-cell junctions are labeled with an antibody against Armadillo, the Drosophila ortholog of beta-catenin (Figure 2A). Specific defects included missing secondary and tertiary pigment cells, doubled secondary pigment cells, and asters (four pigment cells surrounding bristles at ommatidial vertices). To quantify these observations, we performed cell counts for each non-neuronal cell type visible on the apical surface of the retinal epithelium in pupal eyes: cone cells, primary pigment cells, secondary pigment cells, tertiary pigment cells, and bristles. We did not observe a change in the number of primary pigment cells (Figure 2B) or cone cells (Figure 2C). Cone cell markers N-cadherin and Cut were expressed in the normal pattern (Figure 2D). Secondary pigment cells decreased from 6 cells per ommatidium to 5.3 cells per ommatidium, ∼12% reduction (Figure 2E). Tertiary cells decreased from 3 cells per ommatidium to 2.1 cells per ommatidium, ∼30% reduction (Figure 2F). Finally, bristle complexes decreased from 3 per ommatidium to 1.8 bristle complexes per ommatidium, which is ∼40% reduction (Figure 2G). This decrease in cell number during development corresponds to the smaller appearance of adult eyes and could reflect decreased proliferation or increased cell death. We previously published that GMR>DNhe2 expression increases stochastic proliferation in eye imaginal discs (Grillo-Hill et al., 2015), so we hypothesized that reduced cell number results from increased cell death.

FIGURE 2: Decreased numbers of secondary pigment cells, tertiary pigment cells, and bristle complexes with overexpression of DNhe2. (A) Pupal retinae immunolabeled for Armadillo (Arm, Drosophila beta-catenin) to highlight cell-cell junctions in w1118 and GMR>DNhe2 flies. Schematic diagrams (below) are tracings of micrographs. (B) Cell counts of primary pigment cells (highlighted in blue) in w1118 and GMR>DNhe2. (C) Cell counts of cone cells (highlighted in blue) in w1118 and GMR>DNhe2. (D) Expression of cone cell markers N-cadherin (N-cad, green) and Cut (magenta) in w1118 and GMR>DNhe2. (E) Secondary pigment cell counts (highlighted in blue) in w1118 (mean count = 6) and GMR>DNhe2 (mean count = 5.3). (F) Tertiary pigment counts (highlighted in blue) in w1118 (mean count = 6) and GMR>DNhe2 (mean count = 2). (G) Bristle complex counts (highlighted in blue) in w1118(mean count = 3) and GMR>DNhe2 (mean count = 1.8). Data are graphed as median values with interquartile ranges. Statistical significance was determined by unpaired t tests with Welch’s correction; N = 30 ommatidia from 5 individual flies for both genotypes.

Genetic interaction studies support DNhe2-induced autophagic cell death

We next investigated which mode of cell death (necrosis, apoptosis, or autophagy) removes cells with GMR>DNhe2 expression. Necrotic tissue in the Drosophila eye shows disrupted membranes and a lack of architecture in histological sections. We ruled out necrosis as our previous histological analysis of adult retinae in GMR>DNhe2 flies found that cell bodies are intact, and membrane-rich rhabdomeres are present and correctly patterned, allowing for the previously described planar cell polarity defects (Simons et al., 2009; Grillo-Hill et al., 2015). This suggests that apoptosis or autophagy is responsible for the missing cells. The secondary pigment cells, tertiary pigment cells, and bristle complexes cell types are collectively called interommatidial cells and are known to be selectively eliminated through apoptosis in larval and pupal development.

We predicted that if apoptosis is responsible for the reduction in cell number, then inhibiting apoptosis should rescue the GMR>DNhe2 rough eyes, restoring normal patterning and missing cells. We tested for genetic interactions with anti-apoptotic genes, including buffy, H99 deletion and overexpression of the viral caspase inhibitor p35 (Figure 3A). Previous studies show that overexpression of the anti-apoptotic gene buffy produces flies with wild-type eyes and blocks caspase-dependent cell death (Quinn et al., 2003). Overexpression of buffy alone using the GMRGAL4 driver showed no effect on retinal patterning and did not show a genetic interaction with GMR>DNhe2 (Figure 3A). The H99 deletion removes three genes, grim, reaper (rpr), and head involution defective (hid) (White et al., 1994). The genes grim, rpr, and hid inhibit the inhibitor of apoptosis proteins (IAP) proteins, which in turn inhibit caspases, hence the H99 deficiency indirectly blocks apoptotic cell death via IAP inhibition of caspases (Yu et al., 2002). H99 heterozygous flies do not display visible patterning errors. GMR>DNhe2 flies that were heterozygous for H99 showed a moderate suppression of the reduced eye size but retained patterning defects (Figure 3A). Finally, we tested p35, a viral caspase inhibitor and anti-apoptotic protein that completely inhibits apoptosis (Hay et al., 1994). Expression of p35 alone appeared wild-type, and when p35 and DNhe2 were coexpressed, the rough eye phenotype was suppressed (Figure 3A), causing the adult eye to appear larger and more organized. We saw this suppression consistently using three different p35 transgenic fly lines.

FIGURE 3: Caspase inhibition does not completely restore missing interommatidial cells. (A) Light micrographs showing adult eyes from the following genotypes (left to right): top row: GMRGAL4; GMR>buffy; GMRGAL4; H99; GMR>p35; bottom row: GMR>DNhe2; GMR>DNhe2+UAS-buffy; GMR>DNhe2; H99; GMR>DNhe2+UAS-p35. (B) Confocal micrographs of mid-pupal eyes immunolabeled for Arm to show cell-cell junctions in control (w1118), GMR>p35 alone, and GMR>DNhe2+p35. (C) Quantification of interommatidial cell number in w1118 (mean = 15 cells), GMR>DNhe2 (mean = 11.4 cells), GMR>p35 (mean = 25.3 cells), and GMR>DNhe2+p35 (mean 22.2 cells). Data are graphed as median values with interquartile ranges. Statistical significance was determined by unpaired t tests with Welch’s correction; N = 30 ommatidia from 6 individual flies for all genotypes except GMR>DNhe2 (25 ommatidia from 5 flies). Cell count data for GMR>DNhe2 are from data shown in Figure 1F.

We predicted that if apoptosis is the pH-sensitive mode of cell death, then inhibiting caspases should restore the missing cells observed in GMR>DNhe2 pupal eyes. We quantified cell number with p35 expression alone, or with GMR>DNhe2. In pupal eyes, expression of p35 alone did not disrupt patterning (Figure 3B, middle), and increased the number of interommatidial cells from 15 in control to 25.3, suggesting that normally ∼10 cells are removed through apoptosis (Figure 3, B and C). Coexpression of p35 and DNhe2 (Figure 3B, right) increased cell number to 22.2, compared with 11.4 in GMR>DNhe2 alone, which is a difference of ∼11 cells. These data show inhibition of caspases through expression of p35 does not completely rescue the missing cells induced by overexpression of DNhe2, as there are ∼3 fewer cells in GMR>DNhe2+p35 (22.2 cells) compared with GMR>p35 alone (25.3 cells). These data suggest that apoptosis does not cause the reduction in cell number with GMR>DNhe2 expression, since coexpression of p35 did not revert cell number to wild-type levels.

Autophagic cell death is increased with DNhe2 overexpression

We performed genetic interaction studies with known autophagy genes (Figure 4A). We tested mutations in several genes that facilitate autophagy: Atg1, which induces phagophore formation; Atg7 which facilitates vesicle completion; and Atg8a which regulates autophagosome maturation. The Atg13 loss-of-function allele showed no effect on eye development alone, but phenotypically suppressed the GMR>DNhe2 rough eye phenotype (Figure 4A). We decreased expression levels of Atg7 and Atg8a via RNAi, which showed similar effects. These interactions suggest autophagy may be enhanced with GMR>DNhe2 expression. We quantified effects on adult eye size using high-resolution photography and analysis of area (Figure 4B). We found that GMR>DNhe2 flies heterozygous for Atg13 showed a restoration of eye size (129.9 µm2, compared with 74.28 µm2 for GMR>DNhe2 alone shown in Figure 1), and were indistinguishable from their respective controls (Figure 4C).

FIGURE 4: Genetic interactions, phenotypic suppression, and marker analysis support autophagic cell death with GMR>DNhe2 expression. (A) Light micrographs showing adult eyes from the indicated genotypes. (B) High-resolution images of adult fly eyes used for eye size quantification for the following genotypes: w1118; GMRGAL4; GMR>DNhe2; GMRGAL4; Atg13; GMR>DNhe; Atg13. (C) Mean measured adult eye sizes were shown in Figure 1 for w1118(125.83 µm2, N = 7); GMRGAL4 (142.49 µm2, N = 9), and GMR>DNhe2 (74.28 µm2, N = 6), and indicated by showing median values with interquartile ranges. Mean measured adult eye sizes were GMRGAL4; Atg13 (146.4 µm2, N = 15); GMR>DNhe2; Atg13 (129.9 µm2, N = 19). (D) Confocal micrographs of 32H apf pupal eyes of the indicated genotypes, labeled with Armadillo to outline cells. (E) Quantification of interommatidial cell number of the indicated genotypes. Mean values from Figure 1F were: GMR>DNhe2 (mean = 11.42 cells, N = 25 counting areas from 5 flies). Measured mean values were: w1118(15.1 cells, N = 51 counting areas from 11 flies); Atg13 (14.5 cells, N = 30 counting areas from 8 flies); GMR>DNhe2; Atg13 (15.1 cells, N = 30 counting areas from 8 flies). (F) Confocal micrographs of wandering third larval eye imaginal discs expressing GFP::p62 from control or GMR>DNhe2-expressing flies. Panels show single-channel images for DAPI (blue), GFP::p62 (green), an overlay, and a maximum projection through the entire tissue. (G) Quantification of GFP::p62 intensity from control or GMR>DNhe2-expressing flies. Data are graphed as median values with interquartile ranges. Statistical significance was determined by unpaired t tests with Welch’s correction; N = 3 individual flies (3 Z-slices averaged per fly).

To quantify the effects on cell number, we counted interommatidial cells at the end of pattern formation in pupal eyes in flies heterozygous for Atg13 alone, or with expression of GMR>DNhe2. Flies heterozygous for Atg13 showed no defects in patterning or cell counts compared with w1118 control flies. Flies expressing GMR>DNhe2 and heterozygous for Atg13 appeared rescued with respect to cell number; however, some patterning errors in interommatidial cells and cone cells were evident (Figure 4D). Flies heterozygous for Atg13 are comparable to w1118 control flies. Flies heterozygous for Atg13 and expressing GMR>DNhe2 showed a nearly complete rescue of cell number, with an average cell count of 14.5 compared with 11.42 with GMR>DNhe2 alone, and 15 in control w1118 flies (Figure 4E).

To determine whether autophagy levels are increased, we used a ubiquitously-expressed p62 protein with an N-terminal-GFP tag (Kiss et al., 2020) alone or with GMR>DNhe2. p62 is an adaptor protein that recruits proteins targeted for lysosomal degradation, and accumulates when autophagy is inhibited or decreases when autophagy is induced (Bjørkøy et al., 2009, Chapter 12). p62 has proven to be a reliable marker for autophagy (Filimonenko et al., 2007). Wandering third larval instar eye imaginal discs were dissected, fixed, and labeled, and confocal images were collected of tissues expressing GFP::p62 alone or with GMR>DNhe2. GFP::p62 labeling is visible as distinct puncta throughout the third larval instar eye imaginal tissue, both in single Z-slices as well as in a Z-stack projection throughout the entire tissue (Figure 4F). We observed a significant decrease in GFP::p62 fluorescence intensity with expression of GMR>DNhe2 compared with w1118 control, with a normalized mean integrated density values of 0.2597 compared with 0.6200 in w1118 control (Figure 4G). Together, these data strongly support increased autophagic cell death with GMR>DNhe2 expression.

CLOSING THOUGHTS

Our research has shown for the first time that overexpression of DNhe2 results in reduced cell number, likely through autophagic cell death. Our established Drosophila models overexpress the sodium-proton exchanger DNhe2 in the developing eye, which results in increased pHi and a rough, disorganized, smaller eye in adult flies. We showed reduced interommatidial cell number with overexpression of DNhe2. Genetic interaction studies with apoptotic genes showed incomplete phenotypic suppression and did not rescue cell number. However, genetic interactions with autophagy genes suppressed the GMR>DNhe2 rough eye phenotype and rescued missing cells. Finally, expression of molecular markers supports increased autophagy with increased DNhe2 expression. These data support a new model where increased pHi is sufficient to induce autophagic cell death, which has implications for basic biological functions such as proteostasis, and for diseases with dysregulated autophagy like cancer.

One interesting consequence of our studies is the unraveling of pHi-regulated cellular processes during morphogenesis of the developing Drosophila eye. Our genetic interaction studies previously identified pH-regulated proteins like beta-catenin, where coexpression of the Drosophila orthologue Armadillo showed complete phenotypic suppression of the GMR>DNhe2-induced rough eye phenotype. In this study, we observed that distinct aspects of the GMR>DNhe2 phenotype can be genetically dissected, as removing one copy of Atg1 results in phenotypic suppression and restoration of the missing interommatidial cells. However, patterning errors are still visible in the pupal eyes, suggesting abnormalities in cone cell differentiation or survival and in planar cell polarity. These defects contrast with phenotypes seen with coexpression of p35, where extra interommatidial cells are present but have a minimal impact on tissue patterning due to reduced apical cell profiles. This supports the idea that increased pHi impacts different signaling pathways during Drosophila eye development, and that these effects can be disaggregated through genetic interaction studies and subsequently characterized through cell biological studies.

Finally, our data shed insights into the burgeoning field of autophagy research. Many studies in the autophagy field currently focus on specific protein or organelle targets that are degraded through autophagy. It is not known how these findings interface with the less-studied phenomenon of autophagic cell death, also called autosis; the classification of autophagy as a regulated mode of cell death is itself still controversial (Liu and Levine, 2015; Nah et al., 2020; Bai et al., 2023). While the same proteins and biological processes are required for these processes, it is unclear whether they represent a continuum, or distinct processes. Homeostatic autophagy may become increased or dysregulated beyond a threshold, resulting in autophagic cell death. Alternately, distinct signaling cues or signal strength modulation upstream of autophagosome formation may impact the ultimate outcome of surviving cellular stresses or induction of cell death. To further complicate matters, there are complex interconnections between autophagy proteins and induction of cell death through apoptotic or necrotic pathways (Maiuri et al., 2007; Nikoletopoulou et al., 2013; Saleem, 2021; Sorice, 2022). Hence, our model showing increased autophagy at higher pHi in the developing Drosophila eye may lend novel insight in these questions, especially as the Drosophila eye was a significant model system in elucidating apoptotic signaling.

Autophagy is known to be increased in cancer. Overall rates of cell death are decreased in tumors, which is thought to be partially due to inhibition of acid-activated caspases, preventing apoptosis (White et al., 2017). We hypothesize that higher pHi common to cancer cells induces autophagy to permit cells that are stressed or dying from metabolic dysfunction, oxidative stresses, or inflammation to fuel tumor growth by providing building blocks to the surrounding, healthier tumor cells.

MATERIALS AND METHODS

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Drosophila stocks

Flies were maintained on Nutri-Fly Bloomington Formulation media (Genesee, catalogue no. 66-113) and maintained at 25°C unless otherwise stated. Drosophila stocks were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN): w1118 (#3605); w; Kr/CyO, mef2>CD8::RFP (#26882Caspase inhibitor fly strains: w*; P{w[+mC] = UAS-p35.H}BH1 (UAS-p35-1, #5072); w*;P{w+mC= UAS-p35.H}BH2 (UAS-p35-2, #5073); w*; P{w+mC= GMR-p35}X-1, and (GMR>p35, #5774); Df(3L)H99, kniri-1 pp/TM3, Sb1 (H99, #1576); w*; P{w+mC= UAS-Buffy.S}E1 (UAS-Buffy, #32059); w*; Atg13 P{ry+t7.2= neoFRT}82B/TM3, Sb1 Ser1 (Atg13, #60732); y1 sc* v1 sev21; P{y+t7.7 v+t.1.8= TRiP.HMS01358}attP2/TM3, Sb1 (Atg7hms.RNAi, #34369); y1 v1; P{y+t7.7v+t1.8= TRiP.JF02787}attP2 (Atg7jf.RNAi, #27707); y1 sc* v1 sev21; P{y+t7.7 v+t1.8= TRiP.HMS01328}attP2 (Atg8a.RNAi, #34340). Additional stocks used: w;GMRGAL4 (outcrossed to a single copy when used as a control; gift from R. Cagan,); tub>GFP::p62 3.7M (GFP::p62; gift from G. Juhasz); w1118; GMR>DNhe2/CyO (Grillo-Hill et al., 2015).

Genetic interaction studies

Genetic interaction studies were performed in duplicate or triplicate sets of crosses. P0 flies were discarded when F1 larvae were visible. Adult eye phenotypes were documented using a stereomicroscope equipped with a Zeiss AxioCam MRc5 camera using Micro-Manager to acquire images and FIJI (NIH Image J, National Institutes of Health, Bethesda, MD) to adjust display settings.

High-resolution images of adult Drosophila eyes

Flies were generated through genetic crosses or taken from stocks, and the appropriate genotypes were selected by phenotypic markers. Flies were anesthetized with CO2 and placed into 70% ethanol in microcentrifuge tubes. Flies were imaged within 6 h, or stored at 4°C and imaged within 24 h. Flies were prepared for imaging by point mounting. Briefly, flies were glued laterally to the tip of a small piece of triangular cardstock, and pinned with a #2 or #3 entomological pin. For each pinned specimen, one eye was imaged. Images were acquired on a Canon EOS 6D Mark II camera, with a Canon EF 70-200 mm USM II telephoto lens with an attached Mitutoyo 20X Apo Microscope Objective. Focus stacked images of each specimen were produced using a Stackshot Controller and Rail system.

Adult eye size was quantified using ImageJ (National Institutes of Health, Bethesda, MD). An outline of the adult fly eye was made with the freehand selection tool. In the “Analyze” menu, we selected “Measure” to quantify area, mean, minimum, and maximum values. Statistical analyses were performed using Prism (Graphpad Software). Outliers were removed and statistical analysis used one-way ANOVA or Welch’s t test. Scaling was performed according to the imaging software, where 1 pixel = 3016 mm.

Larval and pupal eye dissection, fixation, and imaging

White prepupae were transferred into a humidified plastic dish and incubated at 25°C for 32 h after puparium formation (32H apf). Wandering third instar eye imaginal discs or 32H apf pupal eyes were dissected in PBS then fixed in fresh 4% paraformaldehyde (catalogue no. 15710, Electron Microscopy Sciences, Hatfield, PA) in PBS for 20 min, washed three times 10 min in PBT (PBS + 0.1% Triton X-100 [catalogue no. BP151-500 Sigma-Aldrich, St. Louis, MO]), and incubated at 4°C overnight with mouse anti-Armadillo antibody 1:10 (catalogue no. N2 7A1, Developmental Studies Hybridoma Bank, Iowa City, IO) in PBT + 5% normal goat serum (catalogue no. 642921, MP Biomedicals, Solon, OH). The next day, tissues were washed three times 10 min in PBT, incubated in goat anti-mouse AlexaFluor Plus 555 1:500 (catalogue no. A32727, Thermo Fisher Scientific) and Hoechst 33342 (1:10,000; catalogue no. H3570; Invitrogen, Thermo Fisher Scientific) for 2 h at room temperature in the dark, then washed three times 10 min in PBT, mounted in Prolong Gold or Prolong Glass Mountant (catalogue no. P10144 or no. P36961, Molecular Probes by Life Technologies) and cured overnight at room temperature in the dark then transferred to −20°C until imaging.

Image stacks were collected on a Zeiss LSM 700 confocal microscope using a 40x oil immersion objective (420462-9900-000, Zeiss Objective EC Plan-Neofluar 40x/1.30 Oil DIC M27); p62::GFP imaging or 63x oil immersion objective (420782-9900-000, Zeiss Objective Plan-Apochromat 63x/1.4 Oil DIC M27). High-resolution images (2048 × 2048) were acquired using the z-stack function (ZEN Software) on a high-resolution AxioCam microscope camera. Within each set of experiments, images were acquired using identical settings enabled by the “reuse settings” feature of the ZEN software. Images were adjusted and analyzed using FIJI Software (National Institutes of Health, Bethesda, MD). Control and experimental conditions were imaged on the same day.

p62::GFP imaging.

Within each set of experiments, images were acquired using identical settings enabled by the “reuse settings” feature of the ZEN software. The following pixel parameters were measured using FIJI in all image slices in all channels: mean, min, max, integrated density, and median. The slice with the highest integrated density for the Hoechst 33342 label was selected for analysis as this indicates a focal plane with the greatest amount of tissue. The slices immediately above and below were also quantified for technical replicates. The raw integrated intensity value for each channel was also averaged with their two neighboring slice raw integrated values. The GFP channel raw integrated mean intensity value was then normalized to the Hoechst 33342 channel raw integrated mean intensity value to control for tissue area in each slice. Control (tub>GFP::p62 3.7M) normalized values were then compared with the normalized values from the experimental group (w; GMRGAL4, UAS-DNhe2L4 A/tub GFP::p62). Display settings and crop margins for images in figures were identical for both genotypes.

Cell counts.

Cell counts were performed by identifying a central ommatidium and its six nearest neighbors, and drawing a hexagon through the center of the cone cells in the six neighbors. Each cell completely contained in this hexagon counts as one cell, and cells that are partially contained count as one-half cell. Cell counts were performed by at least 3 different people for reproducibility, and any discrepancies were discussed to consensus. One individual eye imaginal disk or pupal eye are taken from each animal.

We thank members of the Grillo-Hill and French labs for advice and useful critiques during monthly fly meetings. We thank Tim Andriese, Randy Kirschner, Kitty (Ngoc-Huong) Nguyen, Marco Parent, Jonny Shaloub, and Librado Veliz for excellent technical support. We are grateful to Dr. Fredrick Larabee for advice, support, and use of the high-resolution camera for adult Drosophila eye imaging. This work was supported by an NIH SC3GM132049 award (B.G.-H.), a CSUPERB New Investigator Grant Award (B.G.-H.), the Biological Sciences Department F. Albert and Dorothy Ellis Graduate Fellowship (J.P.), the San José State University Office of the Provost Undergraduate Research Award (B.D.), CSUPERB Howell Scholar Award (R.A.S), CSUPERB Presidential Award (A.W.), the Biological Sciences Department John and Betty Davison Fellowship (D.O.), and the CSUPERB Faculty-Graduate Student Research Collaboration Program (L.M.). Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. The DSHB Hybridoma Product N2 7A1 Armadillo developed by Eric Wieschaus was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

Abbreviations used:

apf after puparium formation

Atg autophagy-related

DNhe2 Drosophila sodium proton exchanger 2 (Na-H Exchanger)

GFP green fluorescent protein

GMRGAL4 Glass-multimerized reporter Gal4

NHE1 sodium proton exchanger 1 (Na-H Exchanger)

RNAi RNA interference

Reviewer Report

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E24-02-0058) on May 2, 2024.
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REFERENCES

Bai LWu QZhang XZhao Y (2023). Autosis as a selective type of cell death. Front Cell Dev Biol 11 , 1164681.37091978
Billes VKovács TManzéger ALőrincz PSzincsák SRegős ÁKulcsár PIKorcsmáros TLukácsovich THoffmann G, et al. (2018). Developmentally regulated autophagy is required for eye formation in Drosophila. Autophagy 14 , 1499–1519.29940806
Bjørkøy GLamark TPankiv SØvervatn ABrech AJohansen T (2009). Chapter 12 Monitoring autophagic degradation of p62/SQSTM1. In: Methods in Enzymology, Amsterdam, Netherlands: Elsevier. 181–197.
Cagan RLReady DF (1989). The emergence of order in the Drosophila pupal retina. Dev Biol 136 , 346–362.2511048
Cordero JJassim OBao SCagan R (2004). A role for wingless in an early pupal cell death event that contributes to patterning the Drosophila eye. Mech Dev 121 , 1523–1530.15511643
Das GShravage BVBaehrecke EH (2012). Regulation and function of autophagy during cell survival and cell death. Cold Spring Harb Perspect Biol 4 , a008813.22661635
Debnath JGammoh NRyan KM (2023). Autophagy and autophagy-related pathways in cancer. Nat Rev Mol Cell Biol 24 , 560–575.36864290
Filimonenko MStuffers SRaiborg CYamamoto AMalerød LFisher EMCIsaacs ABrech AStenmark HSimonsen A (2007). Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. J Cell Biol 179 , 485–500.17984323
Grillo-Hill BKChoi CJimenez-Vidal MBarber DL (2015). Increased H+ efflux is sufficient to induce dysplasia and necessary for viability with oncogene expression. eLife 4 , e03270.25793441
Hay BAWolff TRubin GM (1994). Expression of baculovirus P35 prevents cell death in Drosophila. Development 120 , 2121–2129.7925015
Khandia RDadar MMunjal ADhama KKarthik KTiwari RYatoo MIIqbal HMNSingh KPJoshi SK, et al. (2019). A comprehensive review of autophagy and its various roles in infectious, non-infectious, and lifestyle diseases: current knowledge and prospects for disease prevention, novel drug design, and therapy. Cells 8 , 674.31277291
Kiss VJipa AVarga KTakáts SMaruzs TLőrincz PSimon-Vecsei ZSzikora SFöldi IBajusz C, et al. (2020). Drosophila Atg9 regulates the actin cytoskeleton via interactions with profilin and Ena. Cell Death Differ 27 , 1677–1692.31740789
Liu YLevine B (2015). Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ 22 , 367–376.25257169
Maiuri MCZalckvar EKimchi AKroemer G (2007). Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8 , 741–752.17717517
Manent JBanerjee Sde Matos Simoes RZoranovic TMitsiades CPenninger JMSimpson KJHumbert PORichardson HE (2017). Autophagy suppresses Ras-driven epithelial tumourigenesis by limiting the accumulation of reactive oxygen species. Oncogene 36 , 5576–5592.28581519
Matsuyama SLlopis JDeveraux QLTsien RYReed JC (2000). Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2 , 318–325.10854321
Mohseni NMcMillan SCChaudhary RMok JReed BH (2009). Autophagy promotes caspase-dependent cell death during Drosophila development. Autophagy 5 , 329–338.19066463
Nah JZablocki DSadoshima J (2020). Autosis: a new target to prevent cell death. JACC Basic Transl Sci 5 , 857–869.32875173
Nikoletopoulou VMarkaki MPalikaras KTavernarakis N (2013). Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta 1833 , 3448–3459.23770045
Ouyang LShi ZZhao SWang F-TZhou T-TLiu BBao J-K (2012). Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif 45 , 487–498.23030059
Pérez-Sala DCollado-Escobar DMollinedo F (1995). Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease. J Biol Chem 270 , 6235–6242.7890761
Proskuryakov SYKonoplyannikov AGGabai VL (2003). Necrosis: a specific form of programmed cell death? Exp Cell Res 283 , 1–16.12565815
Quinn LCoombe MMills KDaish TColussi PKumar SRichardson H (2003). Buffy, a Drosophila Bcl-2 protein, has anti-apoptotic and cell cycle inhibitory functions. EMBO J 22 , 3568–3579.12853472
Ready DFHanson TEBenzer S (1976). Development of the Drosophila retina, a neurocrystalline lattice. Dev Biol 53 , 217–240.825400
Reshkin SJGreco MRCardone RA (2014). Role of pHi, and proton transporters in oncogene-driven neoplastic transformation. Philos Trans R Soc Lond B Biol Sci 369 , 20130100.24493748
Rock KLKono H (2008). The inflammatory response to cell death. Annu Rev Pathol 3 , 99–126.18039143
Saleem S (2021). Apoptosis, autophagy, necrosis and their multi galore crosstalk in neurodegeneration. Neuroscience 469 , 162–174.34166763
Simons MGault WJGotthardt DRohatgi RKlein TJShao YLee H-JWu A-LFang YSatlin LM, et al. (2009). Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization. Nat Cell Biol 11 , 286–294.19234454
Sorice M (2022). Crosstalk of autophagy and apoptosis. Cells 11 , 1479.35563785
Ulmschneider BGrillo-Hill BKBenitez MAzimova DRBarber DLNystul TG (2016). Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation. J Cell Biol 215 , 345–355.27821494
White KGrether MEAbrams JMYoung LFarrell KSteller H (1994). Genetic control of programmed cell death in Drosophila. Science 264 , 677–683.8171319
White KAGrillo-Hill BKBarber DL (2017). Cancer cell behaviors mediated by dysregulated pH dynamics at a glance. J Cell Sci 130 , 663–669.28202602
Wojtkowiak JWGillies RJ (2012). Autophagy on acid. Autophagy 8 , 1688–1689.22874557
Wolff TReady DF (1991a). The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave. Development 113 , 841–850.1726564
Wolff TReady DF (1991b). Cell death in normal and rough eye mutants of Drosophila. Development 113 , 825–839.1821853
Yu S-YYoo SJYang LZapata CSrinivasan AHay BABaker NE (2002). A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye. Development 129 , 3269–3278.12070100
Zhao LCui LJiang XZhang JZhu MJia JZhang QZhang JZhang DHuang Y (2016). Extracellular pH regulates autophagy via the AMPK–ULK1 pathway in rat cardiomyocytes. FEBS Lett 590 , 3202–3212.27531309
