Restoring calcium crosstalk between ER and mitochondria promotes intestinal stem cell rejuvenation through autophagy in aged Drosophila

Zhang, Yao; Ma, Peng; Wang, Saifei; Chen, Shuxin; Deng, Hansong

doi:10.1038/s41467-025-60196-4

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Published: 27 May 2025

Restoring calcium crosstalk between ER and mitochondria promotes intestinal stem cell rejuvenation through autophagy in aged Drosophila

Nature Communications volume 16, Article number: 4909 (2025) Cite this article

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Abstract

Breakdown of calcium network is closely associated with cellular aging. Previously, we found that cytosolic calcium (CytoCa²⁺) levels were elevated while mitochondrial calcium (MitoCa²⁺) levels were decreased and associated with metabolic shift in aged intestinal stem cells (ISCs) of Drosophila. How MitoCa²⁺ was decoupled from the intracellular calcium network and whether the reduction of MitoCa²⁺ drives ISC aging, however, remains unresolved. Here, we show that genetically restoring MitoCa²⁺ can reverse ISC functional decline and promote intestinal homeostasis by activating autophagy in aged flies. Further studies indicate that MitoCa²⁺ and Mitochondria–ER contacts (MERCs) form a positive feedback loop via IP3R to regulate autophagy independent of AMPK. Breakdown of this loop is responsible for MitoCa²⁺ reduction and ISC dysfunction in aged flies. Our results identify a regulatory module for autophagy initiation involving calcium crosstalk between the ER and mitochondria, providing a strategy to treat aging and age-related diseases.

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Introduction

The functional decline of stem cell activities in many tissues is a hallmark of aging. Intestinal stem cells (ISCs) are the only cell type capable of dividing in response to the physiological demands in the adult fly gut^1,2. As in mammals, epithelial turnover in the Drosophila intestine is also maintained by ISCs. During homeostasis, ISCs are mostly quiescent but can initiate a proliferative response to stress conditions such as infection, toxins as well as aging³. During aging, the intestinal epithelium undergoes dysplasia, which is characterized by ISC over-proliferation and mis-differentiation, where ISCs show mixed feature with the enteroblast (EB), the daughter cell^4,5.

Calcium is a versatile second messenger that is critical in regulating fundamental physiological functions. The concentration of Ca²⁺ varies between different subcellular compartments, which are established and maintained by Ca²⁺ channels and pumps^6,7. The ER is the largest intracellular calcium store and Ca²⁺ homeostasis in ER is achieved by channels that release Ca²⁺ from the ER lumen into the cytosol - including IP3Rs and RyRs - and mechanisms that return Ca²⁺ to the ER, such as SERCA pumps and store-operated Ca²⁺ entry (SOCE)^7,8. ER releases Ca²⁺ via IP3R and allows Ca²⁺ to passively enter the mitochondrial outer membrane (OMM) via VDAC1, a high conductance protein⁹. However, the inner mitochondrial membrane (IMM) is impermeable to Ca²⁺, and Ca²⁺ enters through the mitochondrial calcium uniporter (MCU) complex, which consists of the inner membrane channel MCU and regulatory subunits including MICU1 and EMRE^10,11. High Ca²⁺ concentrations at the mitochondrial intermembrane space activate MCU to mediate the entry of Ca²⁺ into the mitochondrial matrix, where Ca²⁺ stimulates the activities of tricarboxylic acid (TCA) cycle, β-oxidation and respiratory complexes to enhance ATP generation¹².

AMPK (AMPactivated protein kinase) is a cellular energy sensor that activated by falling energy status^13,14. In mammalian cell lines, reduction of MitoCa²⁺ by disruption of MERCs or by the Ca²⁺ chelator BAPTA-AM induced a bioenergetic crisis and activates AMPK-mediated (macro)autophagy to cope with energy depletion^15,16,17. Recently, we and others found that subcellular calcium level in cytosol (CytoCa²⁺) and mitochondria serves as critical coordinators during the transition of ISCs from quiescence to proliferation^8,18,19. This adaption failed in old ISCs, which have sustained higher CytoCa²⁺ and lack efficient MitoCa²⁺ uptake and have undergone a Warburg-like metabolic switch involving increased lactate production and reduced ATP levels, contributing to their deregulated proliferative activity^8,18. How MitoCa²⁺ was reduced and whether restoring MitoCa²⁺ can reverse the metabolic profile and age-related defects in ISCs remains largely unknown.

The MERCs, also known as mitochondria–associated membranes (MAMs), are the physical links between the ER and mitochondria, providing an interface for Ca²⁺ transfer^9,20. These structures are dynamic and highly heterogeneous among tissues and species²¹. MERCs establishment is regulated by multiple scaffold and tethering factors that include among others MFN2, IP3Rs-GRP75-VDACs, FIS1-BAP31, VAPB-PTPIP51, Mmm1, and SIGMAR1^22,23. Increasing evidence indicated that MERC is one of the membrane sources for autophagosomes and is the initiation site for autophagy²⁴. In multicellular organisms, the Atg1 complex (including ATG1, FIP200, Atg17 and Atg13) is targeted to the ER and recruits the class III (PI(3)P) kinase complex (including ATG6 and ATG14) for initiation and maturation of pre-autophagosomes upon autophagy induction^25,26. For instance, the omegasome marker DFCP1/ZFYVE1 (double FYVE domain-containing protein 1) and ATG14 was recruited to the MERCs during starvation²⁷. When MERCs were disrupted by MFN2 knockdown, ATG14 is no longer localized in the MERCs and the autophagosome formation is also inhibited²⁸, suggesting that MERCs play an indispensable role for autophagosome formation. A recent study has also shown that IP3R mediated Ca²⁺ transients at the outer surface of the ER triggers autophagosome initiation by promoting the formation of fluid-like FIP200 puncta²⁹. Whether the calcium oscillations generated between the ER and the cytosol, or between the ER and mitochondria via MERCs, are responsible for the initiation of autophagy, and how the oscillations are generated and maintained, remains unclear.

Accumulating evidence indicates that autophagy declines with age and that impaired autophagy is associated with stem cell aging³⁰. In mice, the regenerative capacity of muscle stem cells is maintained by basal autophagy, and its deregulation leads to premature entry into an irreversible senescent state³¹. Similarly, autophagy genes such as Atg5 and Atg7 are necessary for ISC maintenance and gut homeostasis³². In Drosophila, autophagy deficient ISCs show elevated DNA damage and cell cycle arrest during aging and lead to unlimited ISC proliferation or apoptosis^33,34. These results reveal the crucial role of autophagy in preserving proper stem cell function for the continuous renewal of the intestinal epithelium. Indeed, activation of AMPK is linked to improved intestinal homeostasis during aging and extended lifespan in Drosophila³⁵. How autophagy was impaired during aging was largely unknown.

Here, we found that MitoCa²⁺ influx retrogradely activates IP3R in MERCs, generating Ca²⁺ oscillations in MERCs that promote autophagy independent of AMPK. Genetic restoration of mitoCa²⁺ levels re-establishes the MitoCa²⁺-IP3R-MERCs feedback loop and delays ISC aging by autophagy.

Results

Deficient MitoCa²⁺ uptake contributes to ISC dysfunction in aged Drosophila

Our previous results showed that MitoCa²⁺ was reduced in aged ISCs, while CytoCa²⁺ remained higher^8,18. To further trace intracellular calcium pattern in ISCs during aging, genetically encoded calcium reporters targeted to mitochondria (UAS::MitoGCaMP3) or cytosol (UAS::GCaMP5) were expressed specifically in ISCs by combining esg::Gal4^ts with Su(H)::Gal80 (ISC-Gal4^ts). In ex vivo live-imaged midguts, CytoCa²⁺ level exhibited a progressive increase from around 30 days of age, whereas MitoCa²⁺ level decreased gradually (Fig. 1a and Supplementary Fig. 1a, b), implied that the breakdown of Ca²⁺ homeostasis started at middle age (The lifespan of flies under conventional conditions is estimated to be 60–70 days³⁶). The simultaneous monitoring of CytoCa²⁺ and MitoCa²⁺ in individual ISCs (UAS::MitoGCaMP3 for mitochondria, UAS::RCaMP1 for cytosol) revealed that both CytoCa²⁺ and MitoCa²⁺ in ISCs from young animals (3 d old) were significantly increased upon infection using Erwinia carotovora carotovora 15 (Ecc15), a mild enteropathogen that induces a transient proliferative response in the gut epithelium³⁷. In contrast, in ISCs from aged animals (30 d old), CytoCa²⁺ levels were further increased by infection, but MitoCa²⁺ levels remained lower than those in young animals (Fig. 1b). This suggested that the MitoCa²⁺ response to external stimuli is impaired in aged ISCs.

The components of mitochondrial calcium uniporter (MCU) complex are conserved in Drosophila, such as the pore-forming MCU (CG18769) and the negative gatekeeper MICU1 (CG4495)^38,39. The ISC specific overexpression of MCU (MCU^OE) or the knockdown of MICU1 (MICU1^RNAi) was sufficient to increase MitoCa²⁺ in young ISCs and also to restore MitoCa²⁺ in old ones (Fig. 1c and Supplementary Movie 1-6). Conversely, the overexpression of MICU1 (MICU1^OE) or the knockdown of MCU (MCU^RNAi) was sufficient to decrease MitoCa²⁺ in young ISCs (Supplementary Fig. 1c and Supplementary Movie 7, 8). Notably, MCU overexpression also resulted in a reduction in elevated CytoCa²⁺ in aged ISCs (Supplementary Fig. 1a). Furthermore, MCU overexpression was sufficient to restore MitoCa²⁺ levels, which were reduced and insensitive to Ecc15 infection in aged ISCs (Fig. 1d and Supplementary Fig. 1d). These results suggested that MitoCa²⁺ levels in ISCs are controlled by the MCU complex.

We then examined whether genetically restoring MitoCa²⁺ was sufficient to rescue aged ISCs dysfunction. NRE-eGFP is a genetic reporter of Notch signaling⁴⁰, which expressed exclusively in enteroblast cells (EB), while the percentage of NRE-eGFP positive ISCs significantly increased in aged midguts, indicating that the aged ISCs underwent mis-differentiation and displayed mixed feature of ISC and EB cells⁴, which can be restored by MCU^OE (Fig. 1e and Supplementary Fig. 1e). Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely used to study the self-renewal and differentiation abilities of ISCs in Drosophila^1,2,41. Two different strategies were exploited to monitor ISC activity: clones were induced in young animals (3 d) and the clone number was checked 7days (3 + 7) or 25 days (3 + 25) after clone induction (ACI), reflecting the ability of ISCs pool to form and maintain these clones. Alternatively, clones were induced in aged animals (25 d) and then checked 7 days ACI to check the clone forming ability of aged ISCs (Fig. 1f). Intriguingly, both the clone formation and maintenance capacity of ISC declined in aged animals, and which can be restored by MCU^OE (Fig. 1g). Taken together, these results indicated that old ISCs lack efficient MitoCa²⁺ uptake and the dysfunctions in aged ISCs can be improved by restoring MitoCa²⁺ levels.

Restoration of MitoCa²⁺ delays ISC aging and promotes intestinal homeostasis in aged flies

We then examined the role of MitoCa²⁺ in ISC aging and intestinal function. Cell cycle indicated by the FUCCI system (fluorescent ubiquitination-based cell cycle indicator)⁴² showed that the ratio of ISCs in G1 vs in G2/M stage was significantly increased in aged ISCs, indicating cell cycle dysregulation (Fig. 2a and Supplementary Fig. 2a). Consistent with previous studies^18,34, lactate level (indicated by Laconic, a FRET based Lactate reporter) and DNA damages (indicated by gamma-H2AX staining) were increased in aged ISCs (Fig. 2b and Supplementary Fig. 2b, c). Intriguingly, cell cycle dysregulation, lactate level as well as gamma-H2AX accumulation were all restored in aged ISCs with MCU^OE or MICU1^RNAi by the TARGET system⁴³ (Fig. 2a–c and Supplementary Fig. 2b–d) and also by a drug (RU486) inducible ISC/EB specific driver 5961::GS⁴⁴ (Supplementary Fig. 2e). Conversely, fewer number and reduced clone size (cell number within the clone) were observed in MICU1^OE (Supplementary Fig. 3a). Moreover, the knockdown of MCU in young ISCs (10 d) exhibited similarities to aged ISCs, including cell cycle dysregulation and DNA damage accumulations (Supplementary Fig. 3b, c). Age-related ISC dysfunction impairs midgut digestive functions, such as the loss of gut acid-base homeostasis and the decline of food intake and excretion^45,46. Intriguingly, ISC-specific overexpression of MCU was sufficient to restore the deterioration of gut pH homeostasis (Fig. 2d) and mitigated the decrease of food intake (Fig. 2e) and excretion (Fig. 2f) in aged guts. Together, these results indicated that restoring MitoCa²⁺ levels is sufficient to delay ISC aging and restore gut homeostasis in Drosophila.

Autophagy is activated by MitoCa²⁺ to delay ISC aging

MitoCa²⁺ level is essential for bioenergetics and our previous results showed that ATP level was reduced in aged ISCs¹⁸. Severe ATP depletion could lead to cell apoptosis or necrosis⁴⁷. Previous studies have shown that genetic inhibition of β-oxidation leads to ISC necrosis⁴⁸. Neutral lipid content (stained by LipidTOX) was largely unchanged by genetically inhibiting MitoCa²⁺ level in quiescent ISCs as well as in active ISCs upon infection (Supplementary Fig. 4a). The percentage of apoptotic (indicated by Cleaved Caspase-3 staining) or necrotic ISCs (indicated by Propidium iodide (PI) staining) was also largely unchanged in MCU^OE or MICU1^OE in aged ISCs (Supplementary Fig. 4b, c). We then tested whether MitoCa²⁺ delay ISC aging by regulating autophagy. Indeed, the number of autophagosomes (mCherry-Atg8 positive puncta) and acidic lysosomes (Lysotracker positive staining) in ISCs were significantly increased by MCU^OE or by MICU1^RNAi (Fig. 3a, b and Supplementary Fig. 5a, b). The mCherry-Atg8 positive vesicles in MCU^OE ISCs readily colocalized with the lysosome (indicated by GFP-Lamp1), implied that these autophagosomes were readily fused with lysosome (Supplementary Fig. 5c). Lysosomal activity can be assessed using two fluorescent dye indicators: Magic Red Cathepsin B (Magic Red) and DQ-BSA^49,50. More puncta of Magic Red and DQ-BSA were observed in ISCs by MCU^OE (Fig. 3c, d and Supplementary Fig. 5d, e).

Further track the autophagic flux by the tandem GFP-mCherry-Atg8a reporter in which GFP is rapidly quenched in an acidic environment⁵¹, showed that the percentage of autolysosomes (mCherry⁺/GFP^-, red puncta) was significantly increased in MCU^OE ISCs (Fig. 3e and Supplementary Fig. 5f). Consistently, accumulation of autophagosomes (mCherry⁺/GFP⁺, yellow puncta) were also increased in MCU^OE ISCs under Chloroquine (CQ, an inhibitor of autophagosome-lysosome fusion⁵²) treatment (Fig. 3f and Supplementary Fig. 5g). On the other hand, both of the increased mCherry-ATG8a- and Lysotracker-positive puncta induced by starvation were reduced in MCU^RNAi or MICU1^OE ISCs (Fig. 3g, h and Supplementary Fig. 5h, i). Furthermore, although these autophagosomes can readily colocalize with GFP-Lamp1 (Supplementary Fig. 5c), GFP signals of GFP-mCherry-Atg8a were retained in MICU1^OE ISCs (Supplementary Fig. 5f). Ref(²)P is the ortholog of P62 in Drosophila, which is a conserved autophagy substrate^53,54. The number of Ref(²)P-GFP puncta in ISCs was significantly reduced by MCU^OE, whereas it was increased by MICU1^OE (Fig. 3i and Supplementary Fig. 5j). Transmission electron microscope (TEM) images also indicated that the number of autophagosomes and/or autolysosomes in ISCs was significantly increased by MCU^OE or MICU1^RNAi (Fig. 3j and Supplementary Fig. 5k). These data indicated that autophagic activity was sensitive to MitoCa²⁺ in ISCs of fly intestine. In addition, overexpression of MCU in indirect flight muscles by IFM::Gal4 also promoted autophagy (indicated by increased mCherryAtg8+ puncta) in muscles (Supplementary Fig. 6a). Treatment of CQ can completely block MCU^OE induced ISC rejuvenation on the number of clones induced in old age (25 + 7) or maintenance of clones induced at young age (3 + 25) (Fig. 3k and Supplementary Fig. 6b). DNA damage suppressed by MICU1^RNAi in aged ISCs was blocked when autophagy was disrupted by Atg1^RNAi (Fig. 3l and Supplementary Fig. 6c). On the other hand, increasing autophagy by ISC specific Atg17 overexpression, a component in the ATG1 complex essential for phagophore formation⁵⁵, was sufficient to suppress DNA damages in aged ISCs (Fig. 3m and Supplementary Fig. 6d-f). Together, these results indicated that MitoCa²⁺-mediated anti-aging effect is dependent on autophagy.

MitoCa²⁺ promotes autophagy by activating IP3R-mediated Ca²⁺ release from the ER

We then attempted to elucidate the mechanism by which MitoCa²⁺ stimulates autophagy. Autophagosome initiation in ISCs, as indicated by antibody staining against endogenous Atg8a^56,57 (Fig. 4a), as well as by genetic reporters for Atg13 in the Atg1 complex, Atg6 in the PI(3)P kinase complex, Atg9 for autophagosome membrane source⁵⁸ and ZFYVE1 in omegasomes, was robustly increased by MCU^OE (Fig. 4b and Supplementary Fig. 7a-d). Endocytosis (indicated by the levels of Rab5 and Rab7, markers for early and late endosomes respectively), was mostly unaffected by MCU^OE in ISCs (Supplementary Fig. 7e–g). AMPK is a central regulator of autophagy¹³. However, AMPK activity, indicated by AMPK-SPARK (a phase separation dependent genetic reporter of AMPK activity)⁵⁹ and downstream mTOR activity (shown by phosphor-4EBP staining) was largely normal in MCU^OE ISCs (Supplementary Fig. 8a, b). Consistently, the cell size of ISCs was largely unchanged by genetically regulating MitoCa²⁺ levels (Supplementary Fig. 8c). Meanwhile, although autophagic activity in ISCs (indicated by immune-staining against endogenous p62⁶⁰) was found to be both sensitive to AMPK and MitoCa²⁺ levels, epistatic analysis indicated that the inhibition of AMPK activities suppresses autophagy mediated by MitoCa²⁺, and vice versa (Supplementary Fig. 8d). Excessive ROS level can activate autophagy through JNK(c-Jun N-Terminal Kinase)⁶¹. However, no obvious increase of mitochondrial ROS (H₂O₂) level (shown by a genetic ROS sensor MitoRoGFP2_Orp1¹⁸), while JNK activity (indicated by TRE-RFP⁶²) was even reduced by MCU^OE in ISCs (Supplementary Figs. 8e, f).

IP3R, instead of RyR, is the main calcium release channel in the ER of Drosophila ISCs^7,8 and Itpr encodes the sole homolog of IP3R in fly genome⁶³. MitoCa²⁺ in ISCs was inhibited by Itpr knockdown (Itpr^RNAi), whereas increased by Itpr overexpression (Itpr^OE) (Fig. 4c and Supplementary Fig. 9a), indicating that MitoCa²⁺ level was coupled with IP3R-mediated Ca²⁺ release from the ER.

Since IP3R was shown to trigger Ca²⁺ oscillations on the ER outer membrane and induce autophagosome biogenesis²⁹, we then explore whether MitoCa²⁺ can promote autophagy by eliciting Ca²⁺ oscillations on the ER outer membrane (Fig. 4d). ER-GCaMP6-210, a sensitive reporter of Ca²⁺ in the ER lumen (ERCa²⁺)⁶⁴, was utilized to reflect the activity of IP3R. Surprisingly, increasing MitoCa²⁺ in ISCs by MICU1^RNAi was sufficient to decrease ERCa²⁺ levels, which was blocked when Itpr was simultaneously silenced, indicating that increasing MitoCa²⁺ promoted Ca²⁺ release from the ER matrix by activating IP3R (Fig. 4e). Meanwhile, we generated an Itpr-HA knock-in transgene by CRISPR-Cas9 mediated homologous recombination (Supplementary Fig. 10a) to test whether its protein level or subcellular localization were altered by MitoCa²⁺ level. Indeed, Itpr-positive signals (indicated by anti-HA staining) form obvious puncta which are closely associated with KDEL-GFP (Supplementary Fig. 10b) in ISCs. Intriguingly, these puncta were significantly increased by MCU^OE in ISCs, further indicated that MitoCa²⁺ promotes IP3R activity (Supplementary Fig. 10c). Autophagy induced by starvation or MICU1^RNAi in ISCs (quantified by the number of autophagosomes and Lysotracker) was also diminished by Itpr^RNAi (Fig. 4f, g and Supplementary Fig. 9b–g), while overexpressing Itpr promoted ISCs autophagic activity (Fig. 4h and Supplementary Fig. 11a, b). On the other hand, DNA damage in aged ISCs suppressed by MICU1^RNAi was blocked by Itpr^RNAi (Fig. 4i and Supplementary Fig. 11c), and Itpr^OE was sufficient to suppress DNA damage in aged ISCs (Fig. 4j and Supplementary Fig. 11d). These results suggested that an increase in mitochondrial Ca²⁺ promotes autophagy and delays ISCs aging by retrogradely activating Ca²⁺ release from the ER via IP3R.

MERCs-mediated calcium crosstalk between the ER and mitochondria involved in MitoCa²⁺-mediated autophagy in ISCs

MERCs as the physical links between the ER and mitochondria, not only serve as a ground for dynamic Ca²⁺ transfer from the ER to mitochondria⁶⁵, but also for autophagic regulation²⁸. We then tested whether MERCs is involved in the regulation of MitoCa²⁺-mediated autophagy. The physical contacts between mitochondria and ER, visualized by DsRed-targeted mitochondrial outer membrane (OMM) and KDEL-GFP labeled ER, were observed in ISCs and were enhanced by MCU overexpression (Supplementary Fig. 12a). To monitor MERCs in ISCs, we generated a transgenic fly expression MAMtracker-Green (MAM-GFP), a dimerization-dependent GFP (ddGFP) based reporter⁶⁶, to detect reversible changes in MERCs structure under the control of UAS-Gal4 system. When overexpressed by ISC-Gal4^ts, the MAM-GFP positive puncta were adjacent and partially colocalized with mitochondria and ER and autophagosomes (Supplementary Fig. 12b–e). Meanwhile, the number of MAM-GFP positive vesicles in ISCs is sensitive to genetic manipulations of known MERCs components. For instance, genetically knocking down Grp75 (CG8542), or Mmm1 (CG13838, a conserved component of MAM also participate in phospholipid trafficking in yeast⁶⁷) was sufficient to reduce MERCs structure, while overexpressing Grp75 or human VAPB (hVAPB) was sufficient to increase MERCs (Fig. 5a), indicating that the MERCs structures are controlled by the conserved tethering complex in Drosophila and MAM-GFP is a sensitive tool to quantify the MERCs dynamics in ISCs. As expected, MitoCa²⁺ in ISCs was reduced when MERCs were genetically disrupted by Grp75^RNAi or Mmm1^RNAi, but increased when MERCs were augmented by Grp75 or hVAPB overexpression (Fig. 5b and Supplementary Fig. 13a), indicating that MitoCa²⁺ levels are controlled by MERCs in ISCs. Surprisingly, increasing MitoCa²⁺ by MCU^OE promoted MERCs formation, as indicated by the number of MAM-GFP and structures observed under TEM microscope (Fig. 5c–e and Supplementary Fig. 13b). Moreover, Itpr knockdown profoundly suppressed the number of MAM-GFP puncta induced by MICU1^RNAi, whereas Itpr^OE was sufficient to increase MERCs (Fig. 5f and Supplementary Fig. 13c), indicating that increasing MitoCa²⁺ promoted MERCs structures by activating IP3R. Intriguingly, autophagy induced by MitoCa²⁺ was suppressed by Mmm1^RNAi or Grp75^RNAi (Fig. 5g, h and Supplementary Fig. 14a, b), while increasing MERCs by overexpressing Grp75 or hVAPB was sufficient to promote autophagy (Fig. 5i, j and Supplementary Fig. 14c, d). Increasing MERCs by expressing an artificial tether, Tether-RFP⁶⁸, was also sufficient to increase autophagy in ISCs as indicated by Atg8a positive puncta (Fig. 5k). Taken together, these data indicated that MERCs-mediated calcium crosstalk is critical for MitoCa²⁺-mediated autophagy in ISCs.

MERCs are critical for MitoCa²⁺-mediated rejuvenation of aged ISCs

We then investigated the role of MERCs in MitoCa²⁺-mediated rejuvenation of aged ISCs. Genetical upregulation of MERCs was sufficient to restore MitoCa²⁺ levels, which were reduced and insensitive to Ecc15 infection in aged ISCs (Fig. 6a, b and Supplementary Fig. 15a, b). Furthermore, DNA damages in aged ISCs suppressed by MCU^OE were blocked by Mmm1^RNAi (Fig. 6c and Supplementary Fig. 16a). Lactate levels suppressed by MICU1^RNAi in aged ISCs were also increased by Grp75^RNAi (Fig. 6e and Supplementary Fig. 16d). Intriguingly, DNA damages, lactate levels and mis-differentiation were all restored in aged ISCs by hVAPB^OE or Grp75^OE (Fig. 6d–f and Supplementary Fig. 16b–d). Moreover, overexpression of Tether-RFP was also sufficient to reduce DNA damages in aged ISCs (Fig. 6g). Taken together, these data indicated that MERCs are critical for MitoCa²⁺-mediated rejuvenation of aged ISCs.

Breakdown of ER-mitochondria calcium crosstalk triggers ISCs aging due to defective autophagy

We then explore how MitoCa²⁺ was reduced in aged ISCs. Expression levels of components in the MCU complex, such as MCU, MICU1 and EMRE, were largely unchanged in the whole intestine at different ages, as shown by RT-qPCR (Supplementary Fig. 17a). Furthermore, we found that the expression levels of some calcium-associated channels (PMCA, Stim, Orai, IP3R, RyR, VDAC, MCU, MICU1 and EMRE) and mitochondria-ER contact tethers (Mfn2, Grp75, VAPB, Mmm1) were largely unchanged in old ISCs compared to young ISCs by RNA-seq dataset analysis (GSE157794, Supplementary Fig. 17b). Then we hypothesized that dysfunctional IP3R and/or MERCs lead to decreased MitoCa²⁺ in aged ISCs. Intriguingly, compared to young ISCs, despite no significant changes in ERCa²⁺ levels, ERCa²⁺ responded poorly to starvation in aged ISCs (Fig. 7a and Supplementary Fig. 17c), indicating that the activity of IP3R was impaired in aged ISCs. Meanwhile, the number and intensity of MAM-GFP puncta in aged ISCs were lower than those in young ISCs under both feeding and starvation conditions (Fig. 7b, c and Supplementary Fig. 17d), suggesting that the structure of MERCs was damaged in aged ISCs. These results showed that the breakdown of IP3R-mediated Ca²⁺ crosstalk in MERCs is critical for MitoCa²⁺ reduction in aged ISCs.

**Fig. 7: The breakdown of the ER-mitochondria calcium crosstalk triggers ISCs aging due to defective autophagy.**

On the other hand, more p62 aggregates were observed in aged ISCs than those in young ones (Fig. 7d and Supplementary Fig. 18a, b). The number of mCherryATG8a+ puncta in aged ISCs were lower than those in young ISCs under both feeding and starvation conditions (Fig. 7e and Supplementary Fig. 18c). Notably, unlike young ISCs, starvation failed to induce an increase of mCherryATG8a+ puncta in aged ISCs (Fig. 7e and Supplementary Fig. 18c). These results showed that autophagy was impaired and autophagic machinery was less sensitive to starvation in aged ISCs. Surprisingly, although autophagic activity was reduced, AMPK activity (indicated by AMPK-SPARK positive puncta) was actually increased in aged ISCs (Fig. 7f and Supplementary Fig. 19a), which may be due to ATP level drop caused by reduced MitoCa²⁺ in aged ISCs¹⁸. Indeed, increasing MitoCa²⁺ by overexpressing MCU downregulated AMPK activity in aged ISCs (Fig. 7f and Supplementary Fig. 19a), suggesting that autophagic failure in aged ISCs is not due to the suppression of AMPK.

Interestingly, increasing MitoCa²⁺ by overexpressing MCU was sufficient to restore IP3R activity, MERCs as well as autophagy in aged ISCs (Fig. 7a–e, and Supplementary Fig. 17c, d, 18a–c). Similarly, overexpression of Itpr or hVAPB was sufficient to promote autophagic activity in aged ISCs (Fig. 7g and Supplementary Fig. 19b). These results indicated that MitoCa²⁺, IP3R and MERCs form a positive feedback loop and the breakdown of the loop triggers ISC aging through autophagic failure, even with elevated AMPK activity (Fig. 7h).

Discussion

Inter-organelle communication in eukaryotic cells is spatially and temporally organized and display a transient dynamic pattern responds to pathophysiological conditions⁶⁹. MERCs, as the platforms between ER and mitochondria, actively involved in processes such as lipid trafficking, calcium crosstalk, redox homeostasis, and autophagy⁷⁰. This is corroborated by the growing body of evidence indicating that MERCs play a pivotal role in regulating the aging process and age-related diseases⁷¹. However, the mechanisms governing the number, composition, and function of MERCs, as well as the factors that disrupt their regulation during aging, remain largely unelucidated.

Here we found that subcellular Ca²⁺ homeostasis is disturbed in ISCs of aged flies: Ca²⁺ levels in the ER lumen and cytosol are maintained at higher levels, while those in the mitochondria are reduced. Compromised autophagy is a common feature of aging across diverse species⁷². In mammalian cell lines, the pro- or anti-autophagic effect of Ca²⁺ depends on the context, kinetics, amplitude or spatial extent. For example, many studies have shown that increased Cytosolic Ca²⁺ signals can activate AMPK through CaMKKβ by phosphorylating pThr172^73,74. Activated AMPK then promotes autophagy by inhibiting mTORC1 or controversially by phosphorylating ULK1^75,76. While other studies have also shown that the elevation of CytoCa²⁺ increases protein phosphatase4, which inactivates AMPK through dephosphorylation of pThr172⁷⁷. Reduced MitoCa²⁺ can activate AMPK due to energy deprivation⁷⁸. A recent study showed that a temporal decrease in ATP during mitosis would activate AMPK, which can activate MCU through phosphorylation⁷⁹. In this scenario, the AMPK/MCU couple helps to maintain MitoCa²⁺ level and energy status. However, we found AMPK activity was even increased in aged ISCs, the impaired autophagy and poor sensitivity to starvation was due to the disruption of the positive feedback loop consisting of MitoCa²⁺, MERCs via IP3R. Our results further indicated that AMPK and the MitoCa²⁺/IP3R/MERCs cascade are mutually required for autophagy induction. These results indicated that these two modules function in parallel to regulate autophagy induction in ISCs.

Based on the results, we propose a model in which the Ca²⁺ homeostasis in MERCs is maintained by a balance between Ca²⁺ release from the ER and Ca²⁺ uptake by mitochondria. Forced MitoCa²⁺ uptake would cause temporal drop of Ca²⁺ in MERCs, which activates IP3R and cause replenishment of Ca²⁺ in MERCs and forms calcium oscillations (Fig. 7h). How IP3R detect local Ca²⁺ drops in MERCs? IP3Rs are unusually large proteins, comprising tetramers of closely-related subunits, and each with about 2700 amino acid residues⁸⁰. In addition to IP3, Ca²⁺ was shown also to regulate IP3R with a biphasic concentration dependence⁸¹; modest increases in CytoCa²⁺ levels enhancing responses to IP3, while higher levels of CytoCa²⁺ were inhibitory for IP3R⁸². It’s possible that forced MitoCa²⁺ influx cause Ca²⁺ drop in the microdomain, which was sensed by IP3R and lead to its activation. IP3R activity is also regulated by its subcellular localization and post-translational modifications⁸³. For instance, Cdk5, a kinase localizes in MERCs, phosphorylates IP3R1 at Ser⁴²¹ and regulates ER Ca²⁺ transfer to the mitochondria in mouse embryonic fibroblasts (MEFs)⁸⁴. Therefore, MitoCa²⁺ may also regulate IP3R activity through local Ca²⁺-dependent kinases or phosphatases in MERCs. Here, we found that Ca²⁺ oscillations in MERCs mediated by mitochondrial Ca²⁺ and IP3R form a positive feedback loop to regulate autophagy. Breakdown of this loop is a driven force for ISC aging and genetically restoring the loop is sufficient led to ISC rejuvenation through AMPK-independent autophagy. Given that the components are conserved, it is plausible that the MERCs-specific Ca²⁺ oscillations also function as a module for autophagy regulation in mammals. Our results provide a strategy to tackle aging and aging-related diseases.

Methods

Fly strains and reagents

Flies were cultured at 25 °C and 60% humidity with a 12 h light/dark cycle on standard yeast/molasses-based food unless otherwise indicated (recipe for 1 L food: 13.8 g agar, 22 g molasses, 75 g malt extract, 18 g dry inactivated yeast, 80 g corn flour, 10 g soy flour, 6.25 ml, propionic acid, 2 g methyl 4-hydroxybenzoate, 7.2 mL ethanol and water up to 1 L).

The following strains were obtained from the Bloomington Drosophila Stock Center: w¹¹¹⁸ (BL3605), tub::GAL80ts (BL7108), UAS-mCherry (BL25774), UAS-nls-GFP (BL4775), UAS-tdTomato (BL36327), UAS-MitoDsRed (BL93056), UAS-20XUAS-RCaMP1f (BL52210), UAS-tdTomato-P2A-GCaMP5G (BL80079), UAS-MCU (BL16357), FRT40A (BL5759), UAS-GFP.E2f1.1-230,UAS-mRFP1.CycB.1-266 (BL55099), NRE::EGFP.S (BL30727), UAS-MitoRoGFP2_Orp1 (BL67667), UAS-GFP-mCherry-Atg8a (BL37749), UAS-mCherry-ATG8a (BL37750), UAS-Itpr^RNAi (BL25937), UAS-Itpr (BL30742), UAS-ER.GCaMP6-210 (BL83293), UAS-Hsc70-5.FLAG.HA.V (BL95230), UAS-hVAPB.HA (BL82230), UAS-GFP-Lamp1 (BL42714), UAS-GFP-Rab5 (BL43336), UAS-GFP-Rab7 (BL42705), UAS-ATG17(BL91226), UAS-GFP-KDEL (BL9899), UAS-RFP-KDEL (BL30909), UAS-AMPK^CA (BL32110), UAS-AMPK^DN (BL32112). UAS-MICU1^RNAi (v110456), UAS-MCU^RNAi (v110781), UAS-Hsc70-5^RNAi (v47745) were obtained from Vienna Drosophila Stock Center (VDRC). UAS-MICU1.ORF.3xHA (F000962) were obtained from FlyORF. UAS-ATG1^RNAi (THU2357), UAS-mCD8-GFP (THJ0080), UAS-Mmm1^RNAi (THU5442) were obtained from TsingHua University Fly Center. esg::GAL4 was a gift from S.Hayashi, Gbe::GAL80 from S.X.Hou, UAS-MitoGCaMP3 from F.Kawasaki, hsFlp;FRT40,tub::GAL80;tub::GAL4,UAS-GFP from B.Ohlstein, UAS-Laconic from O.Morris, UAS-GFP-Ref(²)P from T.Neufeld, TRE-RFP from D.Bohmann, UAS-AMPK-SPARK from H.Huang, UAS-GFP-ATG6, UAS-GFP-ATG9, UAS-GFP-ZFYVE1, UAS-Tether-RFP from C.Tong, UAS-mCherry-ATG13 from S.J.He. A full list of fly genotypes used in each figure panel is available in Supplementary Table 1.

Drosophila TARGET system, aging and MARCM induction

We used the TARGET system⁴³ and the Gene Switch system⁴⁴ for conditional expression of UAS-linked transgenes. Conditional expression in ISCs + EBs was achieved by combining ISC/ EB driver esg::Gal4 (esg::G4) with Gal80^ts (G80^ts), a temperature-sensitive Gal4 inhibitor driven by a tubulin promoter (Tub::G80^ts). For conditional expression in only ISCs, esg::Gal4 and Tub::Gal80^ts were combined with Su(H)GBE-G80, which drives the expression of a temperature-insensitive version of Gal80 in EBs. When using the TARGET system, flies were cultured at permissive temperature (18 °C) to avoid transgene expression before being shifted to the restrictive temperature of 29 °C for the indicated time to induce transgene expression. For experiments using our aging model, flies were kept at 18 °C for the indicated time before shifting to 29 °C for 4 days to induce transgene expression. Food was changed every 2–3 days.

For experiments with the 5961 GeneSwitch system, flies were raised for 3 days on normal food post eclosion and then kept for 4 days on food supplemented with RU486 at 5 mg/ml (mifepristone; Calbiochem #475838) dissolved in ethanol, or with ethanol alone (control).

For MARCM clone induction, flies were heat-shocked at 37 °C for 1 h. Following heat-shock, flies were kept at 25 °C and dissected after the indicated time. For starvation treatments, flies were transferred to fresh vials containing 5% sucrose at 29 °C for 8 h prior to dissection. In all experiments, only female posterior midgut was analyzed.

Cloning and transgenic fly generation

MAMtracker-Green (MAM-GFP) was amplified from pMAMtracker-Green, which was a gift from Koji Yamanaka, using primers Fw: GAATTCatggctattcaactcagg and Rev: CTCGAGttacttcatgaaaagacc. Amplified fragments were then cloned into pUAST-attB vector using EcoRI and XhoI restriction enzyme sites. For Itpr-HA knock-in transgenic fly, the CRISPR-Cas9-based strategy was utilized to generate the homology-mediated knock-in allele of Itpr (CG1063, the fruit fly IP3R homolog). Briefly, the sgRNA sequence in the CDS of CG1063 was designed by CRISPOR and cloned into the pEASY-Blunt vector. Three set of primers were designed for the donor plasmid: the primer pair for the homologous arms spanning the stop codon of CG1063 and the primer for the linker plus 3xHA sequence. The sequences were then sequentially cloned into the pMD™18-T vector. In this way, the donor plasmid was knocked into the CG1063 coding sequence just before the stop codon in frame. The sgRNA and donor construct were then microinjected into fly (BL78781) embryos. Positive transgenic lines were verified by sequencing.

The primers for sgRNA CG1063 sg-F 5’ tacagactcactgaaacggcGTTTTAGAGCTAGAAATAGC 3’ and CG1063 sg-R 5’ gccgtttcagtgagtctgtaCGACGTTAAATTGAAAATAGG 3’. The primers for donors CG1063 Lpart-FCGACGATATCTCTAGctaattaaaattataactgactttcagttactttaccact, CG1063 Lpart-RAGTCCGGActgaaacggcaggaggc, CG1063 linker+3x HA-F gtttcagTCCGGACTCAGATCTGGCAGC, CG1063 linker+3x HA-R cagactcaAGCGTAGTCTGGGACGTCG, CG1063 Rpart-F CTACGCTtgagtctgtaagaaatagaatagaatactaattttggcac and CG1063 Rpart-R ATGACCATGATTACGCCTTCAATCCACTCCACGC. The transformation injection was performed in the Core Facility of Drosophila Resource and Technology, CAS (Shanghai).

Ecc15 infection and Chloroquine treatment

Bacteria Ecc15 were cultured overnight at 30 °C in LB medium. A concentrated bacterial pellet (A_600 nm of ~200), which was centrifuged from 1-ml overnight culture media, was resuspended 1:1 with 5% sucrose to yield an Ecc15 infection solution. Flies were starved at 29 °C for 2 h and then transferred to fresh vials covered with a filter paper (WhatmanTM) and either 150 μL of Ecc15 infection solution or 5% sucrose (mock) solution. Flies were infected for 16 h before dissection for Ca²⁺ imaging.

As previously described⁸⁵, for CQ treatment (Chloroquine diphosphate salt solid, #C6628, Sigma-Aldrich), tubes containing food supplemented with 100 μM CQ were used. Briefly, for MARCM clones, flies were starved at 25 °C for 2 h to synchronize feeding and transferred to tubes containing food supplemented with 100 μM CQ for 2 days before heat-shock. Following heat-shock, flies were kept at 25 °C and transferred to tubes containing supplemented 100 μM CQ every 2days and dissected after the indicated time.

Bromophenol blue assay

The bromophenol blue assay was used to detect the pH change in the midgut as previously described⁴⁵. Briefly, 100 µl of 2% Bromophenol blue sodium (Sigma, B5525) was added to the food surface and several holes were poked to allow the Bromophenol blue solution completely absorbed by the food. Images were taken immediately after 16 h.

Cafe assay and fly excretion measurement

For Café assay, a 5 µl capillary containing liquid food (10% yeast, 10% sucrose, and blue dye) was inserted into a cotton plug of 6 cm long vial, and 1 cm high 1% agar was at the vial bottom to keep the moisture. The food intake was recorded every 12 h and the capillary was changed every 24 h. For fly excretion measurement, flies were dry starved for 2 h and put into Bromophenol blue vial food for 24 h, and then the deposits on the vial wall were imaged and quantified.

RNA isolation and quantitative real-time PCR

RNA was isolated from guts of 15 flies using TriZol reagent (#15596026 Life technologies). Around 20 ug RNA then were reversely transcribed using 5X All-In-One RT Mastermix With Accurt Kit (#G592, Applied Biological Materials) according to the manufacturer’s instructions. All of the reactions were performed in triplicate in a CFX96TM Real-Time System (Bio-Rad Laboratories). The following primers pairs were used: MCU, 5′-GTCTCGCCCTGCGTTTGG-3′ and 5′-CGAAGCTTCTGTGCTGCTG-3′; MICU1, 5′-GTGGCCATGGTCAATCTTTC-3′ and 5′-TTGTTGCTGAGTTGGTTGTCA-3′; EMRE, 5′-ACATGTCCAGCGTGTACTTTC-3′and 5′-GGTATGACGGCACAGAAGATG-3′; Rp49, 5′-GCCGCTTCAAGGGACAGTATCTG-3′and 5′-AAACGCGGTTCTGCATGAG-3′. Expression was normalized with Rp49 and quantification was performed using the comparative CT method.

Transmission electron microscopy

Guts were dissected in PBS, fixed with 2.5% glutaraldehyde, and washed three times with 0.1 M phosphate buffer at 4 °C, post-fixed with 1.0% OsO4 for 1 h at 4 °C, washed with phosphate buffer, dehydrated in ethanol, and embedded in Spurr resin. Thick section was stained with Toluidine blue and ultra-thin sections of the embedded guts were double-stained with uranyl acetate and lead citrate and examined with a JEM1400 transmission electron microscope (TEM).

Immunostaining and florescence microscopy

Female fly midguts were dissected in PBS and fixed for 20 min at room temperature in 4% formaldehyde. After fixation, the samples were washed with PBS containing 0.5% bovine serum albumin and 0.1% Triton X-100 (PBST) for 1 h. Appropriate primary antibodies were added to the samples and incubated at 4 °C overnight. Midguts were washed and then incubated with secondary antibodies for 4 h at room temperature. Samples were mounted in Mounting Medium (#S2100, Solarbio). The following antibodies or dyes were used: rabbit anti-Histone H2AvD pS137 (1:200; #600-401-914, Rockland); rabbit anti-Ref(²)P (1:500; #ab178440, Abcam); rabbit anti-Atg8a (GABARAP⁵⁷) (1:500, #13733S Cell Signaling Technology); rabbit anti-HA (1:250; #3724 T, Cell Signaling); rabbit anti-cleaved Caspase 3 (1:100; #Y407558, Applied Biological Materials); rabbit anti-phospho-4EBP1 (1:200; #2855S, Cell Signaling); rabbit anti-GFP (1:500; #PABG1, Chromotek); Alexa-488-conjugated donkey anti-rabbit secondary antibody (1:1000;#A21206, Molecular Probes); Goat-α-rabbit-IgG(H + L)-Cy3 secondary antibody (1:1000; #A10520, Molecular Probes); Goat-α-rabbit-IgG(H + L)-Cy5 secondary antibody (1:1000; #A10523, Molecular Probes); and Hoechst 33342 (0.1 μg ml^-1, 6 min incubation; #H1399, Invitrogen). All images were taken on a Zeiss Axio Imager M2 fluorescence microscope equipped with a 40× Plan-Apochromat oil-immersion objective lens (Zeiss) or an Olympus IXplore SpinSR confocal microscope equipped with a 100×1.5 NA oil-immersion objective lens (Olympus) and processed using Adobe Photoshop, Illustrator and Image J.

LipidTOX, Lysotracker, propidium iodide, Magic Red and DQ-BSA staining

LipidTox staining was performed as previously described⁸⁶. In brief, fixed guts were washed 3 times for 5 min in PBST, then directly stained with LipidTOX™ solution (1:200, # H34476, Invitrogen) diluted in PBST, mounted in Mounting Medium with Hoechst and analyzed on a confocal microscope.

For Lysotracker staining, guts were dissected in AHLS and incubated in AHLS containing 0.5 μM Lysotracker Red (#C1046, Beyotime) for 5 min at room temperature. They were then mounted similarly to all other live-imaging experiments and imaged immediately by a two-photon laser microscope.

For propidium iodide staining, guts were dissected in PBS and then stained in 1.5 μM PI (#P1304MP, Invitrogen) for 15 min at room temperature. The guts were then fixed for 20 min in 4% formaldehyde, washed three times in PBS, mounted in Mounting Medium with Hoechst and analyzed on a confocal microscope.

For Magic Red and DQ-BSA staining, guts were dissected in AHLS and incubated in AHLS containing Magic Red (1:250, #ICT-941, Immunochemistry) or DQ-BSA (4 μg ml^–1, #D12051, Invitrogen) for 10 min at room temperature. They were then mounted similarly to all other live-imaging experiments and imaged immediately by a two-photon laser microscope.

Ex vivo Drosophila intestine imaging setup

Flies were dissected in Adult Hemolymph-like Saline (AHLS) culture media containing 2 mM CaCl2, 5 mM KCl, 5 mM HEPES, 8.2 mM MgCl2, 108 mM NaCl, 4 mM NaHCO3, 1 mM NaH2PO4, 5 mM Trehalose and 10 mM Sucrose. Guts were rapidly transferred to a microscope slide with thin adhesive spacers (#GBL654008, Sigma-Aldrich), embedded in 1% low melting agarose (in AHLS) and submerged in AHLS. All live-imaging experiments were imaged using an Olympus FVMPE-RS two-photon microscope equipped with two lasers for fluorescence excitation. Only the middle posterior midgut region (region R4) was analyzed.

Image acquisition and quantifications

For Cyto[Ca²⁺], Mito[Ca²⁺] and ER[Ca²⁺] imaging experiments except Fig. 1B, identical sized z stacks covering a single layer of the Drosophila epithelium were imaged using an Olympus FVMPE-RS two-photon microscope equipped with two lasers for fluorescence excitation. An InSight X3-OL laser (Spectra-Physics) tuned to 530–550 nm was used to excite TdTomato/DsRed/mCherry, and a MaiTai eHPDS-OL laser (Spectra-Physics) tuned to 488 nm was used to excite GCaMP3/5/6. The laser beams were focused using a 25× 1.05 NA water-immersion objective lens (Olympus). GCaMP3/5/6 emission was captured at 495–540 nm and TdTomato/DsRed/mCherry emission at 575–645 nm. For Cyto[Ca²⁺] and Mito[Ca²⁺] imaging experiments in Fig. 1b, images were taken on a Zeiss Axio Imager M2 fluorescence microscope equipped with an apotome. The laser beams were focused using a 40× Plan-Apochromat oil-immersion objective lens (Zeiss).

For the ratiometric MitoRoGFP2_Orp1 biosensor, experiments were performed based previous publications^18,87. Freshly dissected midgut tissues were incubated with 20 mM NEM (N-ethyl maleimide, #E387, Sigma-Aldrich) (in AHLS) for 10 min before imaging. Probe fluorescence was excited sequentially using the 405 nm and 488 nm laser lines and emission was detected at 495–540 nm.

For Laconic FRET imaging experiments, following excitation using the 440 nm laser line, emission was sequentially detected at 460–500 nm and 520–560 nm.

For AMPK-SPARK imaging experiments, EGFP was excited at 488 nm and emission was detected at 495–540 nm, mCherry was excited at 545 nm and emission was detected at 575–645 nm.

All quantifications were done manually using FIJI (NIH ImageJ) software. When sensors were expressed using esg::G4 in the absence of Su(H)GBE::G80ts, regions of interest (ROIs) were manually drawn so as to only segment smaller cells that likely represent ISCs and early-stage EBs. Larger esg::G4 positive cells, which are probably late-stage EBs that are soon approaching differentiation into ECs, were excluded from analysis.

For MARCM clone analysis, the clone formation and maintenance capacity of ISCs was measured by quantifying the number of MARCM clones containing at least 3 cells in the posterior midgut.

For ISC cell cycle analysis using the FUCCI system, the nucleus of GFP, RFP, GFP/RFP and non-fluorescent labeled ISC indicated cell residing in G1, S, G2 and G0 phase, respectively. The number of different fluorochrome-tagged nuclei was manually quantified across the whole posterior midgut.

For ISC differentiation analysis, ISCs and EB cells were labeled with mCherry and GFP, respectively. The mis-differentiation of ISCs was measured by quantified the percentage of ISCs labeled with mCherry and GFP.

For γH2AvD measurements, γH2AvD foci per nuclei of ISCs was counted manually. Mean intensity quantifications were made in individual ISCs of GFP channels normalized to nearby background.

For p-4EBP1, Ref(²)P, TRE-RFP and GFP-MAM measurements, mean intensity quantifications were made in individual ISCs of GFP or mCherry channels normalized to nearby background.

For mCherry-ATG8a, Lysotracker, GFP-Ref(²)P, GFP-ZFYVE1, GFP-ATG6, GFP-ATG9, mCherry-ATG13 and GFP-MAM puncta measurements, number quantifications were counted in individual ISCs of GFP or mCherry channels.

For Cyto[Ca²⁺], Mito[Ca²⁺] and ER[Ca²⁺] measurements, mean intensity quantifications were made in the ROIs of RCAMP1, MitoGCaMP3, GCaMP5/TdTomato, MitoGCaMP3/TdTomato, MitoGCaMP3/MitoDsRed or ERGCaMP6/mCherry channels.

For ratiometric measurements of MitoRoGFP2_Orp1, mean intensity quantifications were made in the ROIs of both 405 nm and 488 nm excitation channels.

For ratiometric FRET measurements of Laconic, mean intensity quantifications were then made in the ROIs of both the 460–500 nm and 520–560 nm emission channels.

For ratiometric measurements of AMPKSPARK, mean intensity quantifications were made in the ROIs of EGFP/mCherry channels.

For Magic Red and DQ-BSA staining, the number and intensity of puncta were quantified in ISCs which labeled by GFP.

Statistical analysis

Statistical analysis is performed using GraphPad Prism 8. Data derived from different genetic backgrounds or different conditions were compared using Student’s two-tailed unpaired t-test, one-way ANOVA followed by Tukey’s multiple comparison test, two-way ANOVA followed by Tukey’s multiple comparison test as indicated in the figure legends. All data were presented as mean ± SEM. The significance was set at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = non-significant (P > 0.05). All experiments were performed in biological triplicates.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All materials are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank Bloomington Drosophila Stock Center, Vienna Drosophila Stock Center, Tsinghua Fly Center and Dr. Margaret S Ho, Dr. Hai Huang, Dr. Chao Tong, Dr. Heinrich Jasper for stocks and reagents. We thank Dr. Hong Zhang for helpful discussions. This work was supported by a National Key Research and Development Project [2018YFA0107100], National Natural Science Foundation of China [grant no. 31871371 and 32071147] to H.D.

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Yangzhi Rehabilitation Hospital, Sunshine Rehabilitation Center, Frontier Science Center for Stem Cell Research, School of Life Sciences and Technology, Tongji University, Shanghai, 20092, China
Yao Zhang, Peng Ma, Saifei Wang, Shuxin Chen & Hansong Deng

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Yao Zhang
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Peng Ma
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Saifei Wang
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Shuxin Chen
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Hansong Deng
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Y. Z. and H. D. conceived and designed the study. H.D. prepared the first draft of the paper. Y.Z., P.M., S.W., S.C. contributed to the experimental work. Y.Z. conducted the statistical analysis of the data.

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Correspondence to Hansong Deng.

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Zhang, Y., Ma, P., Wang, S. et al. Restoring calcium crosstalk between ER and mitochondria promotes intestinal stem cell rejuvenation through autophagy in aged Drosophila. Nat Commun 16, 4909 (2025). https://doi.org/10.1038/s41467-025-60196-4

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Received: 23 August 2024
Accepted: 15 May 2025
Published: 27 May 2025
DOI: https://doi.org/10.1038/s41467-025-60196-4