# ER-endosome contacts generate a local environment promoting phagophore formation

Juliane da Graça, Charlotte Thiola, Myckaëla Rouabah, Ida Chiara Guerrera, Naïma El Khallouki, Maryse Romao, Carla Alemany, Dobrawa Amiri, Sarah Dubois, Ashvini Srimoorthy, et al.

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► To cite this version:

Juliane da Graça, Charlotte Thiola, Myckaëla Rouabah, Ida Chiara Guerrera, Naïma El Khallouki, et al.. ER-endosome contacts generate a local environment promoting phagophore formation. Cell Reports, 2025, 44 (7), pp.115993. 10.1016/j.celrep.2025.115993. hal-05393619

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Distributed under a Creative Commons Attribution 4.0 International License
**Cell Reports**

# ER-endosome contacts generate a local environment promoting phagophore formation

## Graphical abstract

<figure><img src="image_3.png" /></figure>

## Authors

Juliane Da Graça, Charlotte Thiola, Myckaëla Rouabah, ..., Francesca Giordano, Graça Raposo, Etienne Morel

## Correspondence

etienne.morel@inserm.fr

## In brief

Da Graça et al. identify ER-endosome contact sites as critical hubs for autophagosome biogenesis, where starvation triggers sequential endosome tethering to ER exit sites. This spatiotemporal coordination regulates $Ca^{2+}$ signaling and phase separation, leading to the formation of nano-vesicles that seed phagophore initiation.

## Highlights

* ER-endosome contact sites drive phagophore formation during early response to starvation

* Rab5/11 endosomes dynamically tether to ERESs, with local $Ca^{2+}$ release and phase transition

* Initiation of phagophore assembly at ER-endosome contacts requires Rab3/RAB3GAPs

* Dense membrane material is detected at ER-endosome interface during autophagic response

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Article

# ER-endosome contacts generate a local environment promoting phagophore formation

Juliane Da Graça,¹,⁷ Charlotte Thiola,¹ Myckaëla Rouabah,¹ Ida Chiara Guerrera,² Naïma El Khallouki,³,⁴

Maryse Romao,⁵,⁶ Carla Alemany,¹ Dobrawa Amiri,¹ Sarah Dubois,¹ Ashvini Srimoorthy,¹ Francesca Giordano,³,⁴

Graça Raposo,⁵,⁶ and Etienne Morel¹,⁸,*

¹Université Paris Cité, INSERM UMR-S1151, CNRS UMR-S8253, Institut Necker Enfants Malades, 75015 Paris, France

²Proteomic Platform Necker, INSERM US24 Structure Fédérative de Recherche Necker, Université Paris Cité, Paris, France

³Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France

⁴INSERM U1280, 91198 Gif-sur-Yvette Cedex, France

⁵Institut Curie, PSL Research University, CNRS, UMR144, Structure and Membrane Compartments, 75005 Paris, France

⁶Institut Curie, PSL Research University, CNRS, UMR144, Cell and Tissue Imaging Facility (PICT-IBiSA), 75005 Paris, France

⁷Present address: Interfaculty Institute of Bioengineering and Global Health Institute, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland

⁸Lead contact

*Correspondence: etienne.morel@inserm.fr
https://doi.org/10.1016/j CELREP.2025.115993

## SUMMARY

Autophagy starts with the formation of a double-membrane vacuole called the autophagosome, initiated by a transient structure known as the phagophore. Previous studies reported that phagophore biogenesis primarily occurs at endoplasmic reticulum (ER) omegasome subdomains, but other evidence suggests that the phagophore derives from recycling endosomes. Our study demonstrates the importance of ER-endosome interactions, revealing the dynamic mobilization of endosome-ER contact sites (EERCSs) in response to starvation. We characterize a sequential tethering of Rab5 and Rab11 endosomes to omegasomes, facilitating phagophore biogenesis. Detailed analyses reveal that EERCS-associated molecular machinery creates a confined environment that promotes local $Ca^{2+}$ accumulation and liquid-liquid phase separation at ER exit sites. This environment primes de novo phagophore formation through a Rab3a-RAB3GAP1/2-mediated nano-vesicle fusion. We propose that EERCS mobilization generates transient cytoplasmic confinement, fostering localized accumulation of components for phagophore biogenesis. Our study reveals a novel role for the ER-endosome interface in the nutrient deprivation response, emphasizing organelle coordination during autophagy initiation.

## INTRODUCTION

Maintaining cellular homeostasis is essential to prevent pathological deviations and cell death. Autophagy, a key process activated by stressors like starvation, enables cytoplasmic recycling and supports survival,¹,² and its dysregulation is linked to numerous diseases.³⁻⁷ Autophagy begins with the de novo formation of phagophores, which mature into autophagosomes that sequester cytoplasmic components for lysosomal degradation.³⁻⁷ In response to starvation, mTORC1 complex signaling activity is blocked, and the ULK complex (with core proteins ULK1, ATG13, FIP200, and ATG101) is then recruited at transient and specific subregions of the endoplasmic reticulum (ER) known as omegasomes. Omegasomes are characterized by an omega-ring shape, by the presence of DFCP1 protein,⁸ and by an enrichment of phosphatidylinositol-3-phosphate (PI3P) lipid.⁹,¹⁰ The presence of a PI3P pool at the omegasome is

mainly mediated by the PI3KC3 complex (including VPS34, VPS15, ATG14L, and Beclin1), which is associated with other regulating proteins such as AMBRA1 and VMP1 (vacuolar membrane protein 1; linked not only to autophagy initiation but also to various membrane contact sites¹¹⁻¹⁵). This allows for the subsequent targeting of PI3P-binding WIP12 protein accompanied by ATG (autophagy-related) regulators such as ATG5-12/ ATG16L1, which directly participate in LC3 lipidation on the phagophore.¹⁶ These recruitment events—marking phagophore nucleation—are linked to local phase separation¹⁷,¹⁸ and ER $Ca^{2+}$ release.¹⁹ The expansion of nascent phagophores requires lipid input via ATG2A, a lipid transporter tethering the ER and phagophore,²⁰,²¹ and ATG9, a scramblase that reshapes the bilayer.²¹,²² Multiple organelles, including mitochondria, the plasma membrane, and the Golgi, seem to contribute to this lipid delivery, particularly through membrane contact sites.¹⁵,²³⁻²⁶ Despite the emergence of this detailed literature, the

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<figure><img src="image_6.png" /><figcaption>Figure 1. Mobilization of endosome-ER contacts upon acute starvation</figcaption></figure>

(A) Proximity ligation assay (PLA) with anti-EEA1 and anti-VAPB antibodies in HeLa cells under basal (ctrl) or starvation (15 or 60 min) conditions. Scale bar: 5 µm.

(B) Quantification of PLA puncta per cell. Mean ± SEM, 5 experiments; one-way ANOVA: *p = 0.0260.

(C) Electron micrographs of ssKDEL-HRP-expressing HeLa cells in basal or 15-min starvation; left: cell overview (1 µm); the insets (a–d) show endosome-ER contact sites (250 nm).

(D) Quantification of endosome-ER contacts per cell. Mean ± SEM, 7 cells; Welch's test: *p = 0.0362.

(E) Shortest endosome-ER distances (nm). Means ± SEM, *n* = 22–47 contacts; Welch's test: *p = 0.0434.

(F) Electron microscopy of endosomal membrane deformation at contact sites after 15-min starvation. Scale bar: 100 nm.

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phagophore formation sequence is still largely misunderstood, especially regarding the origin of the phagophore seeding membrane(s).

In eukaryotes, endosomes ensure the transport of nutrients and signaling molecules to intracellular stations and show diverse morphologies—vesicular, tubular, and multivesicular.²⁷,²⁸ Their identity is defined by Rab GTPases—regulated by GEFs (guanine exchanging factors) and GAPs (GTP hydrolysis-activating proteins)²⁹—such as Rab5 (on early endosomes) and Rab11 (on recycling endosomes²⁹). Given its critical role in intracellular trafficking and signaling, the endosomal pathway is highly sensitive to stress,²⁷,²⁸,³⁰ and we recently reported the dynamic recruitment of early endosomal membranes toward sites of phagophore biogenesis in response to starvation.³¹ Numerous studies have highlighted the importance of endosomal proteins in autophagic processes,³²⁻³⁵ including the key GTPases Rab5 and Rab11,³⁶,³⁷ and essential autophagic regulators, such as ATG9A and ATG16L1, are directly associated with endosomal trafficking.³⁸ However, the direct contribution of endosomal membranes to phagophore biogenesis remains unclear. Importantly, endosome-ER contact sites (EERCSs) actively participate in the regulation of both endosomes and ER morphodynamics,³⁹⁻⁴¹ but these contacts have not been properly explored in the early steps of autophagic processes so far.

Because extensive literature underscores the pivotal role of the ER in phagophore assembly, other seminal studies have highlighted the direct contribution of endosomal membranes in this process.

This difference in the dual origin of pre-autophagosomal membranes could suggest that both types of organelles may cooperate to promote the conditions for *de novo* biogenesis of the phagophore. In this context, we investigated the potential involvement of endosome-ER membrane interactions in the early stages of phagophore assembly. We show that early and recycling endosomes are sequentially tethered to specialized ER regions to initiate phagophore biogenesis in response to acute starvation. These endosome-ER contacts foster local confinement of cytoplasm, inducing a space-limited release of calcium and promoting protein phase separation at ER exit sites (ERESs). Such a microenvironment favors calcium-dependent Rab3a-RAB3GAP1/2 machinery, facilitating the formation and fusion of dense nano-vesicles, in turn leading to phagophore nucleation.

## RESULTS

### Acute autophagic response correlates with endosome-ER contact mobilization

Nutrient deprivation, commonly referred to as starvation, induces acute autophagosome biogenesis, mobilizing ER mem-

branes and associated contact sites.⁴² To detect and quantify the dynamics of EERCSs, we used a proximity ligation assay (PLA) with a resolution below 40 nm, a commonly used method for the study of membrane contact sites,⁴³ combining an anti-EEA1 antibody to label early endosomes with an anti-VAP-B antibody as a marker for the ER membrane involved in contact sites⁴⁴ (Figures S1A and S1B). This PLA combination proved successful in revealing bona fide EERCSs, as reflected by the localization of EEA1/VAP-B PLA puncta at the interface of Rab5-GFP-positive endosomes and Sec61β-RFP ER membranes (Figure S1C). In HeLa cells, 15 min of starvation is sufficient to increase the number of EEA1/VAP-B PLA puncta, which returned to normal at 1 h (Figures 1A and 1B), suggesting that dynamic and reversible formation and/or stabilization of EERCSs is an acute cellular response to starvation. Conventional electron microscopy (EM) on cells expressing the ER luminal marker KDEL-HRP confirmed the increase in EERCSs at 15 min of starvation (Figures 1C and 1D), which was, interestingly, associated with a 25% increase in the interspace distance between the ER and endosome membranes (from ±20 to ±25 nm; Figure 1E). In addition, we noticed that EERCSs often displayed an alteration of the endosomal membrane straightness at the site of contact in this condition (Figures 1F and 1G). Further expansion microscopy analyses using anti-EEA1, Sec61β-GFP, and VMP1-mCherry as ER-associated contact site markers indicate that endosomes involved in EERCSs during starvation adopt a distinctive "bean-shaped" morphology (Figure 1H). Altogether, these results suggest that starvation-induced EERCSs are morphologically different than regular EERCSs, underlying their involvement in a specific process.

To demonstrate that endosomes can be physically tethered to the ER during starvation, we optimized a protocol for the biochemical purification of EERCSs (Figure 1I). This protocol was adapted from the commonly used biochemical purification of mitochondria-associated membranes (MAMs) described by Wieckowski et al.⁴⁵ Using this fractionation approach, we successfully isolated EERCS-enriched fraction that showed high expression of ER contact site proteins (such as VMP1 and VAP-A) along with endosomal proteins compared to the remaining (non-ER-associated) endosomal fraction (Figure 1J). The signal ratio of endosomal proteins within the EERCS fraction relative to the remaining endosomal fraction (EERCS/endosome ratio) reflects the formation of endosome-to-ER contacts. In agreement, 15 min of starvation led to the increase of the EERCS/endosome ratio of the small GTPase Rab5 protein, a key player for early endosomes, which returned to a normal level at 60 min when the EERCS/endosome ratio of Rab11, previously reported to regulate autophagy,³³,³⁴ increased (Figures 1K and 1L). These findings suggest that both Rab5⁺ (early) and Rab11⁺ (recycling) endosomes can establish contact with the

<footnote>(G) Quantification of membrane deformation by counting straight-line intersections. Means ± SEM, *n* = 22–47 contacts; t test: **p** = 0.0010.</footnote>

<footnote>(H) Expansion microscopy of HeLa cells expressing Sec61β-GFP and VMP1-mCherry with anti-EEA1 under basal or starved conditions. Insets: (a) 3D cell view (2.5 µm), (b) 3D crop of endosome-ER contact (500 nm), (c) orthogonal zxy view (125 nm), and (d) single Z-slice showing contact site (1 µm).</footnote>

<footnote>(I) Schematic of biochemical purification of endosome-associated ER membranes (EERCSs) vs. raw endosomes.</footnote>

<footnote>(J) Representative western blot of fractions from HeLa cells.</footnote>

<footnote>(K) Western blot showing Rab5 and Rab11 levels in EERCSs and endosomes under basal and starvation (15 and 60 min) conditions.</footnote>

<footnote>(L) Quantification of Rab5 and Rab11 EERCS/endosome ratios. Mean ± SEM, 3 experiments.</footnote>
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A

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<figure><img src="image_18.png" /><figcaption>Figure 2. Endosome-ER contacts precede and dictate the site of phagophore formation</figcaption></figure>

(A and C) Images of HeLa cells obtained after confocal microscopy and unbiased machine learning-based workflow using Ilastik and Imaris software from cells expressing Sec61β-RFP with staining for EEA1 and LC3 (A) or Sec61β-RFP and Rab11-GFP with LC3 (C). Scale bars: 5 µm and 1 µm (inset).

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ER during the autophagic response. Furthermore, it implies a potential switch from Rab5⁺ to Rab11⁺ endosomes to establish EERCSs during prolonged starvation. Taken together, our data, obtained through multiple methods, demonstrate a dynamic increase in endosome-ER contacts with specialized characteristics during the early stages of starvation.

## Endosome-ER contacts precede and dictate the site of phagophore formation

To investigate whether the increase in EERCSs during starvation is directly associated with the autophagic sequence, we developed an unbiased machine learning-based workflow using Ilastik and Imaris software to quantify triple proximity events between endosomes, ER, and autophagic markers. Both early (EEA1⁺) and recycling (Rab11⁺) endosomes were dynamically associated with the ER marker Sec61β-RFP and with endogenous LC3 (a marker of autophagic structures) puncta, displaying similar kinetics to those observed previously (see Figures 2A and 2B for EEA1 endosomes and 2C and 2D for Rab11 endosomes). Similar observations were made using ATG14, another marker of the initiation autophagic machinery (Figures S2A-S2D). Using this approach, we quantified up to 20% of total early endosomes per cell (Figure 2B) and up to 70% of total recycling endosomes per cell (Figure 2D) associated with autophagic processes, indicating that only a specific subset of endosomes seems to be involved. These results suggest that the transient increase in EERCS number (as shown in Figure 1) is indeed associated with local autophagic processes.

We further employed DFCP1, a marker of omegasomes, to analyze the dynamic relationship between EERCSs and the sequence of phagophore biogenesis. Live imaging revealed that the appearance of DFCP1-CFP puncta is spatiotemporally preceded by EERCS establishment (labeled with Rab5-RFP and VMP1-GFP) (Figure 2E). Moreover, while nearly 40% of newly formed DFCP1 puncta were preceded by EERCSs under basal conditions, this value increased up to 80% in response to 15 min of starvation (Figure 2F) independently of the cell type (Figures S3A and S3B) or the early autophagic marker (Figures S3C and S3D), confirming the dynamic formation of pre-autophagosomes at EERCSs.

Then, we analyzed the behavior of each individual contact and observed that the appearance of DFCP1 puncta takes approximately 5 s once the Rab5-VMP1 contact is formed (Figures 2E and 2G). Compared to EERCSs present under basal conditions, starvation-induced EERCSs were more stable, as shown by the longer time of apposition between endosome and ER markers

(from 15 to 25 s, respectively [Figures 2E and 2G]). Since both early (Rab5/EEA1) and recycling (Rab11) endosomal populations appeared to be sequentially recruited within EERCSs in response to starvation, we used live imaging to analyze and quantify the proportion of newly formed DFCP1 puncta associated with either Rab5 or Rab11. Our data revealed that DFCP1 puncta appearance was mostly associated with Rab5⁺ structures at 15 min of starvation and then with Rab11⁺ structures after 1 h of nutritional stress (Figures 2H and 2I), highlighting the adaptation of the endosomal system via a switch from Rab5 to Rab11 endosomes during prolonged starvation.

We hypothesized that recruitment/stabilization of Rab5⁺ endosomes at EERCSs during the first 15 min of starvation may divert these endosomes from their basal functions, such as endosomal maturation and cargo sorting, and Rab11⁺ endosomes may act as a relay to facilitate the release of Rab5⁺ membranes from EERCSs. To investigate this, we analyzed endosomal maturation events during starvation by quantifying the presence of Rab7-GTP (the active form of the protein, bound to GTP) on EEA1⁺ endosomes. We observed that the number of EEA1/Rab7 co-distribution events decreased after 15 min of starvation but returned to baseline levels after 60 min (Figures S4A and S4B), suggesting that endosomal maturation is partially altered or delayed during the early stages of the starvation response. To further investigate and confirm that prolonged starvation induces the production of Rab11⁺ endosomes, we quantified the number of Rab11⁺ particles that increased in cells starved for 60 min compared to control cells (Figures S4C and S4D, siCTR [control] conditions, left).

In the context of endosomal maturation and dynamics, the regulation of cargo sorting between Rab5⁺ and Rab11⁺ endosomes has been shown to depend on SNX1 tubules and the FERARI (Factor for Endosome Recycling and Retromer Interactions) complex.<sup>46,47</sup> Moreover, we have previously demonstrated that SNX1 tubules are enhanced upon starvation.<sup>31</sup> Based on these, we hypothesized that SNX1 might play a role in this transition from Rab5⁺ to Rab11⁺ endosomes during the early stages of the autophagic response. To test this, we analyzed the number of Rab11⁺ particles under SNX1 depletion and found that the starvation-induced formation of Rab11⁺ endosomes was inhibited (Figures S4C and S4D), suggesting that the sequential recruitment of Rab5⁺ to Rab11⁺ endosomes for phagophore formation is, at least in part, regulated by SNX1.

Because membrane contact sites are typically induced and stabilized by multiple proteins, their complete inhibition is

(B and D) Quantification of the percentage of EEA1 (B) or Rab11 (D) structures in close proximity to ER and LC3 in basal or starved (15 or 60 min) cells. Means ± SD/SEM; *n* = 27–40 cells; one-way ANOVA: \*\*p < 0.005 (B) and \*\*\*p < 0.0001 (D).

(E) Live imaging of HeLa expressing VMP1-GFP, Rab5-RFP, and DFCP1-CFP after 15-min starvation; arrows indicate endosome-ER contacts (hollow head) and DFCP1 puncta (white head). Scale bar: 1 µm.

(F) Quantification of new DFCP1 puncta preceded by endosome-ER contacts in basal vs. starved cells. Means ± SD, *n* = 33–42 cells; unpaired t test: \*\*\*p < 0.0001.

(G) Timing/stability of EERCS and DFCP1 formation in basal vs. starved cells. Means ± SD/SEM; *n* = 53–218 contacts; two-way ANOVA: \*\*\*p < 0.0001.

(H) Live imaging stills of Rab11-GFP, Rab5-RFP, and DFCP1-CFP at 15-min and 1-h starvation. Scale bar: 2 µm.

(I) Percentage of new DFCP1 puncta preceded by Rab5, Rab11, or Rab5-Rab11 ER contacts under basal or starved conditions. Means ± SD, *n* = 19–27 cells; two-way ANOVA: \*\*\*p < 0.0001.

(J) Confocal images of HeLa stably expressing RFP-FKBP-Rab5 and ERM-HA-FRB after 6-h starvation, ± rapalog, stained for LC3. Scale bar: 5 µm.

(K) Quantification of LC3 puncta per cell ± rapalog. Means ± SEM; *n* = 40–43 cells; unpaired t test: \*p = 0.0444.
<figure><img src="image_19.png" /><figcaption>B</figcaption></figure>

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<figure><img src="image_21.png" /><figcaption>D</figcaption></figure>

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<figure><img src="image_29.png" /><figcaption>Figure 3. Analysis of EERCS proteome using split-TurboID reveals KTN1 tethering in response to starvation</figcaption></figure>

(A) Western blot of co-immunoprecipitation in HeLa cells with anti-VMP1 or control immunoglobulin (Ig)G.

(B) Schematic of split-TurboID Rab5-VMP1 and Rab11-VMP1 constructs; biotinylation occurs only upon ER-endosome tethering.

(C and D) Volcano plots of mass spectrometry from Rab5-VMP1 (C) and Rab11-VMP1 (D) split-TurboID in HeLa cells starved for 6 h with biotin vs. no biotin.

(E) Key proteins identified by mass spectrometry in Rab5-VMP1 and Rab11-VMP1 split-TurboID datasets.

(F) Immunofluorescence of HeLa cells stained for EEA1, KTN1, and DAPI under basal or 15-min starvation conditions; arrowheads indicate EEA1-KTN1 co-localization. Scale bars: 5 µm (large) and 1 µm (zoom).

(G) Quantification of EEA1-KTN1 colocalization events per cell. *n* = 3 experiments; \*p = 0.0139.

(H) Representative western blot of purified ER-associated endosomal membranes (EERCS) vs. total endosomal membranes for KTN1 and VMP1 in basal or starved (15 or 60 min) cells.

(I) Quantification of KTN1 EERCS/light membrane ratio. *n* = 3 experiments; Friedman test: \*p = 0.0412 and ns, not significant.

(J) Proximity ligation assay (PLA) for EEA1 and VAPB in control (siCTRL) or KTN1 knockdown (siKTN1) HeLa cells under basal or 15-min starvation conditions. Scale bar: 5 µm.

(K) Quantification of PLA puncta per cell under indicated conditions. *n* = 166–196 cells; one-way ANOVA: \*\*\*p = 0.0009, \*\*\*p < 0.0001, and ns, not significant.

challenging. Our live imaging data have underscored the dynamic and transient nature of EERCSs in response to starvation. Therefore, we opted to employ an artificial tether based on rapalog-induced dimerization of FRB-FKBP (FKBP [FK506-binding protein]; rapamycin binding domain), as described previously,⁴⁸ to force endosome-to-ER anchoring and inhibit their dissociation (Figure S5A). We subsequently quantified autophagic readouts in cells expressing stabilized EERCSs to elucidate whether they exert a direct impact on autophagosome formation. Stabilizing EERCSs led to a decrease in LC3 (Figures 2J and 2K) and ATG13 puncta (Figures S5B and S5C), indicating that the dissociation of EERCSs is necessary to complete the formation of autophagosomes. Taken together, our data demonstrate that dynamic spatiotemporal modulation of endosome-ER con-

tacts is required to establish hotspots for autophagosome biogenesis.

## Identification of the starvation-specific endosome-ER contactome

To gain insights into the specific role of EERCSs during the early stages of the autophagic process, we conducted unbiased proximity labeling to identify the local proteome of starvation-induced contacts. Both endosomal Rab5 and Rab11 proteins were co-immunoprecipitated with the ER resident and contact site protein VMP1 (Figure 3A). Therefore, we optimized a split-TurboID tool⁴⁹ by fusing the N-terminal part of the biotinylating enzyme with either Rab5 or Rab11, while its C-terminal part was coupled with VMP1 (Figure 3B).
A merge + DAPI, Rab5<sup>RFP</sup>, GCamp6f<sup>Cyb5</sup>

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C merge, Rab5<sup>RFP</sup>, GCamp6f<sup>Cyb5</sup>

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E merge, Rab5<sup>CFP</sup>, ATG13, LC3, GCamp6f<sup>Cyb5</sup>

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F merge, Rab5<sup>RFP</sup>, GCamp6f<sup>Cyb5</sup>

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G EEA1 TPC1 VMP1<sup>GFP</sup>

<figure><img src="image_37.png" /><figcaption>Figure 4. EERCSs induce local and confined calcium release</figcaption></figure>

(A) Confocal images of HeLa cells transiently expressing GCamp6f-Cyb5 and Rab5-RFP under basal (ctrl) or starved (15 min) conditions. Right images show GCamp6f-Cyb5 intensity in fire color scale (black = low, yellow = high). Arrowheads indicate endosome-ER contact sites (EERCSs). Left images show entire cells (scale bar: 5 µm); insets 1 and 2 zoom in on contacts (scale bar: 1 µm).

(B) Quantification of local Ca²⁺ enrichment at EERCSs as GCamp6f-Cyb5 intensity normalized to global probe expression in basal and starved cells. Means ± SD, n = 50 contacts per condition; unpaired t test: ****p < 0.0001.

(C) Live-cell imaging of HeLa cells expressing GCamp6f-Cyb5, Rab5-RFP, and DFCP1-CFP after 15-min starvation. Arrowhead points to endosome-ER contacts involved in phagophore biogenesis. Scale bar: 1 µm.

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Following starvation, we enriched biotinylated proteins from cell lysates, conducted mass spectrometry analyses, and identified a restricted and specific set of proteins in the immediate vicinity (±10–20 nm) of the VMP1-Rab5 and VMP1-Rab11 interactions (Data S1; see Figures 3C for Rab5-VMP1 and 3D for Rab11-VMP1). While our split-TurboID screen identified a low number of autophagic proteins, probably because this technique only detects enriched proteins that are implicated in the process before the massive recruitment of autophagic regulators, we identified—as positive controls—proteins already described to be involved in ER tethering (such as VAPA, VAPB, and MOSPD2<sup>40,50</sup>) and endosome-ER-contact-regulating proteins (such as RRBP1 and PTP1B<sup>48,51</sup>). Interestingly, almost half of the mass spectrometry hits were shared between both types of EERCSs (Figure 3E), pointing to common machinery for both Rab5 and Rab11 endosomes during autophagic processes.

We noticed that both Rab5-VMP1 and Rab11-VMP1 split-TurboID mass spectrometry analyses revealed the ER-resident KTN1 (kinectin1) protein as being present—and enriched—in both interactomes (Figures 3C–3E). Given that KTN1 has been recently shown to bind PI3P on early endosomes,<sup>48</sup> we investigated the behavior and localization of KTN1 in response to starvation and its potential role in EERCS formation. Using immunofluorescence, we observed an increase in the KTN1 signal intensity on EEA1<sup>+</sup> structures during starvation (Figures 3F and 3G), indicating that nutrient deprivation stabilizes early endosomes to KTN1-enriched regions of the ER. Furthermore, we detected an increase in the amount of KTN1 protein within the purified EERCS fraction after 15 min of starvation (Figures 3H and 3I), suggesting a rapid mobilization of KTN1 tethering activity after starvation. We next depleted KTN1 in cells and examined the dynamics of EERCSs using PLA<sup>EEA1-VAPB</sup>. As expected, starvation resulted in an increase and stabilization of EERCSs in siCTRL cells, while this response was abolished in the absence of KTN1 (Figures 3J and 3K). These findings indicate that KTN1 is essential for EERCS mobilization during nutritional stress.

## EERCSs support local and confined calcium release in response to starvation

Among the shared hits of Rab5-VMP1 and Rab11-VMP1 split-TurboID mass spectrometry (Data S1), we noticed proteins related to ER Ca²⁺ channeling (such as ITPR [inositol 1,4,5-trisphosphate receptor] 1 and ITPR3) and Ca²⁺ sensing (such as STIM1 and CISD2) (Figures 3C–3E), suggesting that the EERCS environment is a hotspot for Ca²⁺ local dynamics during the response to starvation. We thus employed a Ca²⁺ probe expressed on the ER surface (GCamp6f-Cyb5), allowing for the

local cytosolic detection of Ca²⁺ release at the surface of the ER.<sup>19</sup> Thus, we measured the specific GCamp6f-Cyb5 fluorescence signal at EERCSs normalized on the baseline signal of the probe along the ER (Figure S6A). We observed that the local Ca²⁺ level was increased within EERCSs during the response to starvation (Figures 4A and 4B). The local GCamp6f-Cyb5 increase did not reflect an ER remodeling effect since the Sec61β ER marker did not show any local pattern alteration (Figures S6B and S6C), confirming the specificity of Ca²⁺ release within the Rab5/ER interface.

Release of ER-stored Ca²⁺ can be mediated by ITPR channels, a process that has been recently suggested to participate in local autophagy initiation.<sup>19</sup> Thus, we reasoned that the starvation-induced local Ca²⁺ burst at EERCSs is associated with omega-some formation. Live imaging of cells co-expressing GCamp6f-Cyb5, DFCP1-CFP, and Rab5-RFP showed that Ca²⁺ bursts occurred after endosome-ER contact and DFCP1 local appearance (Figures 4C and 4D). In addition, we observed in fixed cells the co-distribution of Ca²⁺ bursts and early autophagic machinery markers (LC3 and ATG13) with endosomes (Figure 4E). Similar local Ca²⁺ enrichment was observed at the Rab11/DFCP1 interface at 60 min of starvation (Figures S6D and S6E).

Knocking down ITPR1 and ITPR3 Ca²⁺ channels decreased autophagosome formation, monitored by the quantification of double-positive LC3-ATG13 and ATG16L1-phospho (S278) puncta per cell (Figures S7A–S7F), as previously reported.<sup>19,52</sup> The Ca²⁺ burst induced by starvation was no longer detected in ITPR1/3 knockdown cells (Figures 4F and 4G), suggesting that Ca²⁺ accumulation within EERCSs is mediated by a specific release from the ER. Together, these data indicate that ITPR1/3-mediated Ca²⁺ release within the EERCS interspace is directly linked with autophagic processes.

To decipher how the EERCS environment could induce Ca²⁺ release, we questioned local ITPR activation. A recent study proposed that ER-located IPRs are activated by Ca²⁺ in a feedforward-loop manner, relying on the lysosomal-related calcium channel TPC (two-pore channel) 2.<sup>53</sup> We thus investigated whether the TPC channel located at early/recycling endosomes—here, TPC1, which strongly co-distributed with the EEA1 signal (Figures S7G and S7H)—could play a role in the activation of ITPR1/3-associated Ca²⁺ release within EERCSs in response to starvation. The knockdown of TPC1 (Figures S7I and S7J) significantly decreased the starvation-dependent Ca²⁺ burst at the EERCS interface (Figures 4F and 4G), suggesting that ER-located ITPR channels were activated by endosomal TPC1 within EERCSs. This was reinforced by expansion microscopy of cells stained for EEA1 and VMP1, which showed that the TPC1 signal was concentrated on

<footnote>(D) Fluorescence intensity of GCamp6f-Cyb5, Rab5-RFP, and DFCP1-CFP at EERCS events preceding DFCP1 puncta appearance in starved cells, normalized to min-max of selected regions. Means ± SEM, *n* = 5 events.</footnote>

<footnote>(E) Fixed HeLa cells expressing Rab5-CFP and GCamp6f-Cyb5, stained for LC3 and ATG13, under basal or starved (15 min) conditions. Right images show GCamp6f-Cyb5 intensity in fire color. Arrowheads indicate endosome-ER contacts. Scale bars: 5 µm (cell) and 1 µm (enlargement).</footnote>

<footnote>(F) Confocal images of HeLa cells (control siCTRL or knocked down for ITPR1/3 or TPC1) expressing GCamp6f-Cyb5 and Rab5-RFP under basal or starved (15 min) conditions. Right images show GCamp6f-Cyb5 intensity in fire color. Arrowheads indicate endosome-ER contacts. Scale bars: 5 µm (cell) and 1 µm (insets 1, 2, and 3).</footnote>

<footnote>(G) Quantification of local Ca²⁺ enrichment at EERCS in starved cells with siCTRL, siITPR1/3, or siTPC1 knockdown, measured by normalized GCamp6f-Cyb5 intensity. Means ± SEM, *n* = 55–57 contacts; one-way ANOVA: \*\*\*p < 0.0001.</footnote>

<footnote>(H) Expansion microscopy image of HeLa cells transiently expressing VMP1-mCherry, stained for EEA1 and TPC1 after 15-min starvation. Arrowhead points to TPC1 puncta. Scale bar: 125 nm.</footnote>
the endosomal membrane facing the contact site (Figure 4H). These data suggest that endosomes engaged in contact with the ER membrane display a dedicated morphology that allows for local Ca²⁺ release and accumulation to promote phagophore formation.

To further confirm the role of Ca²⁺ in the dynamics of EERCSs during starvation, we finally employed drugs designed to modulate Ca²⁺ waves. First, we used BAPTA-AM (25 µM), a global Ca²⁺ chelator, to inhibit all Ca²⁺ fluxes, effectively blocking both TPC1 (at endosomes) and ITPR (at ER membranes) activities. In parallel, we used thapsigargin (TG; 100 nM; pre-treatment for 1 h) to inhibit SERCA (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase) activity and deplete ER Ca²⁺ stores, thereby preventing ITPR-mediated Ca²⁺ release during starvation. Both treatments successfully inhibited Ca²⁺ accumulation within EERCSs under starvation, as monitored by GCamp6f (Figures S8A and S8B), confirming the relationship between starvation-induced EERCSs and starvation-induced ER Ca²⁺ release.

## EERCSs favor a Ca²⁺-dependent phase transition and phagophore formation at ERESs

To precisely determine which ER subdomains would preferably be involved in EERCS formation, and thus phagophore formation, we took advantage of the Rab5/Rab11 split-TurboID mass spectrometry common results and identified Sec16A (Figures 3C-3E; Data S1), a key regulator in COP-II (coat protein complex) formation at ERESs.⁵⁴ Given previous findings demonstrating the importance of ERESs (or ERES components) in autophagosome formation,²³,⁵⁵,⁵⁶ we speculated that these specialized ER regions might facilitate endosome recruitment during nutritional stress and relate to phagophore biogenesis. By quantifying the triple co-distribution of Sec16A (ERES), Sec61β (ER), and Rab5 following 15 min of starvation, we revealed a 2-fold increase in the occurrence of concomitant events between ERESs and EERCSs (Figures 5A and 5B), as also shown by live-cell imaging (Figure S9A). Subsequently, EERCSs associated with ERESs co-distributed with the autophagic markers (LC3 and DFCP1) under starvation (Figures 5C and 5D and S9B and S9C, respectively), as we observed for LC3 and Rab11 (Figures S9D and S9E). We also show that the knockdown of KTN1, which we identified as a key ER-to-endosome tether in response to starvation (see Figure 3), resulted in a significant reduction in ERES-endosome contacts during starvation (Figures S10A and S10B for Rab5 endosomes and S10C and S10D for Rab11 endosomes), highlighting the specificity of ERES-EERCS dialogue during the response to starvation.

We thus wondered whether ERES "canonical" functionality could be altered in our experimental conditions. Interestingly, we observed that ERES structures associated with EERCSs in response to starvation lacked Sec31, a critical component of COP-II vesicles (Figures 5E and 5F). This observation suggests that a subset of ERES/Sec16A puncta change their nature when associated with EERCSs and phagophore assembly sites during starvation. This was further confirmed by analyzing the "secretory activity" of ERESs that are mobilized at the endosome interface in response to stress via a synchronized RUSH (Retention Using Selective Hooks) assay using ManII-mCherry⁵⁷ in cells expressing

Sec16A-GFP and Rab5-CFP. Under starvation, ManII cargoes did not accumulate at Sec16A puncta that were already engaged with Rab5 (Figures S11A and S11B), suggesting that starvation-induced endosome contacts may indeed divert the function of a small proportion of ERESs from their typical secretory role to transiently participate in phagophore formation.

Importantly, Sec16A can undergo liquid-liquid phase separation (LLPS) in response to various cellular stresses.⁵⁸,⁵⁹ Such an LLPS phenomenon has been recently observed with the FIP200 protein during autophagosome biogenesis in a Ca²⁺-dependent manner.¹⁹ We observed a marked increase in the intensity of Sec16A puncta and reduced cytosolic distribution after 15 min of starvation (Figures S12A and S12B), suggesting a potential phase-transition phenomenon. To investigate this further, we conducted fluorescence recovery after photobleaching (FRAP) experiments to assess the dynamics of Sec16 puncta recovery in response to cellular nutrient status (control vs. starvation) and, depending on their association with endosomes, whether they were positive for Rab5 (Figure 5G). Under starvation, Sec16A fluorescence intensity recovered faster when associated with EERCSs (Rab5⁺) compared to basal Sec16A puncta (Figures 5G-5I), suggesting that the fluidity of Sec16A condensates is increased upon starvation locally, in the presence of endosomes. To further support the idea that Sec16A can form condensates in specific cytoplasmic regions, we first adjusted the incubation conditions, as lower temperatures are known to increase condensate size, thereby enhancing their detection.⁶⁰ Thus, we elevated—or reduced—temperatures for 3 min before the fixation of starved cells expressing Sec16A-GFP and Rab5-RFP (Figures 5J and 5K). At physiological temperature (37°C), we observed that the size of Sec16A puncta associated with endosomes was slightly larger than those alone. Interestingly, this size difference was abolished at higher temperatures (42°C), while it was further exacerbated at lower temperatures (25°C), suggesting that the phase-transition properties of Sec16A upon starvation are specifically influenced by the local presence of endosomes (Figures 5J and 5K). In addition, we used 1,6-hexanediol (1,6-HD), a drug described to inhibit protein condensation,⁶¹,⁶² using a short and low-dose treatment to avoid cytotoxicity (15 min at 0.1% 1,6-HD). Our data show that 1,6-HD treatment also abrogated the difference in Sec16A puncta size depending on their association or not with endosomes upon starvation (Figures S12C and S12D). This last experiment further demonstrates that Sec16A condensation induced by starvation is associated with the local presence of endosomes.

Since we documented a starvation-specific Ca²⁺ wave (see Figure 4) and because similar Ca²⁺ events have previously been linked with phase separation in the context of autophagosome biogenesis,¹⁹ we further investigated the ERES-EERCS dialogue from this perspective. We show that, after 15 min of starvation, Rab5-Sec16A colocalization events were reduced in cells treated with BAPTA-AM, whereas no change was observed with TG pre-treatment (Figures S13A and S13B), suggesting that endosomal TPC1 Ca²⁺ activity may be critical for establishing and/or stabilizing the contacts themselves. We further analyzed the dynamics of Sec16A-Rab5 colocalizations using
<figure><img src="image_38.png" /><figcaption>Figure 5. EERCSs favor phase transition and phagophore formation at ER exit sites</figcaption></figure>

(A) HeLa cells transiently expressing Sec16A-GFP, Sec61β-RFP, and Rab5-CFP in basal (ctrl) or starved (15 min) conditions. Arrowheads indicate endosome-ER contact sites. Scale bars: 5 µm (cell) and 1 µm (insets).

(B) Quantification of Sec16A-Rab5-Sec61β triple co-distribution events per cell in fixed HeLa cells under basal or starved conditions. Means ± SD, *n* = 34–30 cells; unpaired t test: \*\*\*p < 0.0002.

(C) HeLa cells expressing Sec16A-GFP and Rab5-CFP stained for LC3, under basal or starved (15 min) conditions. Arrowheads indicate endosome-ER contact sites. Scale bars: 5 µm (whole cell) and 1 µm (enlargements).

(D) Quantification of Sec16A-Rab5-LC3 triple co-distribution events per cell under basal or starved conditions. Means ± SD, *n* = 34–30 cells; unpaired t test: \*\*\*p < 0.0001.

(E) HeLa cells expressing Sec16A-GFP and Rab5-CFP, fixed and stained for Sec31, under basal or starved (15 min) conditions. Scale bars: 5 µm (whole cell) and 1 µm (enlargements).

(F) Percentage of Sec16A puncta colocalizing with Sec31 relative to total Sec16A puncta per cell under basal or starved conditions. Means ± SEM, *n* = 39 cells per condition; unpaired t test: \*\*\*p < 0.0001.

(G) Live-cell FRAP imaging of HeLa cells expressing Sec16A-GFP and Rab5-RFP under basal or starved (15 min) conditions. Scale bar: 1 µm.

(H) Average fluorescence recovery curves of Sec16A puncta associated (Rab5+) or not (Rab5−) with Rab5 during FRAP experiments under basal or starved conditions. Inset shows zoom of first 45 s of recovery.

(I) Quantification of recovery efficiency from FRAP experiments, represented as slope of regression lines from normalized fitted curves. Violin plot; *n* = 12–15 Sec16A puncta from 4 independent experiments; Welch's test: \*p = 0.0246.

(J) HeLa cells expressing Sec16A-GFP and Rab5-RFP, starved for 15 min and fixed after 3-min incubation at 42°C, 37°C, or 25°C. White arrowheads indicate Sec16A-Rab5 colocalization sites; green arrowheads indicate Sec16A puncta alone. Scale bars: 5 µm (whole cell) and 1 µm (enlargements).

(K) Quantification of Sec16A puncta area (µm²), depending on Rab5 association or not, in starved HeLa cells fixed after temperature treatments. Means ± SEM, *n* = 130 puncta; two-way ANOVA: \*\*\*p < 0.0001 and ns, not significant.

live imaging during starvation in TG conditions. While Rab5-Sec16A contact durations were similar to those observed for Rab5-VMP1 contacts (see Figures 2E and 2G for Rab5-VMP1), TG treatment resulted in the stabilization of endosome-Sec16A contacts over time (Figures S13C and S13D). These findings indicate that, under starvation, the recruitment of endosomes in EERCSs at Sec16A-positive ER sites depends on local calcium release from the ER. Moreover, ITPR-mediated Ca²⁺ is specifically required for the disassembly of these ER-endosome contacts.

Altogether, our findings show that starvation-induced endosomal recruitment to Sec16A/ERES domains of the ER induces LLPS associated with Ca²⁺, most likely reflecting the formation of a locally confined environment in the ER-endosome inter-space associated with phagophore biogenesis.

### Phagophore formation at EERCSs requires the recruitment of Rab3/RAB3GAP machinery

Alongside their role in LLPS, Ca²⁺ and Ca²⁺-associated channels are known regulators of RAB3GAP1 and RAB3GAP2 proteins,⁶³
which were identified in the split-TurboID screen (Figures 3C–3E; Data S1). Interestingly, cells depleted for both RAB3GAP1/2 showed a decrease in the formation of autophagosomes (as shown by ATG13-LC3 and phospho-ATG16L1 puncta, Figures S14A–S14F), reinforcing their reported role in autophagy regulation.⁶⁴,⁶⁵ RAB3GAP1/2 are regulatory factors for small GTPases Rab3a and Rab18.⁶⁴,⁶⁵ In neuronal cells, RAB3GAP1/2 promote GTP hydrolysis from synaptic vesicle-associated Rab3a upon Ca²⁺ stimulation in order to promote vesicular fusion and synaptic content release.⁶³,⁶⁶ We thus monitored the behavior of Rab3a and Rab18 after 15 min of starvation to decipher which GTPase could be regulated by RAB3GAP1/2 in our experimental conditions. While endogenous Rab3a signal fluorescence increased and displayed a puncta pattern (Figures 6A and 6B) after 15 min of starvation, the Rab18-associated signal was decreased in the same situation (Figures S15A and S15B). To go further, we analyzed the cellular pattern of fluorescent Rab3a-GFP or Rab18-mRuby in cells expressing the ER marker Sec61β. Upon starvation, Rab3a-GFP displayed a switched distribution from diffuse to massive ER localization, as assessed by co-distribution with Sec61β (Figures S15C–S15F). On the other hand, the Rab18-mRuby pattern did not show any changes upon starvation treatment (Figure S15G).

Based on these observations, we focused on Rab3a mobilization, and we observed that Rab3a puncta formation coincides with EERCSs upon nutritional stress in fixed (Figures 6C and 6D) or live (Figures 6E and 6F) conditions. The formation of ER-localized Rab3a puncta preceded starvation-induced Ca²⁺ bursts, which concomitantly decreased the Rab3a signal (Figure 6G), suggesting that Rab3a dynamics is regulated by local ER Ca²⁺ release. Consistent with our previous results, Rab3a puncta were also found to co-distribute with LC3 at EERCSs (Figures 6H and 6I) and additional early autophagic markers (DFCP1 and FIP200, Figures S16A–S16D) in response to starvation. Similar data were obtained for Rab11 and LC3 after 1 h of starvation (Figures S16E and S16F).

The role of the Rab3/RAB3GAPs partnership at synapses is directly related to vesicular formation and membrane fusion.⁶³,⁶⁶ We thus hypothesized that the local EERCS interspace could generate conditions for transient nano-vesicle formation, which could determine the origin of pre-phagophore membranes. To visualize putative undefined membranous structures, we performed EM analyses on starved cells knocked down for RAB3GAPs or Rab3a (Figure 7A). RAB3GAP knockdown slightly increased ER-endosome tethering, while the knockdown of Rab3a strongly disrupted ER-endosome contacts (Figures 7A and 7B). Compared with control-depleted cells, the decrease of RAB3GAP expression resulted in the massive accumulation of electron-dense vesicular material surrounding endosomal membranes in close proximity to the ER (Figures 7A and 7C). Surprisingly, such dense material was totally absent from the endosomal surface in Rab3a-depleted cells (Figures 7A and 7C); these results together suggest that nano-vesicles, presumably bearing Rab3a, are transiently formed in the EERCS interspace in response to starvation and that the absence of Rab3a-regulatory RAB3GAP proteins leads to their accumulation.

As ATG9A vesicles have been proposed to be the seed of pre-autophagosomal membranes,⁶⁷⁻⁷⁰ we analyzed the putative

ATG9A/Rab3a colocalization in our experimental framework. We observed that the triple ATG9-Rab3a-Rab5 co-distribution was increased upon starvation situation (Figures S16G and S16H), indeed arguing for a concomitant mobilization of ATG9A and Rab3a at EERCSs during phagophore biogenesis.

To address more precisely the role of Rab3a and its regulating proteins RAB3GAP1/2 in the autophagic process, we monitored the dynamics of the LC3 signal associated with EERCSs by live imaging in cells depleted of RAB3GAP1/2 or Rab3a proteins and grown in the starved condition. On the one hand, siCTRL cells specifically displayed an increase of LC3-EERCS co-distribution upon starvation, whereas RAB3GAP-depleted cells exhibited a drastic accumulation of both markers, independently of the nutritional status of the cells (Figures 7D and 7E), a phenomenon that could suggest a massive accumulation of lipidated LC3 at these sites. On the other hand, the sole knockdown of Rab3a inhibited the starvation-induced LC3 association at EERCSs (Figures 7D and 7E). Together, these data show that Rab3a and RAB3GAP1/2 are key actors in the starvation-dependent dynamics of EERCSs prior to autophagosome biogenesis. Our results suggest that the confined environment promoted by EERCSs favors Rab3a recruitment and the endosomal-derived formation of nano-vesicles, which, upon ER-mediated Ca²⁺ release and RAB3GAP1/2 activation, fuse together to form the nascent phagophore membrane.

DISCUSSION

The origin of autophagosomal membranes and the mechanisms of de novo phagophore assembly have been long-standing questions. While ER mobilization, particularly via omegasomes, has been emphasized for decades,⁷¹ other studies have highlighted the role of recycling endosomes, marked by Rab11, in autophagosome biogenesis.⁷² Here, we reveal a starvation-induced interplay between ER and endosomes through EERCSs involving the ER-resident protein KTN1, which binds endosomal PI3P.⁴⁸ We show that endosomal involvement in early autophagy occurs in two waves: Rab5+ early endosomes are first recruited, followed by Rab11+ endosomes, possibly enabling prolonged adaptation. EERCSs create localized cytoplasmic confinement and ER Ca²⁺ release, promoting protein phase separation. Within EERCSs, endosomes tether to ERESs, redirecting their conventional role toward autophagosome formation. We also identify Ca²⁺-dependent Rab3a-RAB3GAP1/2 machinery involved in dense nano-vesicle fusion, contributing to pre-phagophore seeding.

The endolysosomal system integrates intra- and extracellular stress signals.²⁷,³⁰,⁷³ We recently showed that early endosomes are transiently reshaped via SNX1/2 during starvation,³¹,⁷⁴ and prior studies also linked endosomal proteins to autophagy.³²⁻³⁴,⁷⁵ Our data now demonstrate a direct contribution of endosomal membranes to autophagosome biogenesis via EERCSs. This progression from Rab5 to Rab11, mediated by SNX1, supports a timely response, although the regulation of this switch remains unclear. Notably, EERCS inhibition in Rab3a-depleted cells correlates with enlarged endosomes containing more intraluminal vesicles, suggesting a delay in their maturation⁷⁶,⁷⁷ to prioritize autophagy.
<figure><img src="image_39.png" /><figcaption>**Figure 6.** Rab3a is recruited at EERCSs upon starvation</figcaption></figure>

(A) HeLa cells transiently expressing Sec61β-RFP, fixed and stained with anti-Rab3A antibody under basal (ctrl) or starved (15 min) conditions. Dashed lines outline individual cells. Scale bars: 5 µm.

(B) Quantification of Rab3A fluorescence intensity per cell in basal and starved HeLa cells. Means ± SD, *n* = 32–35 cells; unpaired t test: \*p = 0.0288.

(C) HeLa cells transiently expressing Rab3A-GFP, Sec61β-RFP, and Rab5-CFP in basal or starved (15 min) conditions. Arrowheads indicate sites of endosome-ER contacts. Scale bars: 5 µm (cell) and 1 µm (zoom).

(D) Quantification of Rab3A co-distribution with EERCS (Rab5<sup>+</sup>Sec61β<sup>+</sup>) per cell under basal or starved conditions. Means ± SD, *n* = 55–48 cells; unpaired t test: \*\*\*p < 0.0001.

(E) Live-cell imaging of HeLa cells expressing Rab3A-GFP, VMP1-mCherry, and Rab5-CFP after 15 min starvation. White hollow arrow indicates endosome-ER contact associated with Rab3a. Scale bar: 1 µm.

(F) Quantification of the percentage of Rab3A puncta associated with EERCS (Rab5<sup>+</sup>VMP1<sup>+</sup>) in basal or starved HeLa cells. Each dot represents one Rab3A punctum, scored as associated (=100) or not (=0) with EERCS; means represent the overall percentage association. Means ± SEM, *n* = 100 puncta per condition; unpaired t test: \*\*\*\*p < 0.0001.

(G) Live imaging of HeLa cells transiently expressing GCamp6f-Cyb5 and Rab3A-RFP after 15-min starvation. Red arrowheads indicate Rab3A puncta; green arrowheads indicate GCamp6f-Cyb5 puncta. Scale bar: 1 µm.

(H) HeLa cells expressing Rab3A-GFP, Sec61β-RFP, and Rab5-CFP, fixed and stained for LC3 under basal or starved (15 min) conditions. Arrowheads indicate Rab3A-EERCS association sites. Scale bars: 5 µm (cell) and 1 µm (zoom).

(I) Quantification of Rab3A-Rab5-LC3 triple co-distribution events per cell in basal and starved conditions. Means ± SEM, *n* = 68–56 cells; unpaired t test: \*\*\*\*p < 0.0001.

EERCSs assemble rapidly—within 15 min of starvation—to support autophagosome biogenesis. Other ER-based contact sites, like those with mitochondria and the plasma membrane,¹⁵,²⁵ also act as autophagy initiation hubs. Organellar

specificity may depend on stress type, timing, cell identity, or subcellular localization. Such flexibility could explain why disrupting a single contact type rarely abolishes autophagy entirely. Our findings reveal EERCSs as confined, transient
<figure><img src="image_40.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_41.png" /></figure>

ER endosome dense material / vesicles

<figure><img src="image_42.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_43.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_44.png" /><figcaption>**Figure 7.** Phagophore formation at EERCSs requires the recruitment of Rab3/RAB3GAP machinery</figcaption></figure>

(A) Electron microscopy images of HeLa cells in starved conditions (15 min), either control (siCTRL) or knocked down for RAB3GAP1 and RAB3GAP2 (siR-AB3GAPs) or Rab3A (siRab3A). Bottom images show manual coloring: ER in blue, endosomes in orange, and dense/vesicular material in purple. Scale bars: 200 nm.

(B) Quantification of the percentage of endosomal structures contacting the ER in starved cells across conditions. Each dot represents a single endosome either associated (=100) or not associated (=0) with the ER; the mean represents the overall percentage. Means ± SEM; *n* = 40–45 endosomes; one-way ANOVA: **p = 0.0094** and ***p < 0.0001.

(C) Quantification of the percentage of endosome-ER contacts associated with dense material under the same conditions as in (B). Each dot represents one endosome-ER contact either associated (=100) or not (=0) with dense material. Means ± SEM; *n* as above. One-way ANOVA: **p(siCTRL vs. siRAB3GAPs) = 0.0083**, **p(siCTRL vs. siRab3A) = 0.0032**, and ***p < 0.0001.

(D) Representative stills from live imaging of HeLa cells in starved conditions expressing Rab5-CFP, VMP1-GFP, and LC3-RFP, comparing control and knockdown conditions. Scale bars: 5 µm.

(E) Percentage of newly appearing LC3 puncta associated with endosome-ER contacts relative to total new LC3 puncta per cell under basal and starved conditions across control and knockdown cells. Means ± SEM; *n* = 11–32 cells; two-way ANOVA: ****p < 0.0001.

microenvironments that support organelle biogenesis. While further work is needed to characterize LLPS at ERESs, our data suggest that EERCSs foster condensate formation akin to “pre-autophagic droplets,”¹⁸ concentrating autophagy regulators.⁷⁸ This may also support the idea that LC3 lipidation requires phase separation,⁷⁹ as recently suggested. Ultimately, EERCSs disassemble to release proto-phagophores and allow subsequent steps like ATG2A-mediated lipid transfer.⁶⁷,⁸⁰⁻⁸² Similar concepts might be extended to mitochondria and plasma membrane contributions, which have primarily been associated with lipid supply.²⁰,²⁴ It remains to be explored whether PI3P is directly transferred from endosomes to the omegasome/pre-phagophore zone via the ANKFY1-ATG2A axis.³⁵

Although autophagosome biogenesis has been extensively studied,⁸³ our present work points out that both the “ER hypothesis” and “endosome hypothesis” are converging. Four major

hypotheses for phagophore initiation exist: (1) clustering of the initial autophagic complexes via liquid-liquid phase transition, possibly at the ER surface¹⁷⁻¹⁹; (2) ATG9 and COP-II vesicle-dependent creation of a proto-phagophore; (3) formation of a Golgi/endosome hybrid compartment (HyPAS)⁸⁴; and (4) recycling endosomes converted into a phagophore.⁸⁵ Here, we show that Rab5 and Rab11 EERCSs promote local Ca²⁺ bursts that induce phase transitions at ERESs. Ca²⁺ signaling regulates multiple autophagy steps, from omegasome to autophagosome formation. Both our study and others¹⁹,⁵² find that ITPR-mediated Ca²⁺ release supports phagophore formation, while SERCA-mediated reuptake is needed for phagophore detachment and autophagosome maturation.⁸⁶ We further show that TPC1 channels on endosomes initiate ITPR activation, resembling TPC2 function at lysosomes,⁵³ emphasizing ER-endosome crosstalk.
ATG9 vesicles are recognized seeds for autophagosome formation⁶⁸ yet likely require local fusion of multiple vesicles. We propose that ATG9+ endosomal nano-vesicles fuse via Rab3a-RAB3GAP1/2 to form proto-phagophores. This aligns with reports of ATG9-Rab3 colocalization in presynaptic vesicles⁸⁷ and the recent identification of ATG9+ nano-vesicle subtypes.⁸⁸ Further studies are needed to define the lipid composition of Rab3a+ vesicles and their relationship to ATG9 structures.

In summary, our study highlights the importance of spatial and temporal membrane dynamics in stress response. We identify fast, reversible ER-endosome tethering as a key mechanism in phagophore formation and emphasize the need to consider organelle dynamics when analyzing contact sites under stress.

## Limitations of the study

Our study reveals that ER-endosome contact sites promote phagophore biogenesis during starvation. While amino acid deprivation strongly induces autophagy, the role of ER-endosome contacts under other stress conditions remains unexplored. Most insights come from HeLa cells, which may not reflect autophagosome formation in differentiated or polarized cells. It is also unknown whether other ER contact sites contribute to phagophore assembly alongside ER-endosome contacts. Despite these gaps, our findings underscore ER-endosome interactions in stress responses and lay the groundwork for future studies on early autophagic processes.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Etienne Morel (etienne.morel@inserm.fr).

### Materials availability

Plasmids generated in this study will be made available upon request.

### Data and code availability

* This paper does not report original code.

* Data reported in this paper are available from the lead contact upon request.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank T. Rapoport (Boston, USA), V. Haucke (Berlin, Germany), M.I. Vacaro (Buenos Aires, Argentina), H. Zhang (Beijing, China), J. Gruenberg (Geneva, CH), T. Schikorski (Bayamón, Puerto Rico), N. Mizushima (Tokyo, Japan), and F. Perez and A. Claude-Taupin (Paris, France) for sharing their tools with us. We are grateful to our colleagues Aurore Claude-Taupin, Cédric Delevoye, and Patrice Codogno from INEM (Paris, France); Carine Joffre from CRCT (Toulouse, France); and Franck Perez and Zeina Chamoun-Morel from Institut Curie (Paris, France) for a critical reading of the manuscript. We thank Necker SFR technical platforms and especially Nicolas Goudin (bio-image analysis) and Meriem Garfa-Traoré and Sébastien Dupichaud (cell imaging). The authors received financial support from INSERM, CNRS, Université-Paris Cité, ANR (ANR-17-CE140030-02, ANR-17-CE13-0015-003, ANR 22-CE14-0019, and ANR 18-CE14-0006 to E.M.; ANR-22-CE11-0024-01 to F.G.; and ANR 10-INBS-04 to G.R.); FRM (to E.M.); and AFM Telethon (Project AFM 23778, to F.G.). J.D.G. is a recipient of a fellowship from the French Ministry of Research and a 4th year PhD FRM scholarship (FDT202304016558).

## AUTHOR CONTRIBUTIONS

J.D.G. performed all the biochemical and imaging experiments, formal analysis, quantifications, data analyses, and manuscript writing; C.T., M.R., C.A., D.A., A.S., and S.D. contributed to some of the biochemical and imaging experiments; I.C.G. performed the mass spectrometry experiments and contributed manuscript writing; N.E.K. and M.R. performed some of the EM experiments; F.G. and G.R. helped with the EM analyses and contributed to manuscript writing; and E.M. supervised the project and the methodology, contributed to formal analysis, and wrote the manuscript.

## DECLARATION OF INTERESTS

The authors have no conflicts of interest to declare.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Cell culture

* Cell transfection

* METHOD DETAILS

* Cell treatments

* Antibodies

* Immunofluorescence and analyses

* Proximity ligation assay (PLA)

* Electron microscopy

* Expansion microscopy

* Biochemical purification of EERCS

* Analyses using machine learning

* Live imaging and RUSH assay

* Over-stabilization of EERCS

* Split TurboID and mass spectrometry analysis

* Data processing following LC-MS/MS acquisition

* Co-immunoprecipitations assays

* FRAP experiment and analyzes

* SDS-PAGE and western blotting

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.115993.

Received: August 14, 2024

Revised: March 20, 2025

Accepted: June 19, 2025

Published: July 10, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Mouse anti-EEA1</td><td>BD TransLab</td><td>Cat#: 610456</td></tr><tr><td>Rabbit anti-VAP-B</td><td>ProteinTech</td><td>Cat#: 14477-1-AP</td></tr><tr><td>Rabbit anti-Actin</td><td>Sigma</td><td>Cat#: A2066</td></tr><tr><td>Mouse anti-Calnexin</td><td>BD Bioscience</td><td>Cat#: 610523</td></tr><tr><td>Rabbit anti-VMP1</td><td>Cell Signaling</td><td>Cat#: 12929</td></tr><tr><td>Rabbit anti-VAP-A</td><td>ProteinTech</td><td>Cat#: 15275-1-AP</td></tr><tr><td>Rabbit anti-Rab5</td><td>Cell signaling</td><td>Cat#: 2143</td></tr><tr><td>Mouse anti-Rab7</td><td>Abcam</td><td>Cat#: ab50533</td></tr><tr><td>Rabbit anti-Rab11</td><td>Cell Signaling</td><td>Cat#: 2413S</td></tr><tr><td>Mouse anti-LC3B</td><td>MBL</td><td>Cat#: 152-3</td></tr><tr><td>Rabbit anti-ATG13</td><td>Cell Signaling</td><td>Cat#: 13468</td></tr><tr><td>Rabbit anti-ATG14</td><td>Sigma</td><td>Cat#: A6358</td></tr><tr><td>Rabbit anti-TPCN1</td><td>ProteinTech</td><td>Cat#: 23758-1-AP</td></tr><tr><td>Rabbit anti- ATG16L1(phospho S278)</td><td>Abcam</td><td>Cat#: ab195242</td></tr><tr><td>Mouse anti-Sec31</td><td>BD Biosciences</td><td>Cat#: 612351</td></tr><tr><td>Mouse anti-IP3RI</td><td>Santa Cruz</td><td>Cat#: sc-271197</td></tr><tr><td>Rabbit anti-RAB3GAP1</td><td>MERCK</td><td>Cat#: SAB4500914</td></tr><tr><td>Mouse anti-Rab3A</td><td>Synaptic System</td><td>Cat#: 107 111</td></tr><tr><td>Rabbit anti-Rab18</td><td>Sigma</td><td>Cat#: SAB4200173</td></tr><tr><td>Rabbit anti-KTN1</td><td>Proteintech</td><td>Cat#: 19841-1-AP</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>pLVX-TetONE-ERM-2xHA-FRB</td><td>Jang et al.<sup>48</sup></td><td>N/A</td></tr><tr><td>pLVX-TetONE-mRFP-FKBP-Rab5A</td><td>Jang et al.<sup>48</sup></td><td>N/A</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>1,6-Hexanediol</td><td>MERCK</td><td>240117</td></tr><tr><td>BAPTA-AM</td><td>Abcam</td><td>ab120503</td></tr><tr><td>Thapsigargin</td><td>Bio-Techne</td><td>1138/1</td></tr><tr><td>Biotin</td><td>Sigma</td><td>B4639</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>Naveniflex Cell kits</td><td>Naveni</td><td>NF.MR.100.RED</td></tr><tr><td>Dynabeads Protein G immunoprecipitation kit</td><td>Invitrogen</td><td>10007D</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>HeLa cells</td><td>ATCC</td><td>CCL2</td></tr><tr><td>MEF cells</td><td>ATCC</td><td>CRL-2991</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>siRNA targeting#1 targeting ITPR1</td><td>Qiagen</td><td>SI00034531</td></tr><tr><td>siRNA targeting#2 targeting ITPR1</td><td>Qiagen</td><td>SI03034024</td></tr><tr><td>siRNA targeting#1 targeting ITPR3</td><td>Qiagen</td><td>SI00034594</td></tr><tr><td>siRNA targeting#2 targeting ITPR3</td><td>Qiagen</td><td>SI00034601</td></tr><tr><td>siRNA targeting#1 targeting TPC1</td><td>Qiagen</td><td>SI00749924</td></tr><tr><td>siRNA targeting#2 targeting TPC1</td><td>Qiagen</td><td>SI02777796</td></tr><tr><td>siRNA targeting#1 targeting RAB3GAP1</td><td>Qiagen</td><td>SI04136986</td></tr><tr><td>siRNA targeting#2 targeting RAB3GAP1</td><td>Qiagen</td><td>SI04246088</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>siRNA targeting#1 targeting RAB3GAP2</td><td>Qiagen</td><td>SI04986436</td></tr><tr><td>siRNA targeting#2 targeting RAB3GAP2</td><td>Qiagen</td><td>SI04241293</td></tr><tr><td>siRNA targeting#1 targeting RAB3A</td><td>Qiagen</td><td>SI00044800</td></tr><tr><td>siRNA targeting#2 targeting RAB3A</td><td>Qiagen</td><td>SI00044786</td></tr><tr><td>siRNA targeting#1 targeting KTN1</td><td>Qiagen</td><td>SI03031973</td></tr><tr><td>siRNA targeting#2 targeting KTN1</td><td>Qiagen</td><td>SI03024413</td></tr></tbody></table>

Recombinant DNA

<table><tr><td>DFCP1-mCherry</td><td>Genecopoeia</td><td>EX-Z3423-M56</td></tr><tr><td>DFCP1-CFP</td><td>Addgene</td><td>58765</td></tr><tr><td>Sec61β-GFP</td><td>T. Rapoport lab</td><td>N/A</td></tr><tr><td>Sec61β-RFP</td><td>T. Rapoport lab</td><td>N/A</td></tr><tr><td>VMP1-GFP</td><td>M.I. Vaccaro lab</td><td>N/A</td></tr><tr><td>VMP1-mCherry</td><td>M.I. Vaccaro lab</td><td>N/A</td></tr><tr><td>GCamp6f-Cyb5</td><td>H. Zhang lab</td><td>N/A</td></tr><tr><td>Rab5-GFP</td><td>J. Gruenberg lab</td><td>N/A</td></tr><tr><td>Rab5-CFP</td><td>J. Gruenberg lab</td><td>N/A</td></tr><tr><td>Rab11-GFP</td><td>C. Delevoye</td><td>N/A</td></tr><tr><td>Rab11-mCherry</td><td>C. Delevoye</td><td>N/A</td></tr><tr><td>Rab11-Cerulean</td><td>Addgene</td><td>55390</td></tr><tr><td>Rab3a-GFP</td><td>Addgene</td><td>49542</td></tr><tr><td>ssHRP-myc- KDEL</td><td>T. Schikorski lab</td><td>N/A</td></tr><tr><td>LC3-RFP</td><td>N. Mizushima lab</td><td>N/A</td></tr><tr><td>Sec16a-GFP</td><td>Addgene</td><td>36155</td></tr><tr><td>Rab18-mRuby3</td><td>Addgene</td><td>166431</td></tr><tr><td>ATG9a-GFP</td><td>A. Claude-Taupin</td><td>N/A</td></tr><tr><td>Rab5-RFP</td><td>This study</td><td>N/A</td></tr><tr><td>Rab3a-RFP</td><td>This study</td><td>N/A</td></tr><tr><td>FIP200-RFP</td><td>This study</td><td>N/A</td></tr><tr><td>Rab5-splitTurboID(N)</td><td>This study</td><td>N/A</td></tr><tr><td>Rab11-splitTurboID(N)</td><td>This study</td><td>N/A</td></tr><tr><td>VMP1-splitTurboID(C)-HA</td><td>This study</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>Icy</td><td>https://icy.bioimageanalysis.org/</td><td>N/A</td></tr><tr><td>Fiji</td><td>https://imagej.net/software/fiji/downloads</td><td>N/A</td></tr><tr><td>Ilastik 1.3.post3</td><td>https://www.ilastik.org/download</td><td>N/A</td></tr><tr><td>Imaris 10.1</td><td>https://imaris.oxinst.com/</td><td>N/A</td></tr><tr><td>GraphPad Prism 10.1.1</td><td>https://www.graphpad.com/</td><td>N/A</td></tr></table>

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Cell culture

HeLa cells from ATCC were grown in MEM, supplemented with GlutaMAX (41090093; Thermo Fisher Scientific), 10% FCS, and non-essential amino acid mix (11140035; Gibco, Thermo Fisher Scientific). MEF cells from ATCC were grown in DMEM (41966; Gibco) supplemented with 20% FCS. All cells were incubated at 37°C with 5% CO₂. Both cell lines have been negatively tested for mycoplasma contamination.

Cell transfection

Transfections of cDNA plasmids were performed at 5 ng/µL at least 24h before the experiments using FuGENE (E2312; Promega) according to manufacturer's recommendations. DFCP1-mCherry was bought from Genecopoeia (EX-Z3423-M56), and
DFCP1-CFP is from Addgene (58765). Sec61β-GFP and Sec61β-RFP cDNA were from T. Rapoport (Boston), VMP1-GFP and VMP1-mCherry were from M.I. Vaccaro (Buenos Aires) and GCamp6f-Cyb5 was from H. Zhang (Beijing). Rab5-GFP and Rab5-CFP were from J. Gruenberg (Geneva), Rab11-GFP and Rab11-mCherry were from C. Delevoye (Paris), Rab11-Cerulean and Rab3a-GFP were bought from Addgene (respectively: 55390 and 49542). ssHRP-myc-KDEL was from T. Schikorski (Puerto Rico), LC3-RFP was from N. Mizushima (Tokyo) and Sec16a-GFP and Rab18-mRuby3 were bought from Addgene (36155 and 166431). ATG9a-GFP was from A. Claude-Taupin (Paris). Rab5-RFP, Rab3a-RFP, FIP200-RFP were made using NEBuilder HiFi DNA Assembly Master Mix (E2621L; New England BioLabs) from Sec61β-RFP and respectively Rab5-GFP, Rab3a-GFP and p3xFLAG-CMV10-hFIP200 (Addgene 24300). Rab5-splitTurboID(N) and Rab11-splitTurboID(N) were made after PCR amplification of Rab5 and Rab11 from Rab5-GFP and Rab11-GFP, while splitTurboID(N) was amplified from pLX304 CMV FKBP-V5-sTurboID (N) (Addgene, 153002). PCR products were recombined using Gibson assembly, integrated into pDNOR221 using Gateway cloning system and then put in pDEST-2xFLAG-Cter (Addgene 118372). VMP1-splitTurboID(C)-HA was made by generating a sequence encoding for VMP1-splitTurboID (C)-HA using Eurofins Genomics services than was inserted into a pDEST-2xFLAG-Cter (Addgene 118372) using Gateway cloning. Knockdown of human proteins were performed using a pool of two siRNAs, bought from QIAGEN, for each protein: ITPR1 (SI00034531 and SI03034024), ITPR3 (SI00034594 and SI00034601), TPC1 (SI00749924 and SI02777796), RAB3GAP1 (SI04136986 and SI04246088), RAB3GAP2 (SI04986436 and SI04241293), Rab3a (SI00044800 and SI00044786), KTN1 (SI03031973 and SI03024413). siRNAs were transfected at 20 nM using Lipofectamine RNAiMax (13778150; Thermo Fisher Scientific) 72h prior experiment.

## METHOD DETAILS

### Cell treatments

For starvation analyses, cells were washed twice with 1× PBS and incubated in Earle's Balanced Salt Solution (EBSS, 24010043; Thermo Fisher Scientific) for the indicated times. For LLPS experiments, cells were either put 3 min before fixation at 42°C, 37°C and 25°C, or treated with 0.1% 1,6-Hexanediol (1,6-HD; 240117; MERCK) with EBSS during 15 min. To study calcium fluxes, cells were either treated with BAPTA-AM 25µm (Abcam; ab120503) in EBSS during 15 min, or pretreated with thapsigargin (TG; Bio-Techne; 1138/1) for 1 h before and during EBSS treatment.

### Antibodies

The EEA1 protein was labeled by anti-EEA1 mouse antibodies from BD TransLab (610456) for both immunofluorescence (IF) and PLA (dilution 1/200) and for western blot (WB) (dilution 1/3000). For PLA, VAP-B protein was labeled by anti-VAPB rabbit antibodies from ProteinTech (14477-1-AP, dilution 1/150). Actin protein was used as a loading control for WB with anti-Actin rabbit antibodies from Sigma (A2066, dilution: 1/10,000). Calnexin was revealed by WB using anti-calnexin mouse antibodies from BD Bioscience (610523, dilution 1/3000), VMP1 was revealed by anti-VMP1 rabbit antibodies from Cell Signaling (12929, dilution 1/3000) and VAP-A was labeled by anti-VAPA rabbit antibodies from ProteinTech (15275-1-AP, dilution 1/3000). WB for endosomal proteins such as Rab5 used anti-Rab5 rabbit antibodies from Cell signaling (2143, dilution 1/1000), for Rab7 we used anti-Rab7 mouse antibodies from Abcam (ab50533, dilution 1/1000) and for Rab11 we used anti-Rab11a rabbit antibodies from Cell Signaling (2413S, dilution 1/1000). The LC3 protein was labeled by anti-LC3B mouse antibodies from MBL (152-3, dilution 1/200). ATG13 was targeted using anti-ATG13 rabbit antibodies from Cell Signaling (13468, dilution 1/150). ATG14 was labeled with anti-ATG14 rabbit antibodies from Sigma (A6358, dilution 1/150). TPC1 was stained using anti-TPCN1 rabbit antibodies from ProteinTech (23758-1-AP, dilution 1/150). The phosphorylated form of ATG16L1 was stained using anti-ATG16L1(phospho S278) rabbit antibodies from Abcam (ab195242, dilution 1/150). Sec31 protein was stained using anti-Sec31mouse antibodies from BD Biosciences (612351, dilution 1/150). ITPR1 and RAB3GAP1 were used to check siRNA efficiency using respectively anti-IP3R-I mouse antibodies from Santa Cruz (sc-271197, dilution 1/1000) and anti-RAB3GAP1 rabbit antibodies from MERCK (SAB4500914, dilution 1/3000). IF staining for Rab3a was done using anti-Rab3a mouse antibodies from Synaptic System (107 111, dilution: 1/150). Rab18 was observed using anti-Rab18 rabbit antibodies from Sigma (SAB4200173, dilution 1/150). KTN1 detection was made using anti-KTN1 rabbit antibodies from (Proteintech; 19841-1-AP) for both immunofluorescence (dilution: 1/150) and western blot (dilution 1/3000).

### Immunofluorescence and analyses

For classical immunofluorescence analyses, cells were seeded onto 12-mm glass coverslips and fixed with 4% PFA pre-warmed at 37°C for 25min. Cells were then washed with PBS and blocked with 10% FCS in PBS for 30min. Incubation with primary antibodies diluted in permeabilization/blocking buffer (PBS with 10% FCS and 0.05% saponin) was done for 2h at room temperature. Coverslips were PBS-washed before and after adding secondary antibodies for 1h in blocking buffer. Coverslips were mounted on microscope slides using Mowiol containing or not DAPI. All images were taken using Zeiss Spinning Disk microscope with a 63× oil-immersion objective. Image analyses for puncta measurement was done using Icy software (https://icy.bioimageanalysis.org/) with Spot Detector plugin. Number of codistribution events were manually quantified using Fiji software (https://imagej.net/software/fiji/downloads) and Cell Counter tool. Analyses of GCamp6f-Cyb5 local increase of signal was done using Fiji software by first drawing a segmented line on ER membrane (length between 1 and 3µm) on sites that show high proximity with endosomes and obtain RGB profile plot
using Color profiler plugin (Color_Profiler. jar, https://imagej.nih.gov/ij/plugins/color-profiler.html). On each RGB plot (X and Y axes) we looked at the X coordinate where Rab5 channel exhibited the highest fluorescence intensity according to Y axe. Keeping the same X coordinate, we looked at the Y value of the GCamp6F-Cyb5 channel and subtracted this value to its minimum intensity on the same RBG plot to normalize all experimental measurements. The same method has been used to analyze ER versus cytosol ratio. Measurements of the percentage of EEA1-TPC1 codistribution was made using Fiji software by using color threshold tool with the value 25–50 to obtain the area of colocalization over total signal. Measurements of the signal intensity of Rab3a and Rab18 were done using Fiji software by calculating the ratio of maximal/minimal signal intensity in each cell.

## Proximity ligation assay (PLA)

Cells were cultured on 12mm glass coverslips, fixed with 4% PFA pre-warmed at 37°C for 25min and wash two times in PBS prior PLA staining. All PLA experiments have been done using Naveniflex Cell kits (NF.MR.100 and NC.MR.100.RED) according to manufacturer's recommendations, with all incubation steps at 37°C. Briefly, cells were first incubated with blocking buffer for 1h, then with primary antibodies diluted in primary antibody diluent for another hour. Cells were washed 3 × 5 minutes with PBS-Tween 0.05%, incubated with probes diluted in probes diluent (1/40) for 1h and washed again. Reaction A was prepared using diluting buffer A with enzyme A (1/40) and incubated with cells for 1h. After washes of 2 × 3 minutes with PBS-Tween 0.05%, cells were incubated for 30 min with reaction B composed of diluting buffer B with enzyme B (1/40). Lastly, cells were washed 2 × 3 minutes with PBS-Tween 0.05%, incubated with reaction C (Diluting Buffer C with enzyme C at 1/40 dilution) for 90min, washed again 3 × 5 minutes with PBS-Tween 0.05% and mounted using Mowiol containing DAPI.

## Electron microscopy

For electron microscopy, two distinct protocols have been used. In the first protocol, used to observe HeLa cells expressing HRP-KDEL, cells were fixed with 1.3% glutaraldehyde in 0.1M cacodylate buffer, washed in 0.1M ammonium phosphate (pH 7.4) buffer for 1h, and the HRP was visualized with 0.5 mg/mL DAB and 0.005% H2O2 in 0.1M ammonium phosphate (pH 7.4) buffer followed by extensive washes with cold water. Cells were post-fixed in 1%OsO4+1.5% K3Fe(CN)6 in 0.1 M cacodylate buffer at 4°C for 1h, washed in cold water, and then contrasted in 0.5% uranyl acetate for 24h at 4°C, dehydrated in an ethanol series and embedded in Epon. Ultrathin sections were contrasted with 4% uranyl acetate and observed under a FEI Tecnai 12 microscope equipped with a OneView 4k Gatan camera.

For the observation of knocked-down cells we used a second protocol of chemical fixation and sample preparation described in Hurbain et al.⁸⁹ Briefly, cells were fixed overnight at 4°C with 2.5% glutaraldehyde diluted in 0.1M cacodylate buffer, incubated with 1% osmium tetroxide and 1.5% Potassium ferrocyanide diluted in 0.1M cacodylate buffer, dehydrated in ethanol and embedded in Epon. Sections were prepared using a Reichert UltracutS ultramicrotome (Leica Microsystems) and contrasted with uranyl acetate and lead citrate. Images were acquired with a Tecnai Spirit (Thermo Fisher Scientific) operating at 80kV associated with QUEMESA CCD camera (Olympus).

## Expansion microscopy

Preparation for expansion microscopy has been done using the same protocol previously described.³¹ Briefly, cells after fixation and regular IF (using doubled concentrated antibodies) were incubated with 0.1 mg/mL Acryloyl-X (A20770; Invitrogen) at 4°C overnight. Coverslips were quickly (1-2 min) incubated at room temperature with monomer solution (PBS 1×, 19% sodium acrylate, 10% acrylamide, and 0.1% N, N'-methylenebisacrylamide). Gelation was performed by putting coverslips with cells facing a 35µL drop of monomer solution supplemented with 0.5% APS and 0.5% TEMED on Parafilm and incubated for 30min at room temperature. Coverslips with gels were transferred into 2 mL of digestion buffer (Tris-acetate-EDTA buffer 1×, 0.5% Triton X-100, and 0.8 M guanidine HCl) supplemented with fresh proteinase K (8 U/ml) and incubated for exactly 30 min at room temperature. Gels were collected and carefully transferred into ~50 mL of deionized water for expansion. Water was changed twice every 30 min, with one wash containing DAPI, and incubated overnight at room temperature in the dark for full expansion. Gels were observed on 35mm glass bottom µ-Dish (IBIDI, 81158) precoated with poly-D-Lysine 0.1mg/mL (Gibco, A3890401).

## Biochemical purification of EERCS

HeLa cells were grown as a single monolayer on two 150mm dishes per condition to ensure high final yield. After treatment cells were washed on ice with cold 1X PBS to stop intracellular trafficking. All further steps were performed with high gentle care on ice and all centrifuges were done at 4°C. Cells were collected with 1X PBS using a plastic rubber and centrifuged at 125g for 5min. Pelleted cells were resuspended in homogenization medium (8% sucrose, 3mmM imidazole pH7.4 and 0.1mM EGTA), centrifuged at 750g for 10min and the new pellet was resuspended with 1 volume of homogenization buffer. The mixture was homogenized using a 22G needle (0.7 mm × 32 mm) and cell lysate were monitored under classic bright field optic microscope to obtain isolated nucleus with floating organelles. Homogenized solution was then centrifuged a first time at 750g for 10 min and a second time at 1 180g for 10min to collect the post nuclear supernatant (PNS). PNS were then resuspended in one volume of 62% sucrose to obtain a final concentration of 40.6% sucrose. Sucrose gradients were prepared by adding different sucrose concentration in the following order: 40.6% PNS, 35% sucrose and 8% sucrose. Gradients were centrifuged for 1h at 150 000g and light membranes, present at 35%–8% interface, were collected. Light membranes were resuspended in 3 volumes of 3mM imidazole, very delicately poured on top of a
Percoll solution (225mM mannitol, 25mM HEPES pH7.4, 1mM EGTA and 30% Percoll) and centrifuged at 95 000g for 30min.
Apparent white band was collected and named "EERCS" and the bellowed fraction, with no apparent band, was collected and
named "endosomes". All fractions were diluted to a final concentration of Laemmli 1X, 25mM DTT and heat at 95°C for 5 min before
western blot analyzes.

Analyses using machine learning

Quantifications using machine learning and Imaris was done as reported previously (25). Briefly, samples were immunostained with classical labeling and images were acquired using a Zeiss Spinning Disk microscope with z stack of 0.08-µm interval slices and 0.07µm pixel size. Then, channels were split using ImageJ 2.1.0, and each channel was independently subjected to ilastik 1.3. post3 software for machine learning detection. Machine learning was based on 31 features with sigma 0.30-1.60 in 3D and 3.50-5.00 in 2D (Gaussian Smoothing, Laplacian of Gaussian, Gaussian Gradient Magnitude, Difference of Gaussians, Structure Tensor Eigenvalues, and Hessian of Gaussian Eigenvalues). Data were exported as binary mask, merged with raw channels using again Im- ageJ 2.1.0 and analyzed in Imaris 10.1. To allow better 3D visualization, segmented channels were detected as surfaces by Imaris 10.1. with no further changes in the settings. EEA1 and Rab11 surfaces were quantified using "classification" tool and based on the shortest distance feature with ER surface and other markers.

Live imaging and RUSH assay

HeLa cells were transfected with indicated cDNAs 36h-48h prior live recording and seeded in an 8-wells IBIDI cell chamber (80826; IBIDI). Imaging was performed at 37°C and 5% CO₂ with Zeiss Spinning Disk Microscope using a 63× oil-immersion objective with 5sec intervals for 1min. EERCS timing and apparition of DFCP1 puncta were manually tracked and measured using Fiji software. Appearance timing of DFCP1 puncta and GCamp6f burst was made using the "create_spectrum_jru_v1" plugin in Fiji (downloaded from https://research.stowers.org/imagejplugins/zipped_plugins.html).

RUSH assay was performed with HeLa cells stably expressing ManII-mCherry (gift from F. Perez, Institut Curie). Cells were washed twice with 1XPBS, incubated 10 min with EBSS then 5 min with EBSS containing 50µM Biotin (B4639, Sigma) and recorded with Zeiss Spinning Disk Microscope as described above.

Over-stabilization of EERCS

To stabilize endosomes-ER contacts we generated HeLa stable cell line with doxycycline -inducible ERM-2xHA-FRB and mRFP-FKBP-Rab5A constructs. Plasmids pLVX-TetONE-ERM-2xHA-FRB and pLVX-TetONE-mRFP-FKBP-Rab5A were a gift from V. Haucke (Berlin) and were made as described in Jang et al.⁴⁸ Lentivirus were produced in HEK293T cells transfected with pREV, pVSVG, pRER and pLVX vectors by calcium phosphate method. Next, HeLa cells were infected with enriched-lentivirus supernatant and selected with puromycin (1µg/mL) 48h later. Cells were induced with doxycycline (1µg/mL) for 18h and sorted as single cell via RFP signal with flow cytometer (BD FACS Aria II SORP). Monoclonal populations were grown and tested for both ERM-2xHA-FRB and mRFP-FKBP-Rab5A expression and analyzed for autophagic readouts. Clonal cells were plated a day before adding doxycycline (1µg/mL) with or without rapalog (0.5µM A/C Heterodimerizer, 635056 Takara) and submitted to EBSS treatment 18h later.

Split TurboID and mass spectrometry analysis

HeLa cells were transfected with both pDEST-HA-splitTurboID(C)-VMP1 and pDEST-V5-splitTurboID(N)-Rab11 or pDEST-V5-splitTurboID(N)-Rab5 24h prior treatment. Cells were washed 2x with PBS then treated for 6h with EBSS supplemented with 50µM Biotin (B4639, Sigma) or without Biotin addition for negative control. Biotinylated proteins were purified following the protocol of Cho et al.⁴⁹ Briefly, cells were lysed using RIPA buffer (899001, Thermo scientific) with proteases and phosphatases inhibitor cocktail (78443; Pierce) and centrifuged at 15 300g for 10min at 4°C. Supernatants were incubated overnight with streptavidin beads (88817, Pierce), previously washed with RIPA buffer. Beads were then washed 2x with RIPA buffer, once with 1M KCl, once with 0.1M Na₂CO₃, once with 2M urea in 10mM Tris-HCl pH8, twice with digestion buffer (75mM NaCl, 1mM EDTA, 50mM Tris-HCl pH8) and finally resuspended in Laemmli 4X supplemented with 100mM DTT and 2mM Biotin for elution. S-TrapTM micro spin column (Protifi) digestion was performed according to manufacturer's instructions. Briefly, samples were supplemented with 20% SDS to a final concentration of 5%, reduced with 20mM Tris(2-carboxyethyl) phosphine hydrochloride and alkylated with 50mM chloracetamide for 10min at 95°C. Aqueous phosphoric acid was then added to a final concentration of 2.5% following by the addition of S-Trap binding buffer (90% aqueous methanol, 100mM TEAB, pH7.1). Mixtures were then loaded on S-Trap columns, washed five times, and digested with 1.5µg of trypsin (Promega) at 47°C for 2h. After elution, peptides were vacuum dried and resuspended in 2% ACN, 0.1% formic acid in HPLC-grade water prior to mass spectrometry analysis. The tryptic peptides were injected on a nanoElute (Bruker Daltonics) HPLC system coupled to a timsTOF Pro (Bruker Daltonics) mass spectrometer. HPLC separation (Solvent A: 0.1% formic acid in water, 2% acetonitrile; Solvent B: 0.1% formic acid in acetonitrile) was carried out at 250nL/min using a packed emitter column (C18, 25 cm × 75µm 1.6µm) (Ion Optics) and a 40min elution gradient. Mass-spectrometric data were acquired using the parallel accumulation serial fragmentation (PASEF) acquisition method in data-dependent acquisition mode (DDA). The measurements were carried out over the m/z range from 100 to 1700Th. The range of ion mobilities values were from 0.8 to 1.3 V s/cm2(1/k0). The total cycle time was set to 1.17sec and the number of PASEF MS/MS scans was set to 10.
## Data processing following LC-MS/MS acquisition

Files were processed with the MaxQuant software 2.0.1.0 and searched with Andromeda search engine against the UniProtKB/Swiss-Prot Homo Sapiens database (release 02/2021, 20408 entries). To search parent mass and fragment ions, we set an initial mass deviation of 4.5 ppm and 20 ppm respectively. The minimum peptide length was set to 7 amino acids and strict specificity for trypsin cleavage was required, allowing up to two missed cleavage sites. Carbamidomethylation was set as fixed modification, whereas oxidation (Met), N-term acetylation and biotinylation were set as variable modifications. Match between runs was allowed and LFQ minimum ratio count was set to 2. The false discovery rates (FDRs) at the protein and peptide level were set to 1%. Scores were calculated in MaxQuant as described previously.⁹⁰ The reverse and common contaminants hits were removed from MaxQuant output. Proteins were quantified according to the MaxQuant label-free algorithm using intensities.⁹⁰,⁹¹

Five independent streptavidin immunoprecipitations replicate were analyzed with Perseus software (version 1.6.15.0).⁹² The protein intensities were transformed in log2, and proteins identified in at least 4 replicates in at least one group were submitted to statistical test (volcano plot, FDR = 0.05 and S0 = 0.1) after imputation of the missing value by a Gaussian distribution of random numbers with a standard deviation of 30% relative to the standard deviation of the measured values and 1.8 standard deviation downshift of the mean.

## Co-immunoprecipitations assays

HeLa cells were collected using lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.5% mM EDTA, and 0.5% Nonidet P-40) supplemented with protease and phosphatase inhibitor cocktail (78443; Pierce) and centrifuge at 15 300g for 5min at 4°C. Supernatant were incubated overnight with Dynabeads Protein G (10007D, Invitrogen), previously washed, and coupled with anti-VMP1 antibodies for 2 hours. Then, beads were washed 3 times with washing buffer from Dynabeads Protein G immunoprecipitation kit, resuspended in Laemmli 4X with 100mM DTT and heated at 95°C for 5min before western blot analyses.

## FRAP experiment and analyzes

HeLa cells were transfected with Sec16a-GFP and Rab5-RFP cDNAs 36h–48h prior live recording and seeded in an 8-wells IBIDI cell chamber (80826; IBIDI). Live and FRAP experiments were done on Leica SP8 SMD microscope with 63× oil-immersion objective and FRAP module. Each image was taken every 800ms, cells were first recorded for 20 images, bleached on specific ROI with A488 laser at 100% intensity for 10 images and then recorded for 180 images after bleaching. Both GFP and RFP channels were recorded. Analyses were done using Fiji software (https://imagej.net/software/fiji/downloads) selecting Sec16a-GFP puncta before and after bleaching then used the following plugins: 1) create spectrum jru v1; 2) combine all trajectories jru v1; 3) normalize trajectories jru v1; 4) batch FRAP fit jru v1. All these plugins have been created by Stowers Institute for Medical Research (Jay_Plugins.jar) and are available at https://research.stowers.org/imagejplugins/zipped_plugins.html. Each resulting normalized and fitted curves were pooled to obtain average curves or analyzed individually. Then, we measured the slopes of regression on each normalized fitted curve with GraphPad Prism 10.1.1 to obtain the recovery efficiency.

## SDS-PAGE and western blotting

To check siRNA efficiency cells were transfected with siRNA 72h prior lysis using Laemmli1X (60 mM Tris–HCl pH = 6.8, 2% SDS, 10% glycerol, bromophenol blue, supplemented with 100 mM DTT) and samples were boiled for 15 min at 95°C. For siRNA, biochemical purification and immunoprecipitation, samples were separated by SDS–PAGE, transferred onto PVDF membranes and blocked with PBS-Tween 0.1% and milk 5%. Western blot analysis was performed with specific primary antibodies diluted in PBS-Tween 0.1%–BSA 5% overnight at 4°C. Secondary HRP-conjugated anti-rabbit IgG (AP308P; Chemicon) and HRP-conjugated anti-mouse IgG (1706516; Bio-Rad) were diluted in PBS-Tween 0.1% and milk 5% and incubated for 2 h at room temperature with PVDF membranes. Bands were visualized using a chemiluminescent HRP substrate (WBKLS0500; MERCK) and a ChemiDoc MP Imaging System (Bio-Rad). Quantification of band intensities was carried out using Fiji software, and protein levels are normalized on actin.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses were performed by using GraphPad Prism 10.1.1 (*p* < 0.05, **p** < 0.01, ***p** < 0.001, and ****p** < 0.0001) with test indicated in the corresponding legend. Unpaired student t Test was used to analyze the means between two unmatched groups following a Gaussian distribution. Welch's correction was applied when standard deviations of the dataset where not assumed to be equal. One or two-way ANOVA were performed to analyze the statistical significance between multiple groups affected by one or multiple factors respectively. Friedman test was used for paired non-parametric data from multiple groups for data that did not follow a normal distribution. Groups with unequal variances between them were analyzed using Brown-Forsythe and Welch ANOVA test. On graphs each symbol (circle, triangle, star, square, reversed triangle, hexagon, and diamond) represents one experiment. In panels showing Western blotting or immunofluorescence analysis, unless stated otherwise, images are representative of three independent experiments with similar results. All statistical analyses were performed from at least three independent experiments.