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ER-to-Golgi trafficking through a dynamic intermediate cis-Golgi tubular network in Arabidopsis

Nature Cell Biology volume 27, pages 424–437 (2025)Cite this article

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Abstract

Endoplasmic reticulum (ER)-to-Golgi trafficking is a central process of the secretory system of eukaryotic cells that ensures proper spatiotemporal sorting of proteins and lipids. However, the nature of the ER–Golgi intermediate compartments (ERGICs) and the molecular mechanisms mediating the transition between ERGICs and the Golgi, as well as the universality of these processes among eukaryotes, remain undiscovered. Here we identify a reticulated tubulo-vesicular network, labelled by MEMBRIN proteins, that is mostly independent of the Golgi, highly dynamic at the ER–Golgi interface and crossed by ER-induced released luminal cargos. We find that plant ERGICs become stabilized by the interaction they establish with pre-existing Golgi and gradually mature into Golgi cisternae, this process being dependent on C24-ceramide sphingolipids. Our study is a major twist in the understanding of the Golgi, as it identifies that the ERGICs in plants comprise a Golgi-independent and highly dynamic tubular network from which arise more stable Golgi-associated pre-cisternae structures.

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Fig. 1: The cis-Golgi contains several subpopulations, some of which display a high proportion of structures that remain independent of the Golgi both in time and in 3D.
Fig. 2: MEMB12 cis-Golgi is highly dynamic and displays transient interactions with the medial-Golgi.
Fig. 3: The cis-Golgi becomes stabilized in the vicinity of pre-existing Golgi.
Fig. 4: MEMB12 cis-Golgi displays transient interactions with the ERES and is crossed by the luminal cargo secGFP early after inducible release from the ER, which then crosses SYP32 cis-Golgi.
Fig. 5: MEMB12 cis-Golgi is a tubular network that is mostly independent of the Golgi and displays local intertwined connections with the medial-Golgi.
Fig. 6: C24-ceramide sphingolipids are involved in the generation of the MEMB network and its maturation into the medial-Golgi.
Fig. 7: C24-ceramides regulate the dynamic interaction of MEMB12 with the Golgi.
Fig. 8: C24-ceramides regulate the passage of the luminal cargo secGFP through MEMB12, and a revised model of the ER/Golgi interface in plant cells.

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Data availability

Data supporting the findings of this work are available in this paper and its Extended Data figures and Supplementary Information. All data generated and analysed in this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank I. Hara-Nishimura and H. Ueda for sharing published material. Imaging was performed at the Bordeaux Imaging Center (BIC), part of the National Infrastructure France–BioImaging supported by the French National Research Agency (ANR-10-INSB-04). We are grateful to the BIC team for their expertise, support and access to super-resolution microscopes, as well as their help in quantification. We thank Y. Jaillais and P. Moreau for critical reading of the manuscript and helpful comments. This work was supported by French National Research Agency grants nos. ANR CALIPSO (ANR-18-CE13-0025), ANR FATROOT (ANR-21-CE13-0019) and ANR DIVFUSE (ANR-22-CE92-0038-01) to Y.B., a PhD fellowship from the French government and distributed through the doctorate school in life and health sciences of Bordeaux to L.F.

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  1. These authors contributed equally: Louise Fougère, Magali Grison.

Authors and Affiliations

  1. Laboratoire de Biogenèse Membranaire, Université de Bordeaux, CNRS UMR5200, Villenave d’Ornon, France

    Louise Fougère, Magali Grison, Patricia Laquel, Matheus Montrazi & Yohann Boutté

  2. Bordeaux Imaging Center (BIC), Université de Bordeaux, INSERM, CNRS UAR3420, Bordeaux, France

    Fabrice Cordelières, Mónica Fernández-Monreal & Christel Poujol

  3. Faculty of Core Research, Natural Science Division, Ochanomizu University, Tokyo, Japan

    Tomohiro Uemura

  4. Live Cell Super-Resolution Imaging Research Team, RIKEN Center for Advanced Photonics, Wako, Saitama, Japan

    Akihiko Nakano

  5. Institute for Human Life Science, Ochanomizu University, Tokyo, Japan

    Yoko Ito

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Contributions

L.F. performed all experiments in Figs. 1–4, 7 and 8 and Extended Data Figs. 1e–h, 3–5, 7a,b and 8. M.G. established the τ-STED and ExM in Arabidopsis and performed all experiments in Figs. 5 and 6 and Extended Data Figs. 6 and 7c–f. P.L. and M.M. participated in the generation and selection of plant genetic material. F.C. created the DTC plugin and helped in the quantification of time-lapse acquisition as well as τ-STED quantification. M.F.-M. participated, with M.G., to establish ExM in Arabidopsis. C.P. participated, with M.G., to establish the τ-STED microscopy in Arabidopsis. T.U. provided the unpublished SYP31 and SYP32 fluorescent marker line and provided scientific input. A.N. provided scientific input and initial materials to this study. Y.I. performed the experiments in Extended Data Fig. 1a–d, set up the initial Airyscan live acquisition and quantification37, performed the cloning and solved the initial issues of the RUSH system in Arabidopsis, and conceptualized the research. Y.B. conceptualized and designed the research, obtained financial support, supervised all aspects of this study and wrote the manuscript. The figures were created by L.F. and M.G. All authors read and provided input on the manuscript.

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Correspondence to Yohann Boutté.

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Extended data

Extended Data Fig. 1 The SYP31-positive Golgi Entry Core Compartment (GECCO) exists in Arabidopsis root epidermal cells.

Localization of either GFP-SYP31 (a, b) or α-MEMB (e, f) in Arabidopsis epidermal root cells of the gnl1 mutant complemented with a BFA-sensitive version of GNL1 (GNL1sens) expressing the trans-Golgi marker ST-mRFP (c, d, g, h). (a, c, e, g) In absence of BFA, SYP31, MEMB11/12 and ST localize to Golgi-like structures. (b, d, f, h) In presence of BFA, SYP31 (b) or MEMB11/12 (f) remain in dotty-structures while ST (d, h) is redistributed to the ER-network. (a–d) conventional confocal microscopy, (e–h) airyscan microscopy. All scale bars are 10 µm. The experiments were repeated 3 times.

Extended Data Fig. 2 Schematic representation of the DTC image J plugin and the quantification pipeline of dynamic interaction.

(a) The plugin first detects each individual compartments in both channels for all time points and calculate their centroids. (b) The tracking of individual compartments is performed one step at a time by following the iterative presence of a compartment at tn within its 412 nm (9 pixels) radius zone at tn+1. (c) An association is detected if a compartment of one channel is crossing the association-zone of a compartment of the opposite channel. Oppositely, a compartment of one channel that does not cross any association zone of the opposite channel is independent. (d) The association zone is defined for each couple of markers by calculating the mean distance between two compartments of opposite channels (each centre is labelled by a blue cross) when they were the closest possible without being co-localized. (e) Each compartment is followed for a certain time before it disappears from the field of acquisition, this defines the track time. When a compartment of a channel is crossing the association zone (light purple) of a compartment of the opposite channel, an association event is detected. This association can last for a certain amount of time before it leaves the association zone (dissociation event), this defines the association time. Over a track time, several association/dissociation events can occur. (f–h) For a given track, three behaviours were classified, either a compartment of one channel remains associated to another of the opposite channel during the whole time of the track (f), remains independent during the whole time of the track (g) or show transient interaction possibly involving several association/dissociation events (h).

Extended Data Fig. 3 Validation of the quantitative data obtained from the time-lapse airyscan acquisition of Fig. 2.

(a, e, i) Time-lapse airyscan acquisition in root epidermal cells of either mCherry-MEMB12 x NAG1-EGFP (a), mCherry-SYP32 x NAG1-EGFP (e) or mRFP-SYP31 x NAG1-EGFP (i). The time series show an independent MEMB12-, SYP32- or SYP31-compartment that undergo an association with the medial-Golgi. The white star in a indicates a tubular structure. (b, f, j) Upper edge control: individual MEMB12- (b), SYP32- (f) or SYP31- (j) compartments could be tracked for an average track time of 22 sec for MEMB12 and SYP32 and 18 sec for SYP31. (c, d, g, h, k, l) Lower edge controls: one channel was flipped either horizontally or vertically, or rotated by 180°. Whether for MEMB12 (c), SYP32 (g) or SYP31 (k) the number of Golgi-associated compartments drastically decreased when one channel is rotated or flipped while the number of Golgi-independent compartments is strongly increased. For all markers tested, the time of association between either MEMB12 (d), SYP32 (h) or SYP31 (l) with the medial-Golgi strongly decreases when one channel is rotated or flipped. Data are presented as scatter plots with median represented as a line, n = 40 cells for all set of data. In c, d, g, h, k, l, ****P values were all <0.0001. All statistical tests were performed by non-parametric Mann-Whitney two-sided test. All scale bars are 1 µm. Source numerical data are available in source data.

Source data

Extended Data Fig. 4 Validation of the quantitative data obtained from the time-lapse airyscan acquisition of Fig. 3.

(a) Upper edge control: individual MEMB12-compartments could be tracked for an average track time of 15-17 sec. (b, c) Lower edge controls: one channel was flipped either horizontally or vertically, or rotated by 180°. (b) The number of SYP31-associated MEMB12-compartments drastically decreased when one channel is rotated or flipped while the number of SYP31-independent MEMB12-compartments is strongly increased. (c) The time of association between MEMB12 and SYP31 strongly decreases when one channel is rotated or flipped. Data are presented as scatter plots with median represented as a line, n = 40 cells for all set of data. In b, c, ****P < 0.0001. All statistical tests were performed by non-parametric Mann-Whitney two-sided test. Source numerical data are available in source data.

Source data

Extended Data Fig. 5 Validation of the quantitative data obtained from the time-lapse airyscan acquisition of Fig. 4 and additional secGFP-FKBP-RUSH images.

(a) Upper edge control: individual MEMB12-compartments could be tracked for an average track time of 22 sec. (b, c) Lower edge controls: one channel was flipped either horizontally or vertically, or rotated by 180°. (b) The number of ERES-associated MEMB12-compartments drastically decreased when one channel is rotated or flipped while the number of ERES-independent MEMB12-compartments is strongly increased. (c) The time of association between MEMB12 and the Sec16A-like MAG5 ERES marker strongly decreases when one channel is rotated or flipped. (d–g) Confocal images of root epidermal cells stably expressing both secGFP-FKBP-RUSH construct and mCherry-MEMB12 (repeated 3 times). (d) In absence of FKBP ligand, secGFP-FKBP-RUSH localizes to the ER. In presence of FKBP-ligand, sec-GFP localizes to small dotty structures as well as bigger pro-vacuole-like compartments after 10 min of incubation, or bigger vacuoles after 80 min of incubation. (h–j) Transient expression of the secGFP-FKBP-RUSH (h) in epidermal cotyledon cells stably expressing mCherry-MEMB12 (i, merged in j) (repeated 3 times). Vacuole-like compartments are labelled by secGFP-FKBP-RUSH upon incubation with FKBP-ligand. Data are presented as scatter plots with median represented as a line, n = 40 cells for all set of data. In b-c, ****P < 0.0001. All statistical tests were performed by non-parametric Mann-Whitney two-sided test. All scale bars are 4 µm. Source numerical data are available in source data.

Source data

Extended Data Fig. 6 MEMB12 labels a heterogeneous network while NAG1, SYP31 and SYP32 label cisternae, related to Fig. 5.

(a, b) τ-STED morphometric analyses of either mCherry-MEMB12 x NAG1-EGFP (a) or α-MEMB immunostained compartments in NAG1-EGFP expressing plants (b). The circularity and solidity values were lower for α-MEMB than for mCherry-MEMB12 indicative that α-MEMB labels a network while mCherry-MEMB12 labels a tubulo-vesicular network. The binomial distribution of NAG1 values in Fig. 5j–t is explainable by the orientation of the cisterna during the acquisition, either from side or top view. Donut-like structures have an area, circularity and solidity index higher than rice grain structures (n = 176 NAG1-compartments including 88 “rice” and 88 “donuts” in a, n = 3 049 mCherry-MEMB12-compartments in a, n = 238 NAG1-compartments including 150 “rice” and 88 “donuts” in b, n = 1 837 α-MEMB -compartments in b). (c-h) τ-STED images of α-MEMB immunostained compartments (c, f) in mCherry-MEMB12 (d, g) expressing plants. Both α-MEMB and mCherry-MEMB12 label tubulo-vesiculated networks that share some overlap (e, h). (i) Quantification (n = 34 measures) of the co-localization between α-MEMB and mCherry-MEMB12 (e, h) using Pearson or Manders coefficient. As a control, the co-localization was calculated between α-MEMB and the NAG1-EGFP medial-Golgi. (j–r) τ-STED images of either NAG1-EGFP (j, m, p), EGFP-SYP32 expressed under the SYP32 promoter (k, n, q) or mRFP-SYP31 (l, o, r). (s, t) Morphometric analyses of j-r. SYP31, SYP32 and NAG1 label cisternae-like structures of different area, SYP31- or SYP32-compartments is smaller than the NAG1 medial-Golgi (out of 16 cells, n = 315 NAG1-compartments in s including 199 “rice” and 116 “donut” in t, n = 134 SYP32-compartments in s including 68 “rice” and 66 “donut” in t, n = 311 SYP31-compartments in s including 217 “rice” and 90 “donuts” in t. ****P < 0.0001. In b, nsP = 0.9744 (NAG1-rice/α-MEMB). In s, nsP = 0.0849 (solidity). In t (area), **P = 0.0031 (NAG1-rice/SYP32-rice/SYP31-rice) and nsP = 0.5142 (NAG1-donut/SYP32-donut/SYP31-donut). In t (circularity), *P = 0.0305 (NAG1-rice/SYP32-rice/SYP31-rice) and **P = 0.0041 (NAG1-donut/SYP32-donut/SYP31-donut). In t (solidity), nsP = 0.5590 (NAG1-rice/SYP32-rice/SYP31-rice) and ***P = 0.0003 (NAG1-donut/SYP32-donut/SYP31-donut)). Data are presented as violin plots with median represented as a line. For pair-wise comparison we used the non-parametric Mann-Whitney two-sided test. For multiple comparison in s and t, the Brown Forsythe test was used. Scale bars are 1 µm. Source numerical data are available in source data.

Source data

Extended Data Fig. 7 τ-STED morphometric analyses of the ceramide synthase loh1−/− loh3+/− sesquimutant and the ceramidase acer-1 mutant of Fig. 6.

(a, b) Live cells airyscan acquisition in root epidermal cells of mCherry-MEMB12 x NAG1-EGFP showing that mCherry-MEMB12 compartments are strongly altered in loh1−/− loh3+/− as compared to the wild-type Col-0, NAG1 completely disappeared in loh1−/− loh3+/−. (c-f) τ-STED morphometric analyses show that the remaining MEMB-structures are only slightly or not significantly altered in their morphology, whether for α-MEMB (c, e) or mCherry-MEMB12 (d, f) either in the loh1−/− loh3+/− sesquimutant (c, d) or in the acer-1 mutant (e, f) (n = 8 331 and 3 802 compartments out of 15-17 cells for Col-0 and loh1−/− loh3+/− in c, n = 5 969 and 1 209 compartments out of 21-24 cells for Col-0 and loh1−/− loh3+/− in d, n = 11 095 and 7 911 compartments out of 27-31 cells for Col-0 and acer-1 in e and n = 3 101 and 2 769 compartments out of 17-25 cells for Col-0 and acer-1 in f. ****P < 0.0001. In c, ***P = 0.0006 (area). In d, ***P = 0.001 (circularity), **P = 0.025 (solidity). In e, nsP = 0.5772 (area), nsP = 0.6654 (circularity), *P = 0.0194 (solidity). In f, **P = 0.011 (NAG1 area), nsP = 0.0849 (MEMB12 circularity), nsP = 0.3293 (NAG1 circularity), nsP = 0.0926 (NAG1 solidity)). Data are presented as violin plots with median represented as a line. All statistical tests were performed using non-parametric Mann-Whitney two-sided test. Scale bars are 1 µm. Source numerical data are available in source data.

Source data

Extended Data Fig. 8 Sphingolipid function in the MEMB12-compartments dynamic interaction with the medial-Golgi as well as the passage of the luminal cargo secGFP through MEMB12 (additional data and controls for Fig. 7).

(a) Time-lapse airyscan acquisition in root epidermal cells of mCherry-MEMB12 x NAG1-EGFP upon metazachlor (Mz) treatment. The time series provide a representative Golgi-independent movement. (b) As compared to the control wild-type condition, the number of association and dissociation within the population of transient interaction between MEMB12 and the medial-Golgi is strongly decreased upon Mz (n = 40 cells, ****P < 0.0001). (c) The association time of interaction between MEMB12 and the medial-Golgi is slightly altered upon Mz (n = 40 cells, *P = 0.0229). (d) The total number of either MEMB12- or medial-Golgi NAG1-compartments is not altered upon whether FB1, Mz or in the acer-1 mutant (n = 40 cells. For MEMB12, nsP = 0.4019 (control/FB1), nsP = 0.8821 (control/Mz), nsP = 0.5057 (control/acer-1). For NAG1, nsP = 0.9276 (control/FB1), nsP = 0.92 (control/Mz), nsP = 0.2105 (control/acer-1)). (e-g) Overall representation of the repartition of Golgi-associated, Golgi-independent or transient interaction between MEMB12 and the medial-Golgi NAG1 marker in either the acer-1 mutant (e), upon FB1 (f) or upon Mz (g) (data coming from the Fig. 7c–h for the acer-1 mutant and FB1 treatment) (n = 40 cells for each dataset. In e (acer-1), *P = 0.013 (associated), nsP = 0.3859 (independent), nsP = 0.8821 (transient). In f (FB1), nsP = 0.061 (associated), **P = 0.0013 (independent), nsP = 0.4127 (transient). In g (Mz), nsP = 0.6153 (associated), nsP = 0.9733 (independent), nsP = 0.6702 (transient)). (h) The velocity of Golgi-associated or Golgi-independent MEMB12-compartments is not altered upon Mz (n = 40 cells, nsP = 0.8146 (associated), nsP = 0.92 (independent)). Data are presented as violin plots with median represented as a line. (i–r) Airyscan acquisition of root epidermal cells stably expressing mCherry-MEMB12 together with secGFP-FKBP-RUSH upon Mz treatment. The timing of incubation with the FKBP-ligand is indicated in the upper-left corner of each images. (s) Quantification of the normalized percentage of co-localization between secGFP-FKBP-RUSH and mCherry-MEMB12 upon time after incubation with the FKBP-ligand. As compared to the control (Figs. 4y, 7n), Mz treatment might delay the passage of the secGFP-FKBP-RUSH luminal cargo through MEMB12 compartments, although statistically non-significant (n = 680 cells out of 38 roots, nsP = 0.0501 (0-5/5-10 min), nsP = 0.3926 (5-10/10-15 min), nsP = 0.7116 (10-15/15-20 min), nsP = 0.2065 (15-20/20-25 min), nsP = 0.5692 (20-25/25-30 min)). All statistical tests were performed by non-parametric Mann-Whitney two-sided test. Scale bars is 1 µm in a and 2 µm in i–r. Source numerical data are available in source data.

Source data

Supplementary information

Reporting Summary

Peer Review File

Supplementary Video 1

Airyscan time-lapse acquisition of mCherry-MEMB12 and NAG1-EGFP in root epidermal cells, related to Figs. 1a,b, 2c–e and Extended Data Fig. 3a. MEMB12 is labelling vesicle-like structures as well as a tubular-like network (white star) that is highly dynamic. MEMB12-structures display both independent movements from the medial-Golgi (white arrow) and Golgi-associated movements (white arrowhead). Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 2

Airyscan time-lapse acquisition of mCherry-SYP32 and NAG1-EGFP in root epidermal cells, related to Fig. 1c,d and Fig. 2f–h. SYP32 is labelling vesicle-like structures that mostly display Golgi-associated movements (white arrowhead) and some Golgi-independent movements as well. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 3

Airyscan time-lapse acquisition of mRFP-SYP31 and NAG1-EGFP in root epidermal cells, related to Fig. 1e,f and Fig. 2i–k. SYP31 is mostly labelling vesicle-like structures that display Golgi-associated movements (white arrowhead). Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 4

3D Airyscan acquisition and IMARIS surface modelling of cis-/medial-Golgi compartments, related to Fig. 1g–o and Fig. 3a–f. 3D Airyscan images and surface modelling of either mCherry-MEMB12 (a,b), mCherry-SYP32 (c,d) or mRFP-SYP31 (e,f) together with NAG1-EGFP in root epidermal cells. MEMB12-structures are more fragmented and dispersed than SYP31 that are closer to the Golgi. SYP32 shows an intermediate pattern between MEMB12 and SYP31. Colour code: green is the medial-Golgi labelled by NAG1, red is the cis-Golgi (MEMB12, SYP32 or SYP31) associated to the Golgi, magenta is the cis-Golgi independent from the Golgi.

Supplementary Video 5

Single-channel Airyscan time-lapse acquisition of MEMB12-mCherry in root epidermal cells. MEMB12 labels a highly dynamic tubular network (white star) that is locally wrapped or packed into denser clustered structures. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 6

Another single-channel Airyscan time-lapse acquisition of MEMB12-mCherry in root epidermal cells. MEMB12 labels a highly dynamic tubular network (white star) that is locally wrapped or packed into denser clustered structures. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 7

Airyscan time-lapse acquisition of MEMB12-mCherry and SYP31-EGFP in root epidermal cells, related to Fig. 3h,i. MEMB12 is labelling vesicle-like structures as well as a tubular network (white star) that is highly dynamic. MEMB12-structures display both independent movements from SYP31-compartments (white arrow) and SYP31-associated movements (white arrowhead). MEMB12 labels a highly dynamic tubular network that is locally wrapped or packed into denser clustered structures close to SYP31-compartments. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 8

Airyscan time-lapse acquisition of MEMB12-mCherry and the ERES Sec16-like MAIGO5-EGFP marker in root epidermal cells, related to Fig. 4a–c. MEMB12-structures transiently associate (white arrows) with ERES structures labelled by MAIGO5-EGFP. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 9

3D expansion microscopy (ExM) and IMARIS surface modelling of the MEMB12-network, related to Fig. 5u–ag. 3D Airyscan ExM images and surface modelling of MEMB12-mCherry together with NAG1-EGFP in root epidermal cells. The Golgi-independent MEMB12-network display intertwined connection with the medial-Golgi and is often found to surround it.

Supplementary Video 10

Airyscan time-lapse acquisition of MEMB12-mCherry and NAG1-EGFP in root epidermal cells upon fumonisin B1 (FB1) treatment, related to Fig. 7a. The amount of Golgi-association/dissociation in the population of MEMB12-compartments that transiently interact with the medial-Golgi is decreased upon FB1. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 11

Airyscan time-lapse acquisition of MEMB12-mCherry and NAG1-EGFP in root epidermal cells of the ceramidase acer-1 mutant, related to Fig. 7b. MEMB12-mCherry compartments are stabilized in the vicinity of the Golgi in the acer-1 mutant. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Video 12

Airyscan time-lapse acquisition of MEMB12-mCherry and NAG1-EGFP in root epidermal cells upon metazachlor (Mz) treatment, related to Extended Data Fig. 8a. The amount of Golgi-association/dissociation in the population of MEMB12-compartments that transiently interact with the medial-Golgi is decreased upon Mz. Scale bar, 2 µm; the time in seconds is indicated at the upper right.

Supplementary Tables 1–3

Sequences of the primers used to genotype the acer-1 and the loh1−/− loh3+/− sesquimutant (Supplementary Table 1). DNA sequences of the secGFP-FKBP-RUSH system (Supplementary Table 3). Program for single and double immunostaining (Supplementary Table 4).

Supplementary Table 2

Excel macro used for DTC dynamic analyses.

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Fougère, L., Grison, M., Laquel, P. et al. ER-to-Golgi trafficking through a dynamic intermediate cis-Golgi tubular network in Arabidopsis. Nat Cell Biol 27, 424–437 (2025). https://doi.org/10.1038/s41556-025-01624-x

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