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

Louise Fougère, Magali Grison, Patricia Laquel, Matheus Montrazi, Fabrice Cordelières, Mónica Fernández-Monreal, Christel Poujol, Tomohiro Uemura, Akihiko Nakano, Yoko Ito, et al.

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## To cite this version:

Louise Fougère, Magali Grison, Patricia Laquel, Matheus Montrazi, Fabrice Cordelières, et al., ER-to-Golgi trafficking through a dynamic intermediate cis-Golgi tubular network in Arabidopsis. Nature Cell Biology, 2025, 27 (3), pp.424-437. 10.1038/s41556-025-01624-x. hal-05165357

HAL Id: hal-05165357

https://hal.science/hal-05165357v1

Submitted on 5 Nov 2025

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1 ER-to-Golgi trafficking through a dynamic intermediate cis-Golgi tubular network in Arabidopsis
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4 Louise Fougère¹,#, Magali Grison¹,#, Patricia Laquel¹, Matheus Montrazi¹, Fabrice Cordelières², Mónica Fernández-Monreal², Christel Poujol², Tomohiro Uemura³, Akihiko Nakano⁴, Yoko Ito⁵, Yohann Boutté¹,*

7 # These authors equally contributed to the work

8 * Correspondence should be sent to : yohann.boutte@u-bordeaux.fr

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

10 ² Université de Bordeaux, CNRS, INSERM, Bordeaux Imaging Center (BIC), US4, UAR 3420, F-33000 Bordeaux, France.

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

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

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

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27 Abstract

28 Endoplasmic Reticulum (ER)-to-Golgi trafficking is a central process of the secretory system of
29 eukaryotic cells that ensures proper spatiotemporal sorting of proteins and lipids. However, the
30 nature of the ER-Golgi Intermediate Compartments (ERGIC) and the molecular mechanisms
31 mediating the transition between the ERGIC and the Golgi, as well as the universality of these
32 processes amongst Eukaryotes, remain undiscovered. Here, we identified a reticulated tubulo-
33 vesicular network, labeled by MEMBRIN proteins, mostly independent from the Golgi, highly
34 dynamic at the ER-Golgi interface and crossed by ER-induced release of luminal cargos. We found
35 that the plant ERGIC gets stabilized by the interaction it establishes with pre-existing Golgi and
36 gradually matures into a Golgi-cisternae, this process being dependent on C24-ceramide
37 sphingolipids. Our study is a major twist in the understanding of the Golgi by identifying that the
38 ERGIC in plants is a Golgi-independent highly dynamic tubular network from which arise more
39 stable Golgi-associated pre-cisternae structures.
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The secretory system is an intricate set of membranes that plays fundamental functions to coordinate fluxes and ensure the accurate sorting and targeting of proteins and lipids to different destinations within the cell¹⁻⁵. The first point in the carriageway of this system occurs at the endoplasmic reticulum (ER)-export sites (ERES) that convey proteins to the Golgi apparatus⁶,⁷. In animal cells, the Golgi apparatus is a ribbon of the Golgi units gathered at the centrosome-nucleated microtubules. In plant cells, Golgi units are dispersed within the cytosol and move along actin cables close to the ER⁷⁻¹⁰. In animal cells, the traditional vesicle carriers model was challenged by the recent nano-resolved structure of ERES that was identified as a continuous network of interwoven membrane tubules that connects the ER and emits pearled membrane extensions that lie in the direction of the Golgi apparatus⁴. The ERES-derived transport carriers were previously known to be enriched at the tubulo-vesicular ER-Golgi Intermediate Compartment (ERGIC), but how the ERGIC-to-Golgi transition is ensured remains unadressed⁴,¹¹. In plant cells, due to the close proximity of the Golgi and ERES, it has been suggested that no equivalent of the ERGIC exists⁸,¹². However, it has been suggested that some part of the ER can contact the Golgi apparatus ⁹,¹³⁻¹⁶. In addition, it has been observed that inducing Golgi movement by trapping the Golgi with optical tweezers results in the dynamic remodeling of the ER network, thereby suggesting that the ER and the Golgi are in physical contact¹⁶. In plant cells, it has been shown that while the medial and trans cisternae of the Golgi apparatus disappear upon treatment with the Golgi-destructuring drug brefeldin A (BFA), and their proteins absorbed into the ER, the cis-Golgi Qa-SNARE SYP31 and Retrieval protein1B (RER1B) remain in dotty structures called “Golgi Entry Core Compartment (GECCO)” located in close proximity to the ERES¹⁷. In plants and yeasts, GECCO is thought to be the equivalent of the animal ERGIC⁵,¹⁸⁻²⁰. However, the identity and morphology of these structures as well as their dynamics and the molecular mechanisms involved in their transition to the Golgi remain unknown. In this study, we discovered that the Qb-SNARE MEMBRIN11/12 (MEMB11/12) label a population of compartments that are mostly independent from the rest of the Golgi and display a highly dynamic behavior characterized by transient interactions with the medial-Golgi as well as with the ERES. Importantly, by establishing the lifetime tau-STimulated Emission Depletion (τ-STED) and Expansion Microscopy (ExM) super-resolution methodologies in plants we identified a Golgi-independent tubulo-vesicular network labeled by MEMB11/12 that resembles the Vesicular-Tubular Cluster (VTC)/ERGIC of animal cells while the cis-Golgi marker SYP31/32 labels a more stable Golgi-associated cisterna. By establishing a version of the Retention Using Selective Hooks (RUSH) system in plants to release cargos synchronously from the ER, we could show that luminal cargos are sequentially crossing the MEMB12 cis-Golgi subpopulation and then the SYP32 cis-Golgi subpopulation upon release. We propose that the highly dynamic Golgi-independent MEMB-tubular structures constitute an early station of the ERGIC in plants. Our study further identified a modulation of the dynamics of the ER-Golgi intermediate structures
by local contacts with Golgi cisternae that stabilize the intermediate-network into larger Golgi-associated pre-cisternae structures. Importantly, we identified a functional role of sphingolipids, in particular ceramides containing 24 atoms of carbon (C24-ceramides), in the generation of the MEMB/ERGIC tubulo-vesicular network and the regulation of its dynamics by stabilizing its interaction with the Golgi and favors its maturation in pre-cisternae and then medial-Golgi cisterna. This study proposes a complete reevaluation of the ER-Golgi interface in plant cells by identifying an ER-to-Golgi trafficking *via* a highly dynamic *cis*-Golgi sphingolipid-dependent tubular network and by providing structural and functional identification of the ERGIC in plants.

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## 99 Results

100 MEMBRIN *cis*-Golgi is Golgi-independent and highly dynamic

GECCO was identified in BY-2 tobacco cell culture, upon inhibition of BFA-sensitive ARF-GEFs by BFA treatment¹⁷. In Arabidopsis, given that the ARF-GEF GNOM-LIKE1 (GNL1) is involved in ER-Golgi transport and resistant to BFA, we used the *gnl1* knockout mutant in which an engineered GNL1-BFA sensitive version was introduced beforehand²¹,²². In this genetic background, we either introduced the *cis*-Golgi Qa-SNARE GFP-SYP31 fluorescent marker or check the localization of another *cis*-Golgi marker, the Qb-SNARE MEMBRIN, using an α-MEMB antibody recognizing predominantly MEMB11 and to a lesser extend MEMB12²¹. Our results indicate that both GFP-SYP31 and α-MEMB remain in small dotty structures after BFA treatment (**Extended data 1a, b, e, f**). Contrastingly, the *trans*-Golgi marker ST-mRFP is relocated to the ER upon BFA (**Extended data 1c, d, g, h**). These results indicate that GECCO-like structures exist in the Arabidopsis root model. We next checked whether the *cis*-Golgi could behave, for a portion of it, independently from the rest of the Golgi in native condition, i.e without BFA in wild-type genetic background. To test this hypothesis, we used airyscan microscopy that provides a 1.4-fold increase in lateral resolution (~ 140 nm) as compared with conventional confocal microscopes²³. To compare the localization and dynamics of the *cis*-Golgi to the rest of the Golgi we crossed the *cis*-Golgi markers mCherry-MEMB12, mRFP-SYP31 or its close homolog mCherry-SYP32 with the medial-Golgi marker NAG1-EGFP and performed live cell airyscan acquisitions in root epidermal cells²⁴⁻²⁶. We first performed 5 min time-laps acquisitions and found *cis*-Golgi compartments moving together with the medial-Golgi, consistently with the conventional Golgi stack model (white arrowheads in **Fig. 1a, c, e; Supplementary videos 1, 2, 3**). However, we additionally observed *cis*-Golgi compartments moving independently from the medial-Golgi for all markers tested (white arrows in **Fig. 1b, d, f; Supplementary videos 1, 2, 3**). To be sure that the Golgi-independent compartments were completely separated from the Golgi in 3D, we acquired z-stacks by airyscan microscopy in living root
epidermal cells and performed surface rendering using the IMARIS software to determine the 3D position of the Golgi-independent compartments from the medial-Golgi (Fig. 1g-r). Interestingly, we found numerous cis-Golgi structures clearly separated in 3D from the medial-Golgi (white arrows in Fig. 1g-o), and we also found Golgi-associated cis-Golgi (white arrowheads in Fig. 1g-o; Supplementary video 4). We next evaluated the proportion of the cis compartment that was independent from or associated with the medial-Golgi in our 3D acquisitions and found that Golgi-independent cis can reach up to more than half of the total population of cis-Golgi (Fig. 1p-r). Notably, we found a striking difference between the cis-markers: MEMB12-compartments are mostly independent from the Golgi (Fig. 1p), SYP31-positive compartments are mostly associated with it while SYP32 displays an intermediate behavior closer to MEMB12 than SYP31(Fig. 1q-r). These results challenge the traditional Golgi stack representation and show that the cis-Golgi can be a Golgi-independent structure. Thus, our next question was to address how these independent cis-Golgi behave dynamically. To address this question, we setup a workflow composed of lived airyscan microscopy acquisition, tracking of individual objects in both channels using the Detect Track and Co-localize (DTC) plugin of imageJ (Fig. 2a; Extended data 2a-c)²⁷. This plug-in, coupled with an Excel macro, enables the identification of the interactions occurring between compartments of two opposite channels and calculates their durations (Fig. 2b; Extended data 2d-h). We found that the cis-Golgi compartments could either stay associated with the medial-Golgi over the whole time of tracking (white arrowheads in Fig. 2c, f, i; Supplementary videos 1, 2, 3), remains independent from it (white arrows in Fig. 2d, g, j; Supplementary videos 1, 2, 3), or show transient interactions with the Golgi (Fig. 2e, h, k; Supplementary videos 1, 2, 3; Extended data 3a, e, i). Importantly, in addition to the mCherry-MEMB12-compartments we often and consistently detected a network of mCherry-MEMB12-interconnected tubule-like structures that display a fast kinetics and that are locally wrapped or packed into denser clustered structures that eventually associate with the medial-Golgi (white stars in Fig. 1a, b, g; Fig. 2c, d, e; Supplementary videos 1, 5, 6; Extended data 3a, e, i). Our quantifications showed that MEMB12 mainly displayed transient interactions with the medial-Golgi with an average association time around 15 seconds (Fig. 2l-n). SYP32 and SYP31 show gradually less prominent transient interaction with the medial-Golgi as compared to MEMB12 (Fig. 2l). Consistently, within the transient interaction population, MEMB12 displays about four times more association/dissociation than SYP31 or SYP32 (Fig. 2m, n). Thus, although seemingly intermediate, SYP32 has a dynamic behavior closer to SYP31 than MEMB12 (Fig. 2l). Consistently, both SYP32 and SYP31 have a low proportion of Golgi-independent structures over the whole time of tracking as compared to MEMB12 and have a higher proportion of Golgi-associated structures (Fig. 2l). Altogether, these results suggest that MEMB12 is highly dynamic as compared to SYP31 or SYP32. To validate our quantifications, we estimated for each couple of markers the upper edge (is the average time of tracking
superior to the average time of association?) and lower edge (are the interactions due to random movements in the stochastic background?) of the tracking quantification pipeline and calculated that our values are contained within the frame defined by the upper and lower edge of quantification (for more details see the "Quantitative live tracking of compartment association" part of the online methods, **Extended data 3b-1**).

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### **cis-Golgi stabilization by contacts with pre-existing Golgi**

Using IMARIS surface rendering we found that the *cis*-Golgi subpopulations are variable in size and morphologies (**Fig. 1g-o**). The compartments labeled by mCherry-MEMB12 (**Fig. 3a, b**) that associate with the rest of the Golgi appear more fragmented than those labeled by SYP31 (**Fig. 3e, f**) that are rounder and bigger. Again, SYP32 is intermediate between MEMB12 and SYP31 (**Fig. 3c, d**). We next ranked the *cis*-compartments according to their distance to the medial-Golgi. While the SYP31-population remains close to the medial-Golgi, the majority being within a 2 µm distance (with a minor portion up to 4 µm), the MEMB12-population is homogeneously spread up to 6 µm distance from the medial-Golgi (**Fig. 3g**). SYP32 shows an intermediate distribution but is more similar to SYP31 than MEMB12 (**Fig. 3g**). Interestingly, our results show that the largest SYP31/32 *cis*-compartments are found in close proximity of the Golgi while the smallest are the most distant from the Golgi (**Fig. 3g**). These results show that SYP31/32 *cis*-compartments form larger structures in proximity of the Golgi suggesting that they get stabilized by the Golgi. To address this further, we crossed mCherry-MEMB12 into EGFP-SYP31 and performed airyscan time laps acquisitions (**Fig. 3h, i**). Indeed, we quantified that MEMB12 is mostly transiently associated with SYP31 over the whole time of tracking, with an average time of association between 10-15 sec, consistently to what we observed for the medial-Golgi (**Fig. 3j, k**; **Supplementary video 7**). Our track-time and stochastic background controls validated our data (**Extended data 4a-c**). Importantly, we quantified the velocity of MEMB12-compartments that are either associated with or independent from the Golgi and found that the speed of MEMB12-compartments was lower when it is associated with either SYP31-*cis* or NAG1-medial Golgi (**Fig. 3l-m**). Similarly, the velocity of SYP31 or SYP32 compartments that are independent from the medial-Golgi is faster than the ones associated with it (**Fig. 3n-o**). Altogether, our dynamic and morphometric analysis suggest that the MEMB12-structures are mostly dynamics but gets stabilized and enlarged into bigger structures when they locally contact the *cis*-Golgi or medial-Golgi cisternae. Consistently, in our videos we see that the dynamic MEMB12-structures get locally wrapped into a denser clustered structure at the proximity of either NAG1-medial or SYP31-*cis* cisternae (**Supplementary videos 1, 4, 7**).

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MEMB12 association with ERES and loading of luminal cargos

To decipher whether the MEMB12-structures could be some ER-Golgi intermediate compartments we tested whether the MEMB12-compartments could associate with the ERES Sec16A-like protein marker MAIGO5 (MAG5)-GFP<sup>22,23</sup> in live airyscan time laps of root epidermal cells (Fig. 4a-c, Supplementary video 8). Interestingly, the MEMB12-population mainly displays either transient interactions over the whole time of tracking (average association time around 12 seconds, Fig. 4c-f) or remains independent from the ERES (Fig. 4b, d; Supplementary video 8). The number of MEMB12-structures that remained associated to the ERES over the time of tracking is close to 0 further suggesting that the MEMB12/ERES interactions never get stabilized (Fig. 4a, d). Our track-time and stochastic background controls validated our data (Extended data 5a-c). Together, these results show that the cis-Golgi is able to connect the ERES in a dynamic way. As the ERES are supposed to be the sites of cargo loading, we established in plants a version of the RUSH system that allows to stack a cargo at the ER and synchronously released it upon application of a chemical<sup>4,24</sup> (Fig. 4g, h). The ER-hook we designed is composed from a ER-resident signal, the HDEL peptide<sup>31</sup>, a bait that is a mutated version of the FKBP-rapamycin binding (FRB) domain of the Human kinase TOR able to interact with itself (with a higher affinity for FKBP-ligand)<sup>32</sup>, and the fluorescent protein TagBFP2 (Fig. 4g, h). As a cargo we employed the fluorescent marker secGFP that is widely used to identify ER-to-Golgi trafficking mechanisms in plants<sup>33</sup>, to which we fused a FKBP domain. In transient expression in the *Arabidopsis* cotyledons, we could see an ER-pattern for secGFP-FKBP-RUSH that perfectly matches the ER-pattern of the HDEL-hook (Fig. 4i-k). In stable expression in the Arabidopsis root epidermis, the fluorescence of the HDEL-hook was too weak to be detected but secGFP-FKBP-RUSH was efficiently stacked at the ER network (Extended data 5d).

Upon cargo release by the FKBP-ligand, we observed that secGFP-FKBP-RUSH labels some endomembrane compartments that either closely associate or co-localize with the MEMB12-compartments, both in transient expression in cotyledons (Fig. 4 l-n) and stable expression in roots (Fig. 4o-x). We also noticed that the secGFP-FKBP-RUSH is reaching pro-vacuole-like structures within 10 min and larger vacuoles within 60-80 min after cargo release both in roots (Extended data 5e-g) and cotyledons (Extended data 5h-j). We quantified the trafficking of secGFP-FKBP-RUSH by airyscan microscopy and found that secGFP-FKBP-RUSH labels mCherry-MEMB12-compartments already within 5 minutes after incubation with the ligand (Fig. 4y). After 20 min, the co-localization of secGFP-FKBP-RUSH with mCherry-MEMB12 clearly decreases (Fig. 4y) indicating that the luminal secGFP-FKBP-RUSH had crossed the MEMB12 compartments. We additionally checked when secGFP-FKBP-RUSH is crossing the SYP31/32 compartment. Our observations revealed that secGFP-FKBP-RUSH co-localization with mCherry-SYP32 strongly increases after 20 min of incubation with the FKBP ligand and start to decrease after 35 min (Fig. 4z-ai, aj). Altogether, our results suggest that MEMB12 is an early
secretory compartment that either matures into or interacts with SYP31/32 compartments to sequentially
transfer cargos from a Golgi-independent to a more Golgi-associated and stabilized form of cis-Golgi.

MEMB labels a Golgi-independent tubulo-vesiculated network

In our airyscan time-lapse acquisitions, we not only found dynamic MEMB12-compartment but also a
network of highly dynamic MEMB12-interconnected tubules (white stars in **Fig. 1a, b, g**; **Fig. 2c, d, e**;
**Supplementary videos 1, 5, 6; Extended data 3a**). However, airyscan microscopy lateral resolution is
around 140 nm which might not be sufficient to fully distinguish membrane tubules. Thus, we established
in plants the super-resolution τ-STED technology that uses Fluorescence Lifetime Imaging Microscopy
(FLIM) approach to discriminate the background from the fluorophores²⁵⁻²⁷. In our images, we calculated
that the lateral resolution is 63 nm (+/- 12 nm) in τ-STED while we obtained 166 nm (+/- 19 nm) in
airyscan microscopy (see methods section). We performed two-colors τ-STED microscopy with the
mCherry-MEMB12 marker and the medial-Golgi marker NAG1-EGFP (**Fig. 5a-i**). Strikingly, the medial-
Golgi is a cisterna that looks either flat, like a rice grain, or circular with an edge localization of NAG1-
EGFP, like a donut, depending on the orientation of the cisternae (**Fig. 5a, d, g**). Contrastingly, the
mCherry-MEMB12 labeled a tubulo-vesicular network with small vesicles connected by tubules, similar
to the VTC/ERGIC of animal cells (**Fig. 5b, e, h**). To quantify these observations, we used a
morphometric workflow that calculates three parameters: the area, the circularity and the solidity (the
solidity measures if a structure is carved or branched, the lowest is the value and the more it reveals a
network) of the structures. We found that the area of MEMB12-structures was higher, with a larger
scattering of the values, than for NAG1 (**Fig. 5j**). The circularity of the medial-Golgi was seemingly
spread in two groups, we found out that this was due to the orientation of the cisterna, either like a rice
grain or like a donut (**Fig. 5d, Extended data 6a**). The circularity and solidity values of MEMB12-
structures were lower and more scattered than NAG1 (**Fig. 5j, Extended data 6a**). These results indicate
that MEMB12-structures are more heterogeneous in shape than NAG1-medial-Golgi and quantitatively
confirm that the MEMB12 compartments are a mix of branched tubular network and vesicles (**Fig. 5b, e,
h**). While the MEMB12 network appears mostly independent from the medial-Golgi cisterna, we
consistently see close associations or intertwined connection of the MEMB12 compartments with the
medial-Golgi (**Fig. 5c, f, i**). In proximity of the medial-Golgi we detected either tubulo-vesicular clusters
or more expanded pre-cisternae structures (**Fig. 5f, i**), consistently to what we described earlier by
IMARIS (**Fig. 1g-i; Fig. 3a-b**) or what was described by electron microscopy at the vicinity of the Golgi
stack⁹. Next we addressed whether we could also observe this network in native condition. For this, we
performed an immuno-localization with the α-MEMB antibody that recognizes mostly MEMB11 and to a
lesser extend MEMB12²¹. Our τ-STED results indicated that we could detect a similar tubulo-vesicular network as compared to mCherry-MEMB12 (Fig. 5k-s). To check if MEMB11 and MEMB12 are sitting on the same network we performed co-localization experiments between α-MEMB and mCherry-MEMB12 (Extended data 6c-h). The τ-STED images show a partial co-localization of the two networks that was further confirmed by the quantification using both Pearson and Manders coefficients, as compared to the co-localization control α-MEMB with NAG1-EGFP (Extended data 6i). These results suggest that while MEMB11 and MEMB12 share some potential overlay, the two proteins can localize to two different networks as well. This is reflected in the morphometric analyses we performed on either mCherry-MEMB12 or α-MEMB where we could see that the circularity (reflecting a vesicular shape) of mCherry-MEMB12 is higher than for α-MEMB (Fig. 5j, t; Extended data 6a, b). The solidity (the lowest is the value and the more it reveals a network) of α-MEMB is lower than for mCherry-MEMB12 (Fig. 5j, t; Extended data 6a, b). These results suggest that MEMB11 labels a tubular network while MEMB12 labels a tubulo-vesicular network and bigger pre-cisternae structures. Strikingly, we performed τ-STED on p35S::mRFP-SYP31 and saw a cisterna-like structure, similar to the medial-Golgi although being smaller than NAG1-cisterna (Extended data 6j-t). Cisterna-like structures were also observed in the pSYP32::GFP-SYP32 Arabidopsis line (Extended data 6j-t) and were also smaller than the medial-Golgi (Extended data 6s, t, area). These results are consistent with the IMARIS morphometric analyses (Fig. 1g-r; Fig. 3a-g). Finally, to confirm our findings by yet another independent approach than τ-STED processing, we performed Expansion Microscopy (ExM) to physically expand our samples by a 4X factor and acquire 2D images and 3D-stacks by conventional confocal microscopy. In our ExM images we obtained a lateral resolution of 91 nm (+/- 8 nm). Our results clearly show that the α-MEMB antibody labels a tubular network that is mostly independent from the medial-Golgi (Fig. 5u-w) but that can locally display contact with it (Fig. 5x-ac). Contrastingly, the medial-Golgi is a cisterna, with an accumulation of NAG1-EGFP at the edges of the cisterna, like what we described in τ-STED (Fig. 5a, d, g, k, n, q; Fig. 5x, aa). Furthermore, we acquired 3D ExM stacks, reconstructed them using IMARIS surface rendering and confirmed that α-MEMB labels a Golgi-independent fragmented network while NAG1 is more an isotropic compartment (white arrows in Fig. 5ad-ag; Supplementary video 9). Interestingly, some part of the α-MEMB-labeled network locally surrounds or entangle the medial-Golgi (Fig. 5ad-ag, white arrowheads) and tubular structures (white stars) seems to connect the Golgi-independent and Golgi-associated MEMB-network (Fig. 5ad-ag). Altogether, our τ-STED and ExM results demonstrate that the morphology of MEMB-structures is a Golgi-independent tubulo-vesiculated network that can intertwine the Golgi cisternae.
290 **Sphingolipids regulate the MEMB-tubular network homeostasis**
291 MEMB11/12 are localized on a tubular network. Interestingly, sphingolipids ceramides containing 24
292 atoms of carbon (C24-ceramides) favor the formation of tubular structures through the formation of
293 interdigitated phases²⁸. Thus, we checked the morphology of the MEMB-network by τ-STED microscopy
294 in a double mutant of the Arabidopsis ceramide synthases LOH1 and LOH3 that specifically synthesize
295 C24-ceramides²⁹. The *loh1-2 loh3-2* double homozygote knock-out allele of *LOH1* and *LOH3* is lethal but
296 the sesquimutant (*loh1-2<sup>-/</sup> loh3-2<sup>+/-</sup> or *loh1-2<sup>+/-</sup> loh3-2<sup>-/</sup>*) is viable and displays growth phenotypes such
297 as lateral root development defect²⁹. Our τ-STED results with the α-MEMB antibody show that both the
298 number (per cell) and the density (surface of the compartments per cell area) of MEMB12 objects are
decreased in the *loh1-2<sup>-/</sup> loh3-2<sup>+/-</sup>* sesquimutant (**Fig. 6a-e**). Interestingly, in the *loh1-2<sup>-/</sup> loh3-2<sup>+/-</sup>*
299 sesquimutant we observed a stronger effect on mCherry-MEMB12-labeled structures which was even
300 stronger on the medial-Golgi labeled by NAG1-EGFP (**Fig. 6k-t**). In the *loh1-2<sup>-/</sup> loh3-2<sup>+/-</sup>* sesquimutant
301 our τ-STED images revealed that the medial-Golgi almost completely disappeared (**Fig. 6n, q**) and this is
302 something we also confirmed by airyscan microscopy (**Extended data 7a, b**). The gradual effect we
303 observed suggests a cumulative effect along the ERGIC that is consistent with a sequential maturation
304 from the MEMB-network (α-MEMB) to a more tubulo-vesiculated network with pre-cisternae (mCherry-
305 MEMB12) eventually maturing into a medial-Golgi (NAG1). The morphometric analysis of the
306 remaining MEMB12-structures did not reveal any major change in circularity or solidity in the *loh1-2<sup>-/</sup> loh3-2<sup>+/-</sup>*
307 *loh3-2<sup>+/-</sup>* sesquimutant as compared to the wild-type (**Extended data 7c, d**). Next, we checked the
308 ceramidase *acer-1* mutant that produces more C24-ceramides³⁰. In the *acer-1* mutant, although the effects
309 were less pronounced than in the *loh1-2<sup>-/</sup> loh3-2<sup>+/-</sup>* sesquimutant, we observed a decrease in both the
310 number and density of MEMB2 objects labeled by α-MEMB antibody (**Fig. 6f-j**). This effect was
311 stronger on mCherry-MEMB12-labeled structures and the medial-Golgi labeled by NAG1-EGFP (**Fig.
312 6u-ad**). The morphometric analysis of the remaining MEMB12-structures did not reveal any major
313 change in circularity or solidity in the *acer-1* mutant as compared to the wild-type (**Extended data 7e, f**).
314 These results show that either a reduction or an increase of C24-ceramides result in the reduction of
315 MEMB12-structures suggesting a nonlinear effect of C24-ceramides on membranes, as reported
316 previously²⁸,³¹. Instead, an optimal equilibrium concentration of C24-ceramides³¹ seems to be crucial to
317 keep the MEMB12/ERGIC in check. Moreover, in the *acer-1* mutant, some MEMB12-structures had the
318 typical donut-shape of the medial-cisterna and those actually co-label with the medial-Golgi marker
319 NAG1 (**Fig. 6x-ac, ae**). These results suggest that, in the *acer-1* mutant, the MEMB12-structures are
320 maturing further than what they should. In the wild-type, the MEMB12-structures get stabilized at the
321 vicinity of the Golgi (**Fig. 3g, m**), thus it is possible that C24-ceramides stabilize MEMB12-structures to
322 produce a medial-Golgi cisterna. Thus we performed airyscan dynamic live acquisition of mCherry-
324 MEMB12 and NAG1-EGFP upon alteration of the C24-ceramide composition (Fig. 7a, b; Extended data 8a; Supplementary video 10, 11, 12). We used the ceramidase *acer-1* mutant but did not use the 325 *loh1-2*⁻/⁻ *loh3-2*⁺/⁺ sesquimutant as the medial-Golgi labeled by NAG1 was completely absent (Fig. 6n, q; 326 Extended data 7a, b). Instead, we used FumonisinB1 (FB1), a direct inhibitor of the LOH1 and LOH3 327 enzymes²⁹. As an additional tool, we used Metazachlor (Mz), that specifically inhibits the synthesis of 328 C24 fatty acids that will next be integrated into sphingolipids³²,³³. Upon either FB1 or Mz, we observed a 329 strong decrease of the amount of transient interaction (association/dissociation) between the MEMB12- 330 structures and the medial-Golgi (Fig. 7c; Extended data 8b). This effect was not due to a decrease in the 331 number of MEMB12- or NAG1-compartments upon FB1 or Mz (Extended data 8d). Oppositely, in the 332 *acer-1* mutant, the number of transient interaction is not altered (Fig. 7f; Extended data 8e). 333 334 Interestingly, upon FB1 or Mz, the time of association between MEMB12- and NAG1-compartments as 335 well as the velocity of MEMB12-compartments that are either associated with or independent from the 336 Golgi are not altered (Fig. 7d, e; Extended data 8c, f-h). Oppositely, in the *acer-1* mutant, the time of 337 association is increased and the velocity of Golgi-associated MEMB12-compartments decreased while the 338 velocity of Golgi-independent compartments is not altered (Fig. 7g, h). Altogether, our results show that 339 C24-ceramides are involved in the generation of the MEMB12 tubular-network and its stabilization at the 340 vicinity of the Golgi. If this is true, this would impact the sorting of cargos. Thus, we checked the timing 341 of secGFP-FKBP-RUSH luminal cargo sorting into MEMB12 compartments upon FB1 or Mz treatment. 342 Our results show that, as compared to the control (Fig. 8a-f), there is a time-shift and less prominent 343 cargo-sorting upon FB1 or Mz (Fig. 8g-q; Extended data 8i-s). These results indicate that the sorting of 344 luminal cargos is less efficient or displaced in time when the composition in C24-ceramides is altered 345 which further confirmed the functional role of this sphingolipid specie in trafficking through the 346 MEMB12-tubulo-vesiculated network.

347

348 Discussion

349 Our study revises and transforms our understanding of the architecture and dynamics of the Golgi in a 350 Eukaryotic multicellular system. Our results demonstrate that, in Arabidopsis, the *cis*-Golgi labeled by the 351 Qb-SNARE MEMB11/12 is a highly dynamic network of membrane tubules and vesicles that is mainly 352 independent from either the Golgi or the ERES but can still interacts with them either transiently or more 353 stably. Why was this *cis*-Golgi network never observed before in plant cells? Actually, membrane tubules 354 were suggested to exist between the ER and the plant Golgi for a long time¹²,¹³,³⁴. However, previous 355 immuno-EM experiments using *cis*-Golgi markers were all focused on the Golgi stack and, consistently 356 with our results, all show a pearled membrane structure attached to the *cis*-side of the stack⁹,²¹,³⁵,³⁶. So up
to now our understanding of the Golgi apparatus was biased by a stack-centered view of the Golgi and the governing idea that the *cis*-Golgi is a cisterna moving with the rest of the Golgi. We now propose that the highly dynamic Golgi-independent *cis*-Golgi MEMB11/12-tubulo-vesicular network constitute an early station of the ERGIC in plants (*Fig. 8r, s*). The *cis*-Golgi labeled by the Qa-SNARE SYP31/32 is mostly a stable cisterna-like compartment attached to the medial-Golgi and corresponds to a more mature part of the ERGIC (*Fig. 8r, s*). Interestingly, there is an additional fine gradation in this system, MEMB11 is more tubular than MEMB12 which is a tubulo-vesiculated network with enlarged pre-cisternae, and SYP32 appears as an intermediate cisterna-like compartment between MEMB12 and SYP31 (*Fig. 8r, s*).

Consistently with that revised model, our results show that the MEMB12-compartments are more enlarged and more stable when they get at the proximity of a Golgi-cisterna. Moreover, using the FKBP-RUSH system we show that luminal cargos are crossing the MEMB12-compartments shortly after induced ER-release and then cross SYP32-compartments. We noticed that the vacuole is also stained quickly after ER-release, thus we cannot exclude that luminal cargo follow an additional direct path from the ER as was described earlier for other type of cargos³⁷ or that a proportion of the luminal cargos is mistargeted to the vacuole. Our results indicate that MEMB12-compartments interact transiently with the ERES without being stabilized in agreement with the “kiss-and-run” mechanism that has been proposed in yeast³⁸. Oppositely, MEMB12-compartments dynamically interact with the Golgi and get stabilized at the vicinity of pre-existing Golgi and this is dependent on sphingolipid composition. Previously, it was shown *in vitro* that the acyl-chain length of ceramides induces interdigitated membrane phase and the formation of tubules²⁸,³¹. Consistently, we found that the acyl-chain length of ceramides is involved in the generation of the MEMB12-tubular-network as well as its maturation into a medial-Golgi cisterna. This would most probably occur through the stabilization of the dynamic MEMB12-network as a decrease in C24-ceramides results in the drop of interaction between MEMB12 and the Golgi while an increase in C24-ceramides results in the stabilization of the MEMB12-compartments at the Golgi vicinity. Upon increase of C24-ceramides, MEMB12 is actually even seen on full medial-Golgi cisternae which means that the frontiers between the stabilized MEMB12-pre-cisterna and the medial-Golgi cisterna is controlled by C24-ceramides. Interestingly, the effect of C24-ceramides is nonlinear meaning that a fine-tuned balance in membrane sphingolipid composition participates, probably in combination with other factors, to keep an equilibrium between the flexibility of the MEMB12-tubular network and its stabilization at the vicinity of the Golgi. Our results show that the trafficking of luminal cargos across the ERGIC is dependent on C24-ceramides. However, the question remains open as to whether the cargos are transferred while the pre-cisternae matures from the intermediate dynamic network or whether they are transferred through stabilized membrane contact sites. In animal cells, a wealth of studies has shown how sphingolipids metabolism at the ER/Golgi/trans-Golgi Network nexus impacts on cargo sorting through
membrane contact sites³⁹⁻⁴⁴. However, other studies have shown the role of sphingolipids in protein sorting to vesicular carriers. For example, sphingomyelin with 18 atoms of carbon (SM18) interacts with the p24 complex to initiate COPI-carrier budding and Golgi-to-ER retrograde transport⁴⁵,⁴⁶. Moreover, p24 sorts Glycosyl-Phosphatidyl-Inositol (GPI)-Anchored Proteins (GPI-APs) at specific ERES in COPII-carriers in a C26-ceramides dependent way in yeast or ether-lipids dependent manner in mammals⁴⁷⁻⁴⁹. All these scenarios are not necessarily exclusive since, in animal cells, pearled ER-to-Golgi tubules contain COPII and COPI coats that remain bound to the tubular network rather than forming vesicle carriers⁴,⁵⁰,⁵¹. The results presented in this study open the way to the establishment of a revised model of the Golgi in plants and a revised view of the universality of the corresponding mechanisms across eukaryotes. In this model, some discrete regions of the highly-dynamic tubular MEMB/ERGIC-network are stabilized in a sphingolipid-dependent way into an intertwined membrane sheet, eventually giving rise to a new Golgi-associated pre-cisternae (Fig. 8). In mammalian cells, the ERGIC markers ERGIC-53 and Rab1 localize not only to the structures that are apart from the Golgi ribbon but also to the cis-side of the main Golgi body, suggesting that a similar organization of the ERGIC is shared across kingdoms⁵,⁵²,⁵³.

406

## Acknowledgments

We are grateful to Prof. Ikuko Hara-Nishimura and Dr. Haruko 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 Dr. Yvon Jaillais and Dr. Patrick Moreau for the critical reading of the manuscript and helpful comments. This work was supported by French National Research Agency grants ANR CALIPSO (ANR-18-CE13-0025) and ANR FATROOT (ANR-21-CE13-0019) 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.

417

## Author contribution statement

L.F performed all experiments in Fig. 1, 2, 3, 4, 7, 8 and Extended data 1e-h, 3, 4, 5, 7a-b, 8. M.G established the τ-STED and ExM in Arabidopsis and performed all experiments in Fig. 5, 6 and Extended data 6, 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 the ExM in Arabidopsis. C.P participated with
M.G to establish the τ-STED microscopy in Arabidopsis. T.U provided 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 experiments in Extended data 1a-d, setup the initial airyscan live acquisition and quantification²⁷, 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, got the financial support for, supervised all aspects of this study and wrote the manuscript. The figures were made by L.F and M.G. All authors read and provided inputs on the manuscript.

431

### Competing interests

The authors declare no competing interests

434

### Figure legends

Figure 1. The *cis*-Golgi contains several subpopulations, some of which display a high proportion of structures that remain independent from the Golgi both upon time and in 3D. (a-f) Time-lapse airyscan acquisition in root epidermal cells of either mCherry-MEMB12 (*cis*-Golgi) x NAG1-EGFP (medial-Golgi) (a, b), mCherry-SYP32 (*cis*-Golgi) x NAG1-EGFP (medial-Golgi) (c, d) or mRFP-SYP31 (*cis*-Golgi) x NAG1-EGFP (e, f) showing *cis*-compartments that remain either associated with (white arrowheads) or independent from the medial-Golgi (white arrows). White stars in a, b, g indicate MEMB12 tubular-like structures. (g, j, m) 3D airyscan acquisition of either mCherry-MEMB12 x NAG1-EGFP (g), mCherry-SYP32 x NAG1-EGFP (j) or mRFP-SYP31 x NAG1-EGFP (m) showing that Golgi-independent compartments are not connected to other Golgi in the Z dimension. (g-o) 3D reconstruction (h, k, n) and surface modelling by the IMARIS software (i, l, o) of either mCherry-MEMB12 x NAG1-EGFP (g-i), mCherry-SYP32 x NAG1-EGFP (j-l) or mRFP-SYP31 x NAG1-EGFP (m-o). The medial-Golgi is in green, red and magenta objects are Golgi-associated and Golgi-independent *cis*-Golgi, respectively. (p-r) quantification of IMARIS surface modelling confirming that while SYP31 population is mostly associated to the Golgi, MEMB12 is mostly independent from the Golgi, SYP32 being intermediate between MEMB12 and SYP31 (n= 24 225 Golgi compartments identified out of 12 cells for MEMB12 x NAG1 (p), n= 3 496 Golgi compartments identified out of 12 cells for mCherry-SYP32 x NAG1-EGFP (q) and n= 3 725 Golgi compartments identified out of 12 cells for SYP31 x NAG1 (r)). All scale bars are 1µm. Source numerical data are available in source data.

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Figure 2. MEMB12 *cis*-Golgi is highly dynamic and displays transient interactions with the medial-Golgi. (a) Schematic representation of the Detect Track and Co-localize (DTC) Image J plugin that enables the detection of the centroid of each individual compartments in both channels and calculate the
distance between them (pairwise between opposite channels). A distance of association was defined for each couple of fluorescent markers (see Extended data 2d). (b) Each compartments are followed for a certain time before they disappear from the field of acquisition, this defines the average track time. An association is detected if a compartment of the opposite channel crosses the association zone (light purple) for a certain time during the track time, this defines the transient interaction that last over an average association time. (c-k) Time-lapse airyscan acquisition in root epidermal cells of either mCherry-MEMB12 x NAG1-EGFP (medial-Golgi) (c-e), mCherry-SYP32 x NAG1-EGFP (f-h) or mRFP-SYP31 x NAG1-EGFP (i-k). (c, f, i) Representative time-lapse of Golgi-associated movements for either MEMB12- (c), SYP32- (f) or SYP31-compartments (i). (d, g, j) Representative time-lapse of Golgi-independent movements for either MEMB12- (d), SYP32- (g) or SYP31-compartments (j) away from the medial-Golgi. (e, h, k) Representative time-lapse of transient interaction of either MEMB12- (e), SYP32- (h) or SYP31-compartments (k) with the medial-Golgi. White stars in c, d, e indicate MEMB12 tubular-like structures. (l) Number of MEMB12-, SYP32- or SYP31-compartments (normalized by cell area in µm²) that remain either Golgi-associated or Golgi-independent during the whole time of tracking or that display a transient interaction with the Golgi. MEMB12 is more dynamic and independent from the Golgi than SYP31. SYP32 is intermediate between MEMB12 and SYP31 (for MEMB12/SYP32 and SYP32/SYP31 respectively, **P = 0.0033 and nsP = 0.0991 (associated), ****P = 6.685e-6 and *P = 0.0167 (independent), nsP = 0.1229 and **P = 0.0005 (transient)). (m) Within the population of transient interaction, SYP32 or SYP31 display less events of association and dissociation than MEMB12 (for MEMB12/SYP32 and SYP32/SYP31 respectively, ****P = 1.948e-10 and ***P = 0.0006 (association), ****P = 2.85e-11 and **P = 0.0014 (dissociation)). (n) MEMB12, SYP32 or SYP31 have an association time around 15 sec with the medial-Golgi (nsP = 0.1566 (MEMB12/SYP32), *P = 0.0145 (SYP32/SYP31), nsP = 0.1429 (MEMB12/SYP31)). Data are presented as scatter plots with median, n=40 cells in all set of data. All statistical tests were performed by non-parametric Mann-Whitney two-sided test. All scale bars are 1µm. Source numerical data are available in source data.

Figure 3. The cis-Golgi gets stabilized at the vicinity of pre-existing Golgi. (a-f) 3D reconstruction (a, c, e) and surface modelling by the IMARIS software (b, d, f) of either mCherry-MEMB12 x NAG1-EGFP (a, b), mCherry-SYP32 x NAG1-EGFP (c, d) or mRFP-SYP31 x NAG1-EGFP (e, f). The medial-Golgi is in green, red and magenta objects are Golgi-associated and Golgi-independent cis-Golgi, respectively. (g) ranking of IMARIS surface modelling of MEMB12, SYP32- and SYP31-compartments in function of their distance to the closer medial compartment. The largest part of SYP32- and SYP31-population remain within a 4 µm range distance from the medial-Golgi while MEMB12-population is spread up to 6 µm distance. For SYP32/31, the closer to the medial-Golgi and the bigger are the
492 compartments (n = 9 179 MEMB12-compartments identified out of 12 cells, n = 1 212 SYP32-
493 compartments identified out of 12 cells and n = 800 SYP31-compartments identified out of 12 cells). Data
494 are presented as box-and-whiskers graph, values for the minima, 25% percentile, median, 75 % percentile
495 and maximum are available in **Source data Figure 3** (for MEMB12/SYP32 and SYP32/SYP31
496 respectively, ****P = 2.2e-16 and nsP = 0.1055 (0-2 µm), ****P = 1.446e-5 and nsP = 0.5515 (2-4 µm), nsP =
497 0.1594 (4-6 µm)). (h, i) Time-lapse airyscan acquisition in root epidermal cells of mCherry-MEMB12 x
498 GFP-SYP31. (h) Representative time-lapse of MEMB12-compartments either associated to (white
499 arrowheads) or independent from (white arrows) SYP31-compartments. (i) Representative time-lapse of
500 transient interaction between MEMB12- and SYP31-compartments. (j) Number of SYP31-compartments
501 (normalized by cell area in µm²) that remain either Golgi-associated or Golgi-independent during the
502 whole time of tracking or that display a transient interaction with the Golgi. MEMB12 is mostly
503 displaying transient interaction or stable association with SYP31 (n= 40 cells. ****P = 3.225e-5
504 (associated/transient), ****P = 6.61e-12 (independent/transient), ****P = 1.485e-7
505 (associated/independent)). (k) The association time of MEMB12-compartments with SYP31 is close to 12
506 seconds (n= 40 cells). (l, m) The velocity (µm/sec) of MEMB12-compartments is lower when they
507 associate to either SYP31-compartments (l) or the medial-Golgi (m), as compared to when they are
508 independent from them. (n, o) The velocity (µm/sec) of SYP32- (n) or SYP31-compartments (o) is lower
509 when they associate to the medial-Golgi, as compared to when they are independent from it. (l-o) ****P =
510 6.081e-10 in l, ***P = 0.0003 in m, ****P = 5.499e-13 in n, ****P = 6.529e-9 in o. (j-o) Data are presented
511 as scatter plots with a median line from n=40 cells in all set of data. All statistical tests were performed by
512 non-parametric Mann-Whitney two-sided test. All scale bars are 1µm. Source numerical data are
513 available in source data.

514
515 **Figure 4. MEMB12 cis-Golgi displays transient interactions with the ERES and is crossed by the**
516 **luminal cargo secGFP early after inducible-release from the ER, that then cross SYP32 cis-Golgi.**
517 (a-c) Time-lapse airyscan acquisition in root epidermal cells of mCherry-MEMB12 x Sec16A-like
518 MAG5-GFP (ERES). (a) Representative time-lapse of MEMB12-compartments either associated to the
519 ERES, (b) independent from the ERES or (c) in transient interaction with the ERES. (d) Number of
520 MEMB12-compartments (normalized by cell area in µm²) that are remain either ERES-associated or
521 ERES-independent over the whole time of tracking or that display a transient interaction with the ERES
522 (n= 40 cells, ****P = 9.144e-14 (associated/transient), ****P = 3.53e-14 (associated/independent), nsP =
523 0.6069 (independent/transient)). (e) Within the population of transient interaction, MEMB12 displays
524 equal number of association and dissociation events with the ERES (n= 40 cells, nsP = 0.4478). (f)
525 MEMB12 has an association time around 12 sec with the ERES (n= 40 cells). (g, h) Schema of the
526 secGFP-FKBP-RUSH retained at the ER by the HDEL-hook by FKBP-dimerization and released upon
527 incubation with the FKBP-ligand GPI1485. (i-n) Confocal images of transient transformation in
528 epidermal cotyledon cells of the secGFP-FKBP-RUSH construct (n= 5). (i-k) The secGFP-FKBP-RUSH
529 (i) is retained by the tagBFP2-HDEL-hook at the ER-network (j, merged in k). (l-n) Within 5-15 min
530 after release, secGFP-FKBP-RUSH (l) strongly co-localizes with mCherry-MEMB12 (m) as visible in the
531 merged picture (n). (o-x, z-ai) Airyscan acquisition of root epidermal cells stably expressing either
532 mCherry-MEMB12 (o-x) or mCherry-SYP32 (z-ai) together with secGFP-FKBP-RUSH. The timing of
533 incubation with the FKBP-ligand is indicated in the upper-left corner of each images. (y, aj)
534 Quantification of the normalized percentage of co-localization between secGFP-FKBP-RUSH and either
535 mCherry-MEMB12 (y) or mCherry-SYP32 upon time after incubation with the FKBP-ligand (aj) (n= 774
536 cells out of 47 roots in y, n= 1 217 cells out of 54 roots in aj, from 11 experiments. In y: nsP = 0.7431 (0-
537 5/5-10 min), nsP = 0.2485 (5-10/10-15 min), nsP = 0.6537 (10-15/15-20 min), ****P = 6.016e-5 (15-20/20-
538 25 min) and nsP = 0.4352 (20-25/25-30 min). In aj: nsP = 0.21 (0-5/5-10 min), nsP = 0.0748 (5-10/10-15
539 min), nsP = 0.8362 (10-15/15-20 min), ****P = 9.75e-5 (15-20/20-25 min), nsP = 0.9322 (20-25/25-30
540 min), nsP = 0.4844 (25-30/30-35 min) and nsP = 0.0594 (30-35/35-40 min)). All statistical tests were
541 performed by non-parametric Mann-Whitney two-sided test. Scale bars are 1 µm in a-c, 4 µm in i-n and 2
542 µm in o-x and z-ai. Source numerical data are available in source data.
543

544 **Figure 5. MEMB12 cis-Golgi is a tubular network mostly independent from the Golgi that displays**
545 **local intertwined connection with the medial-Golgi. (a-t) Lifetime τ-STimulated Emission Depletion**
546 (τ-STED) of either mCherry-MEMB12 x NAG1-EGFP (a-i) or α-MEMB immuno-stained compartments
547 in NAG1-EGFP expressing plants (k-s). (j, t) Morphometric quantification of a-i and k-s, respectively.
548 The circularity (reflecting a vesicular shape) of NAG1 was split in two groups, the highest one
549 corresponds to donut-like structures while the lowest group corresponds to rice grain-like structures (see
550 Extended data Fig. 6a, b). The solidity (reflecting network-like structures, the lowest is the value and the
551 more it reveals a network) of NAG1 was high indicative of a homogenous and quiet isotropic structure.
552 Contrastingly, the circularity and solidity of MEMB12 were a spread population with lower values for α-
553 MEMB than for mCherry-MEMB12 indicative that α-MEMB labels a network while mCherry-MEMB12
554 labels a tubulo-vesicular network (in j: out of 16 cells, n= 3 049 mCherry-MEMB12-compartments, n=
555 161 NAG1-compartments, ****P = 2.2e-16 (area and solidity), nsP = 0.09 (circularity). In t: n= 1 837 α-
556 MEMB-compartments, n= 238 NAG1-compartments, ****P = 2.2e-16 (area, circularity and solidity)). (u-
557 ag) 4X Expansion Microscopy (ExM) performed on α-MEMB immuno-stained compartments in NAG1-
558 EGFP expressing plants (n=2 experiments). (u-ac) 2D acquisitions, (ad-ag) 3D acquisitions,
559 reconstruction and surface rendering by IMARIS. For both τ-STED and ExM, MEMB12 is localized on a
tubulo-vesicular network (white arrows and arrowheads) while NAG1 is a cisterna looking a rice grain or a donut, depending on its orientation (note the edge localization of NAG1). The MEMB12 tubulo-vesicular network is mostly independent from the medial-Golgi (white arrows) but intertwined connection are often observed between MEMB12-network and NAG1-cisterna (white arrowheads). The white stars in **ad-ag** indicate tubular connections between Golgi-associated and Golgi-independent MEMB12-network. Connections between MEMB12 and NAG1 can further extend to cisterna-like compartments labelled by MEMB12 that lies close to the medial-cisterna (arrowheads in **r, s, ab, ac**). All statistical tests were performed by non-parametric Mann-Whitney two-sided test. Scale bars are 1µm in **a-i, k-s** and 250 nm in **u-ag**. Source numerical data are available in source data.

**Figure 6.** C24-ceramide sphingolipids are involved in the generation of the MEMB-network and its maturation into the medial-Golgi. Lifetime τ-STimulated Emission Depletion (τ-STED) of either α-MEMB immuno-stained compartments (**a-d, f-i**) or mCherry-MEMB12 x NAG1-EGFP (**k-s, u-ac**) in either the ceramide synthase $loh1^{-/−} loh3^{+/−}$ sesquimutant (**a-d, k-s**) or the ceramidase *acer-1* mutant (**f-i, u-ac**). A decrease in the number of α-MEMB-labelled compartments or their density (surface of the compartments per cell area) is observed in both the $loh1^{-/−} loh3^{+/−}$ sesquimutant (**e**) and *acer-1* mutant (**j**) (n= 15-17 cells in **e**, n= 27-31 cells in **j**. In **e**, **P** = 0.059 (number of compartments) and ****P = 6.062e-5 (α-MEMB density). In **j**, nsP = 0.275 (number of compartments) and *P* = 0.074 (α-MEMB density)). (**k-t**) This effect is stronger on mCherry-MEMB12-labelled compartments and the medial-Golgi labelled by NAG1 in both the $loh1^{-/−} loh3^{+/−}$ sesquimutant (**t**, n= 21-24 cells. For number of compartments, ****P = 7.282e-9 (MEMB12), ****P = 3.284e-9 (NAG1). For density, ****P = 5.3e-13) and *acer-1* mutant (**ad**, n= 17-25 cells. For number of compartments, ****P = 6.734e-5 (MEMB12), ****P = 1.481e-7 (NAG1). For density, ****P = 2.491e-5 (MEMB12), ****P = 8.24e-6 (NAG1)). We could not calculate the density of the medial NAG1-compartments due that they were completely absent in the $loh1^{-/−} loh3^{+/−}$ sesquimutant (**n, q**), this observation was further confirmed in airyscan microscopy (see **Extended data 7a, b**). Strikingly, in the *acer-1* mutant, mCherry-MEMB12 labels the NAG1 medial-cisterna (**x-z**, quantification in **ae**) meaning that the maturation frontiers between the *cis*- and medial-Golgi is controlled by C24-ceramides (n= 16-19 cells in **ae**. ****P = 9.66e-5). All statistical tests were performed by non-parametric Mann-Whitney two-sided test. Scale bars are 1µm. Source numerical data are available in source data.

**Figure 7.** C24-ceramides regulate the dynamic interaction of MEMB12 with the Golgi. (**a, b**) Root epidermal cells expressing mCherry-MEMB12 x NAG1-EGFP (medial-Golgi) upon either the ceramide synthase inhibitor FB1 (**a**) or in the ceramidase mutant *acer-1* (**b**). The time-lapse airyscan acquisitions show Golgi-independent movements of MEMB12 compartments. (**c-h**) Quantification of the dynamic
live tracking either upon FB1 (c-e) or in the *acer-1* mutant (f-h) (n= 40 cells). As compared to the control
wild-type condition (c, f) the number of association and dissociation within the population of transient
interaction between MEMB12 and the medial-Golgi is strongly decreased upon FB1 (c, ****P = 2.698e-6
(association), ****P = 6.539e-6 (dissociation)) while being not altered in the *acer-1* mutant (f, nsP = 0.3118
(Col-0), nsP = 0.2981 (*acer-1*)). (d, g) The association time between MEMB12 and the medial-Golgi is
not altered upon FB1 (d, nsP = 0.2598) but is increased in the *acer-1* mutant (g, ****P = 5.906e-9). (e, h)
The velocity of Golgi-associated and Golgi-independent MEMB12-compartments is not different between
FB1 and the control condition (e, nsP = 0.2361 (control) and nsP = 0.207 (+FB1)) while in the *acer-1*
mutant (h) the velocity of Golgi-associated MEMB12 compartments is decreased (****P = 1.766e-6 (Col-
0), nsP = 0.2722 (*acer-1*)), without altering the velocity of Golgi-independent MEMB12-compartments.
Source numerical data are available in source data.

605

606 **Figure 8. C24-ceramides regulate the passage of the luminal cargo secGFP through MEMB12 and**
607 **revised model of the ER/Golgi interface in plant cells. (a-q) Airyscan acquisition of root epidermal**
608 cells stably expressing mCherry-MEMB12 together with secGFP-FKBP-RUSH in the wild-type control
609 condition (a-e) or upon FB1 (g-p). The timing of incubation with the FKBP-ligand is indicated in the
610 upper-left corner of each images. (f, q) Quantification of the normalized percentage of co-localization
611 between secGFP-FKBP-RUSH and mCherry-MEMB12 upon time after incubation with the FKBP-ligand
612 (n= 774 cells out of 47 roots in f and n= 1 146 cells out of 44 roots in q). Please note that the set of data
613 for the control condition (f) is the same as in Fig. 4o-y (P values in Fig. 4y) but the display pictures (a-e)
614 are different. As compared to the control (f), FB1 treatment (q) delays the passage of the secGFP-FKBP-
615 RUSH luminal cargo through MEMB12 compartments (n=6 experiments. nsP = 0.3948 (0-5/5-10 min),
616 ****P = 1.489e-10 (5-10/10-15 min), ****P = 1.176e-9 (10-15/15-20 min), ****P = 2.208e-11 (15-20/20-25
617 min), nsP = 0.6927 (20-25/25-30 min)). All statistical tests were performed by non-parametric Mann-
618 Whitney two-sided test. Scale bars are 2 µm in a-e and g-p. Source numerical data are available in source
619 data. (r) Up to now, the text-book model stated that in plants the ER-to-Golgi trafficking is ensured
620 through vesicular transport between the ER and the cis-most cisterna of the Golgi stack. The cis-Golgi
621 was thought to be exclusively associated to the Golgi stack. (s) Our revised model now break-through this
622 dogma by identifying a cis-Golgi tubulo-vesiculated network independent from the Golgi stack through
623 which cargos are transferred between the ER and the Golgi. This network is similar to the Vesicular-
624 Tubular Cluster (VTC)/Endoplasmic Reticulum Golgi Intermediate Compartment (ERGIC) of animal
625 cells. The Qb-SNARE MEMBRIN11/12 (MEMB11, MEMB12) label a tubulo-vesicular network that
626 dynamically interacts with the Endoplasmic Reticulum Exit Sites (ERES) from one side and the Golgi on
627 the other side. Interaction with pre-existing Golgi stabilizes the MEMB-network into more stable pre-
628 cisternae that mature into stable cis-Golgi cisternae labelled by the Qa-SNARE SYP32/31. C24-ceramide
629 sphingolipids are involved in both the generation of the MEMB-network and its stabilization at the
630 vicinity of the Golgi.

631
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**Online methods**

**Cloning, plant material and growth conditions.** The following previously published Arabidopsis transgenic fluorescent marker lines were used: pUBQ10:: mCherry-MEMB12 (Wave 127R)⁵⁴, pUBQ10:: mCherry-SYP32 (Wave 22R)⁵⁴, p35S::NAG1-EGFP⁵⁵, Sec16A-like pMAG5::MAG5-GFP²²,²³. The following mutant lines were used: the ceramidase *acer-1* mutant³⁰ and the ceramide synthase *loh1*⁻/⁻
loh3<sup>+/-</sup> sesquimutant<sup>29</sup>. *acer-1* and *loh1<sup>-/-</sup> loh3<sup>+/-</sup> sesquimutant were genotyped using the primers in **Supplementary table 1**. In this study we produced the following transgenic fluorescent marker lines: p35s::GFP-SYP31, p35s::mRFP-SYP31, pSYP32::GFP-SYP32, p35s::ST-mRFP. The generation of the secGFP-FKBP-RUSH line is described in the "Inducible cargo release using the Retention Using Selective Hooks (RUSH) system" part of the online methods. To generate *p35S::GFP-SYP31* and *p35S::mRFP-SYP31*, the DNA fragments coding *p35S::XFP-SYP31-Nos3'* were amplified by PCR from the SYP31 vectors previously published<sup>56</sup>. They were cloned into pENTR/D-TOPO (Thermo Fisher Scientific/Invitrogen), and recombined into pKGW by LR Clonase II (Thermo Fisher Scientific/Invitrogen). To generate *pSYP32::GFP-SYP32*, the *Arabidopsis* genomic SYP32 DNA fragment which contains 2.5 kb 5' and 0.8 kb 3' flanking sequences was amplified by PCR and cloned into pENTR/D-TOPO. The DNA fragment coding GFP was inserted at the 5' side of SYP32 by In-Fusion HD Cloning Kit (Takara), and the whole sequence was recombined into pGWB1 by LR Clonase II. To generate *p35S::ST-mRFP*, first the DNA fragment coding ST was amplified by PCR and cloned into pENTR/D-TOPO, and recombined into pH7RWG2. Next, the DNA coding ST-mRFP was amplified from it and cloned into pENTR/D-TOPO again, and recombined into pGWB2 by LR Clonase II. These constructs were transformed into WT *Arabidopsis* or *gnl1-1/GNL1-Myc* BFA-sensitive *Arabidopsis* line<sup>57</sup> by floral dipping using *Agrobacterium tumefaciens*<sup>58</sup>. Double fluorescent lines were obtained by crossing of the lines above. For all observations, seeds were treated with 0.1% Triton in water during 5 minutes, then washed three times with sterile deionized water and kept 3-4 days at 4°C in water. The seed were then sterilized using a 0.9% chlorine/0.1% Triton solution during 20 minutes and sown on ½ Murashige and Skoogs (MS) agar medium plates (0.8% plant agar, 1% sucrose, and 2.5mM morpholinoethanesulfonic acid pH5.8 with KOH). The seedlings were grown 5 days vertically in long day conditions (150 mE/m<sup>2</sup>/s -1, 16h light/ 8h dark) at 22°C.

**Live cells airyscan and confocal microscopy.** Live time-lapse and Z-stacks acquisitions in **Fig. 1, 2, 3, 4, 7** (except for **Fig. 4i-n**), and **Extended data 1, 3, 5, 7, 8** (except for **Extended data 1a-d, 5h-j**) were done using the Airyscan1 module of a Zeiss LSM 880 using 40X oil-immersion objective (NA 1.3). Live cell airyscan microscopy were performed on time-lapse acquisition for the dynamic tracking of compartments association, 3D-reconstruction using the IMARIS software and cargo trafficking analysis using the Retention Using Selective Hooks (RUSH) system. In all cases, seedlings were mounted between an Epredia™TM SuperFrost Plus slide and a 20x20 mm #1.5 coverslip spaced by double-sided tape, in this small chamber the seedlings were incubated with ½MS liquid medium (1% sucrose, and 2.5mM morpholinoethanesulfonic acid pH5.8 with KOH). Dynamic time-series acquisitions were performed as follow: 150 frames, 1 frame every 2 seconds. 3D Z-stacks series were done with a Z-step of 100 nm through
10 µm thickness. The 561 nm excitation laser was used for mCherry or mRFP while the 488 nm laser was used to imaged GFP, the MBS used was 488/561/633 and the filters used were BP495-550 + LP570. Live cell confocal imaging in **Fig. 4i-n, Extended data 1a-d, 5h-j** was performed with a Zeiss LSM 880 using 40X oil-immersion objective (NA 1.3). The 488 nm excitation laser was used for GFP, the 561 nm laser was used for mRFP and the 405 nm laser was used for the tagBFP2.

**3D Modelling.** 3D reconstruction and analysis was performed using the IMARIS10 software. Prior to IMARIS analysis a median filter (3x3x1) and a subtract background (rolling radius 2, except for p35S::NAG1-EGFP where no subtract background was done) were applied to the Z-stack acquisitions. 3D volumes were created for both channels and then sorted according to the distance between either mCherry-MEMB12, mCherry-SYP32 or mRFP-SYP31 compartments with the nearest p35S::NAG1-EGFP compartment. We then classified them as Golgi-associated or Golgi-independent and calculated their areas.

**Quantitative live tracking of compartment association.** The dynamic analysis was done using the Detect, Track, and Colocalize (DTC) plugin of ImageJ (https://github.com/fabricecordelieres/IJ-Plugin_DTC)⁵⁹. This plug-in identifies the interactions occurring between compartments of two channels and calculates their durations. Images were pre-treated as follow before being analysed with the DTC: individual cells were manually delimited using the polygon selection tool and added to the ROI manager; a Gaussian blur filter (sigma 2) and a subtract background (rolling radius 10, except for p35S::NAG1-EGFP were the rolling radius was 5) were applied to all images. The geometrical centres or centroids were calculated for all compartments in the two channels and for each time points. The compartments were tracked if they were at least detected in two successive frames. The maximum of displacement of a given object between two time-frames was setup to 9 pixels. We setup a reference value for the distance of association between two compartments by measuring, for each couple of markers, the average distance between two compartments when they were clearly associated (see **Extended data 2d**). This distance of association was then applied to the time-lapse acquisitions to distinguish when a compartment in the first channel is associated to another in the second channel. If the distance between one compartment in the first channel and the nearest compartment in the second channel is inferior to the reference distance of association, then we considered that they were associated. Oppositely, if their distance was superior to the reference distance of association, then we considered that they were not associated, e.g. independent. To calculate the upper edge of the tracking quantification pipeline, we checked that the transient interactions we detected are not due to the loss of objects during the tracking. For that we calculated the average duration of one track and compare it to the average association time of its respective marker, in all cases the time track was superior to the association time (Extended data 3b, f, j; Extended data 4a; Extended data 5a). Thus the upper edge
defines that the time track last for a sufficient amount of time to follow a complete interaction sequence.

To obtain the lower edge, we evaluated the stochastic background, in other words: what is the chance to detect an interaction just by chance? To check this, we either turned one channel by 180° or mirrored it vertically or horizontally and keep the other channel still. In all cases when one channel is turned or mirrored, the number of Golgi-associated structures is reduced close to 0 and the number of Golgi-independent structures is much higher (Extended data 3c, g, k; Extended data 4b; Extended data 5b).

Furthermore, the average association time is strongly reduced in all cases we analyzed (Extended data 3d, h, l; Extended data 4c; Extended data 5c). Thus, the interaction events we see are not due to random movements. For the RUSH quantification, we applied as association distance the resolution limit for the AiryScan cell per cell analysed. All values were calculated for each detected compartment, in both channels and each time-frames, the values were then sorted using an excel macro based on Microsoft Visual Basic we established and made available in **Supplementary table 2**.

**Inducible cargo release using the Retention Using Selective Hooks (RUSH) system.** Cloning of the RUSH system for plant expression was based on multisite Gateway® three-fragment vector construction. In the first vector (pDONR P4-P1r) we introduced the mild-expressing UBQ10 promoter (pUBQ10)²⁴, in the second cassette (pDONR P1-P2) we introduced the cargo and in the third cassette (pDONR P2r-P3) we introduced the ER-hook. The three cassette were combined into the pB7m34GW plant expression vector. The luminal cargo sequence was made of an ER-signal peptide (SP) followed by EGFP (optimized for Arabidopsis) and FKBP (Supplementary table 3). The ER-hook sequence was made of a peptide 2A (F2A), an ER-signal peptide (SP) followed by TagBFP2, a linker, FKBP and the ER-retention HDEL peptide (Supplementary table 3). The cargo and hook sequences were obtained by gene synthesis and then integrated into pDONR P1-P2 and pDONR P2r-P3, respectively. We transformed by floral dip the pUBQ10::SP-EGFP-FKBP-P2A-SP-TagBFP2-linker-FKBP-HDEL/ pB7m34GW construct into Arabidopsis thaliana Col-0 plants expressing the pUBQ10::mCherry-MEMB12 marker. We selected 3 homozygote mono-insertion lines and further worked with one line. The selection was based on resistance to Basta (10 µM phosphinotricine final concentration in MS plate). The mono-insertion line selected in Col-0 was crossed in pUBQ10::mCherry-SYP32 transgenic fluorescent line. Live imaging microscopy was performed on 5 days old seedlings that were mounted between a slide and coverslip into ½ MS liquid media containing 200 µM of the synthetic FKBP ligand GPI1485 (MedChemExpress). The glass slides were then vacuumed 3 times for 30 seconds at 300mmHg and then immediately placed under the microscope for acquisition, the t₀ time was defined as when the vacuum was released. The co-localization quantification was based on the limit of resolution measured in our airyscan images, the geometrical centres or centroids were calculated for all compartments in the two channels using the DTC plugin. If the distance between
one compartment in the first channel and the nearest compartment in the second channel is inferior to 166 nm, then we considered that they were co-localized. Oppositely, if their distance was superior to 166 nm, then we considered that they were not co-localized. To setup a baseline of co-localization, we calculated the percentage of co-localization between secGFP-FKBP-RUSH and either mCherry-MEMB12 or mCherry-SYP32 without addition of the FKBP ligand and then used this value to normalize the co-localization values after addition of the FKBP ligand.

**Inhibitor treatments.** Metazachlor (Mz, Merck, Sigma PESTANAL®) treatment was performed on seedlings grown for 5 days on 100 nM Mz-containing ½ MS plates. Fumonisin B1 (FB1, Merck Sigma) treatment was performed on 5-day old seedlings grown on 1.25 µM FB1-containing ½ MS plates. Brefeldin A (BFA, Merck Sigma) treatment was performed on seedlings grown for 5 days on ½ MS plates that were transferred for 1.5 hours into multiwell plates containing liquid ½ MS medium with 50 µM BFA.

**Immuno-staining.** The immuno-localization protocol was adapted from Boutté and Grebe, 2014⁶⁰ and used an Intavis InsituPro VSi robot. After seedling fixation (4% paraformaldehyde dissolved in MTSB: 50mM PIPES,5mM EGTA, 5mM MgSO4 pH 7 with KOH) and root tip dissection on polylysine-coated Epredia® SuperFrost Ultra Plus Gold slides (Fisher Scientific), the slides with the root samples were mounted under a counter slide using 1X MTSB solution and positioned in the InsituPro VSi holder. The program for single and double staining are described in the **Supplementary table 4**. At the end of the program, slides were mounted under a coverslip #1.5 using ProlongGold anti-fade mounting-medium (InVitrogen). The following antibodies and nanobodies were used: rabbit α-MEMB²¹ 1/100, donkey anti-rabbit AlexaFluor AF594 1/300 (InVitrogen), goat anti-rabbit AlexaFluor AF488 1/300 (InVitrogen), goat anti-rabbit ATTO647N 1/300 (Sigma-Aldrich), ChromoTek GFPbooster-ATTO647N 1/200 (Proteintech), ChromoTek RFPbooster-ATTO594 1/200 (Proteintech).

**Lifetime tau-Stimulated Emission Depletion (τ-STED) microscopy and quantification.** Acquisitions were done using a Leica SP8 (DMI6000 TCS SP8 X) equipped with a STED and a FALCON module in sequential mode with a 93X Glycerol immersion objective (NA 1.4). The excitation laser wave lines were 594 nm for the AF594 and ATTO594 and 640 nm for the ATTO647N. The power of the excitation laser was set between 5-15%, the power of the 775 nm depletion laser (used for AF594, ATTO594 or ATTO647N) was set between 30-50%, depending on the expression strength of the protein marker and the fluorophore used. Emission signals were collected between 610-640 nm for AF594/ATTO594 and between 655-690 nm for ATTO647N with a frame accumulation between 10 and 12 for FLIM measurements. Images were automatically processed by the τ-STED module of Leica, LAS X Falcon. Morphometric
analysis of τ-STED images (area, circularity, solidity) were performed on filtered images using a median filter (3x3x2) that were then converted into binary images. Then the analyse particle plugin of ImageJ was applied using the following set up: area from 0.04 to infinity, circularity from 0 to 1, add to ROI manager and exclude on the edges of the image. The ROIs were applied on the original images and ROIs that do not correspond to an individual structure were eliminated. Measurement were set up to obtain the area and shape descriptor of each individual ROI, from the shape descriptor we extracted the circularity and solidity. A circularity value of 1 indicates a perfect circle and a value of 0 indicates a line. The solidity is a ratio of the area by the full convex area of an object. If the full convex area and the area are the same the solidity will be close to 1 while if the object contains indentations, then the full convex area will be superior to the area and thus the solidity ratio will be below 1. The density is the ratio of the sum of all detected ROIs by the area of the cell. When a nucleus is detected, the nucleus area is subtracted from the cell area to get only the cytosolic cell area. The following formulas were used for the calculations:

$$Circularity = 4 \times \pi \times \left( \frac{area}{perimetre^2} \right)$$

$$Solidity = \frac{area}{convex area}$$

$$Density = \frac{somme ROI area}{cell area}$$

Co-localization measurements were performed using the Coloc 2 plugin of FIJI software. Manders and Pearson coefficients has been calculated after subtraction of the background and an auto threshold in the aim to avoid pixels below the image's threshold in the analysis according to the Costes method⁶¹.

**Expansion Microscopy (ExM).** The proExM method was adapted from⁶². Details of the ExM protocol can be find at⁶³. After fixation and immunolabeling root tips are glued to 12 mm round glass coverslips coated with Poly-L-Lysine prior to proExM protocol. Then after digestion, gels containing the roots are detached from the coverslip and carefully dissected to eliminate excess of empty gel. Root-containing gels are transferred to a clean container and expanded in water. Water is renewed several times until plateau is reached. Gels are finally mounted in a coverslip glass previously treated with poly-L-lysine for imaging. Expended root tips were imaged using a Leica SP8 (DMI6000 TCS SP8 X) in sequential mode with a 25X water plunging objective (NA 0.8). The excitation laser wave lines were 594 nm for the AF594 and 647 nm for the ATTO647N. The power of the excitation laser was set between 5-15%, and emission signals were collected between 610-640 nm for AF594 and between 655-690 nm for ATTO647N.
Calculation of the limit of resolution. References were taken in images from τ-STED, ExM and airyscan images to calculate the resolution of the system directly on the image using FIJI. The intensity distributions were fitted to the nearest gaussian and the full width at half maximum “d” value were used to calculate the resolution with the following formula:

$$Resolution = 2 \times \sqrt{2 \times \ln(2)} \times d$$

A minimum of 20 measures have been done for each technics and “d” values were used only if the fitting curve displayed a R²>0,90.

## Statistics & Reproducibility.

General information on statistical methods. No statistical methods were used to pre-determine sample sizes. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Data distributions were not formally tested (except for Extended data 6), therefore for all statistical tests we used non-parametric tests. The comparisons of two data-sets were performed by Mann-Whitney two-sided test (P value > 0.1234 (ns), ≤ 0.0332 (*), ≤ 0.0021 (**), ≤ 0.0002 (***), ≤ 0.0001 (****)). In Extended data 6, the comparison of more than two data-sets were performed by Brown Forsythe test because the data distributions were not Gaussian according to the Kolmogorov-Smirnov test (P value > 0.1234 (ns), ≤ 0.0332 (*), ≤ 0.0021 (**), ≤ 0.0002 (***), ≤ 0.0001 (****)). The statistical tests and data-graphs were performed by GraphPad PRISM. Data collection and analysis were not performed blinded to the conditions of the experiments.

General information on reproducibility. For airyscan live cell dynamic tracking, analyses were performed on 40 cells from 20 roots in 4 independent experiments (Fig 2, 3j-o, 4d-f, 7c-h, Extended data 3, 4, 5a-c, 8b-h). For Extended Data 1, the observation was done in n= 3 independent experiments. For IMARIS 3D reconstruction, analyses were performed on 12 cells from 6 roots in 2 independent experiments (Fig 1p-r, 3g). For RUSH assays, analyses were performed on 774 cells out of 47 roots (Fig. 4o-y, Fig. 8a-f) and 1 217 cells out of 54 roots (Fig. 4z-aj) from 11 independent experiments, on 1 146 cells out of 44 roots (Fig. 8g-q) and 680 cells out of 38 roots (Extended Data 8i-s) from 6 independent experiments. In Fig. 4i-n, observations were done in n= 5 independent experiments. In Extended Data 5d-g and h-j, the observations were done in n= 3 independent experiments. For STED experiments, analyses were performed on 16, 15-17, 27-31, 21-24, 17-25, 16-19 cells from 3 independent experiments (Fig 5a-t, 6e, 6j, 6t, 6ad, 6ae respectively). In Fig. 5u-ag, observations were done in n= 2 independent experiments. For STED, n= 176 NAG1-compartments including 88 “rice” and 88 “donuts” in and n= 3 049 mCherry-MEMB12-compartments in Extended Data 6a, n= 238 NAG1-compartments including 150 “rice” and 88 “donuts” and n= 1 837 α-MEMB -compartments in Extended Data 6b, from 3 independent experiments. The co-localisation analyses in Extended Data 6i, were performed on 34 measures from 3 independent
949 experiments. For STED, n= 315 NAG1-compartments in **Extended Data 6s** including 199 "rice" and 116
950 "donut" in **Extended Data 6t**, n= 134 SYP32-compartments in **Extended Data 6s** including 68 "rice" and
951 66 "donut" in **Extended Data 6t**, n= 311 SYP31-compartments in **Extended Data 6s** including 217 "rice"
952 and 90 "donuts" in **Extended Data 6t** out of 16 cells, from 3 independent experiments. n= 8 331 and 3 802
953 compartments out of 15-17 cells for Col-0 and *loh1*<sup>-/-</sup> *loh3*<sup>+/-</sup> in **Extended Data 7c**, n= 5 969 and 1 209
954 compartments out of 21-24 cells for Col-0 and *loh1*<sup>-/-</sup> *loh3*<sup>+/-</sup> in **Extended Data 7d**, n= 11 095 and 7 911
955 compartments out of 27-31 cells for Col-0 and *acer-1* in **Extended Data 7e** and n= 3 101 and 2 769
956 compartments out of 17-25 cells for Col-0 and *acer-1* in **Extended Data 7f**, from 3 independent
957 experiments.

958
959 **Data availability statement**
960
961 Data supporting the findings of this work are available in this paper and its extended data and supplementary
962 information. All data generated and analysed in this study are available from the corresponding author upon
963 request. Source data are provided with this paper.

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986
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 8</figcaption></figure>
Extended Data Figure 1

<figure><img src="image_11.png" /></figure>
Extended Data Figure 2

a

<figure><img src="image_12.png" /></figure>

b

<figure><img src="image_13.png" /></figure>

c

<figure><img src="image_14.png" /></figure>

d

<figure><img src="image_15.png" /></figure>

<table><thead><tr><th>channels</th><th>distance to other channel</th></tr></thead><tbody><tr><td>mCherry-MEMB12</td><td>6.0 pxl</td></tr><tr><td>medial (NAG1)</td><td>6.0 pxl</td></tr></tbody></table>

e

<figure><img src="image_16.png" /></figure>

f

<figure><img src="image_17.png" /></figure>

g

<figure><img src="image_18.png" /></figure>

h

<figure><img src="image_19.png" /></figure>
Extended Data Figure 3

<figure><img src="image_20.png" /></figure>

MEMB12 x medial (NAG1)

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

SYP32 x medial (NAG1)

<figure><img src="image_22.png" /></figure>

SYP31 x medial (NAG1)
<figure><img src="image_23.png" /><figcaption>Extended Data Figure 4</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>Extended Data Figure 5</figcaption></figure>
<figure><img src="image_25.png" /><figcaption>Extended Data Figure 6</figcaption></figure>
Extended Data Figure 7

<figure><img src="image_26.png" /><figcaption>mCherry-MEMB12
X medial (NAG1)</figcaption></figure>

loh1+ loh3-

<figure><img src="image_27.png" /><figcaption>c α-MEMB area</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>α-MEMB circularity</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>α-MEMB solidity</figcaption></figure>

<figure><img src="image_30.png" /><figcaption>d mCherry-MEMB12
area</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>mCherry-MEMB12
circularity</figcaption></figure>

<figure><img src="image_32.png" /><figcaption>mCherry-MEMB12
solidity</figcaption></figure>

<figure><img src="image_33.png" /><figcaption>e α-MEMB area</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>α-MEMB circularity</figcaption></figure>

<figure><img src="image_35.png" /><figcaption>α-MEMB solidity</figcaption></figure>

<figure><img src="image_36.png" /></figure>

f

<figure><img src="image_37.png" /><figcaption>area</figcaption></figure>

<figure><img src="image_38.png" /><figcaption>circularity</figcaption></figure>

<figure><img src="image_39.png" /><figcaption>solidity</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>Extended Data Figure 8</figcaption></figure>