Eric Hummel

A missense mutation in the Arabidopsis COPII coat protein Sec24A induces the formation of clusters of the endoplasmic reticulum and golgi apparatus.

How the endoplasmic reticulum (ER) and the Golgi apparatus maintain their morphological and functional identity while working in concert to ensure the production of biomolecules necessary for the cells survival is a fundamental question in plant biology. Here, we isolated and characterized an Arabidopsis thaliana mutant that partially accumulates Golgi membrane markers and a soluble secretory marker in globular structures composed of a mass of convoluted ER tubules that maintain a connection with the bulk ER. We established that the aberrant phenotype was due to a missense recessive mutation in sec24A, one of the three Arabidopsis isoforms encoding the coat protomer complex II (COPII) protein Sec24, and that the mutation affects the distribution of this critical component at ER export sites. By contrast, total loss of sec24A function was lethal, suggesting that Arabidopsis sec24A is an essential gene. These results produce important insights into the functional diversification of plant COPII coat components and the role of these proteins in maintaining the dynamic identity of organelles of the early plant secretory pathway.

A Recycling-Defective Vacuolar Sorting Receptor Reveals an Intermediate Compartment Situated between Prevacuoles and Vacuoles in Tobacco

This work examines two mutations of the same motif in a vacuolar sorting receptor that have opposite effects on the protein’s localization, shedding light on the full transport cycle of the receptor. The results support the idea that the prevacuolar compartment matures by gradual receptor depletion from an early prevacuolar compartment to a previously undescribed late prevacuolar compartment. Plant vacuolar sorting receptors (VSRs) display cytosolic Tyr motifs (YMPL) for clathrin-mediated anterograde transport to the prevacuolar compartment. Here, we show that the same motif is also required for VSR recycling. A Y612A point mutation in Arabidopsis thaliana VSR2 leads to a quantitative shift in VSR2 steady state levels from the prevacuolar compartment to the trans-Golgi network when expressed in Nicotiana tabacum. By contrast, the L615A mutant VSR2 leaks strongly to vacuoles and accumulates in a previously undiscovered compartment. The latter is shown to be distinct from the Golgi stacks, the trans-Golgi network, and the prevacuolar compartment but is characterized by high concentrations of soluble vacuolar cargo and the rab5 GTPase Rha1(RabF2a). The results suggest that the prevacuolar compartment matures by gradual receptor depletion, leading to the formation of a late prevacuolar compartment situated between the prevacuolar compartment and the vacuole.

The Syntaxins SYP31 and SYP81 Control ER–Golgi Trafficking in the Plant Secretory Pathway

Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter α‐amylase with the ER‐retained reporter α‐amylase‐HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N‐terminal‐tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C‐terminal‐tagged SYP31 failed to exhibit this effect. Overexpression of both wild‐type and N‐terminal‐tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)–SYP31 was still visible as fluorescent punctae, which, unlike SYP31–GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non‐inhibitory levels, YFP–SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.

The plant ER-Golgi interface.

The interface between the endoplasmic reticulum (ER) and the Golgi apparatus is a critical junction in the secretory pathway mediating the transport of both soluble and membrane cargo between the two organelles. Such transport can be bidirectional and is mediated by coated membranes. In this review, we consider the organization and dynamics of this interface in plant cells, the putative structure of which has caused some controversy in the literature, and we speculate on the stages of Golgi biogenesis from the ER and the role of the Golgi and ER on each other’s motility.

Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

Autophagosome formation requires sequential translocation of autophagy-specific proteins to membranes enriched in PI3P and connected to the ER. Preceding this, the earliest autophagy-specific structure forming de novo is a small punctum of the ULK1 complex. The provenance of this structure and its mode of formation are unknown. We show that the ULK1 structure emerges from regions, where ATG9 vesicles align with the ER and its formation requires ER exit and coatomer function. Super-resolution microscopy reveals that the ULK1 compartment consists of regularly assembled punctate elements that cluster in progressively larger spherical structures and associates uniquely with the early autophagy machinery. Correlative electron microscopy after live imaging shows tubulovesicular membranes present at the locus of this structure. We propose that the nucleation of autophagosomes occurs in regions, where the ULK1 complex coalesces with ER and the ATG9 compartment.

Golgi Regeneration after Brefeldin A Treatment in BY-2 Cells Entails Stack Enlargement and Cisternal Growth followed by Division

Brefeldin A (BFA) treatment stops secretion and leads to the resorption of much of the Golgi apparatus into the endoplasmic reticulum. This effect is reversible upon washing out the drug, providing a situation for studying Golgi biogenesis. In this investigation Golgi regeneration in synchronized tobacco BY-2 cells was followed by electron microscopy and by the immunofluorescence detection of ARF1, which localizes to the rims of Golgi cisternae and serves as an indicator of COPI vesiculation. Beginning as clusters of vesicles that are COPI positive, mini-Golgi stacks first become recognizable 60 min after BFA washout. They continue to increase in terms of numbers and length of cisternae for a further 90 min before overshooting the size of control Golgi stacks. As a result, increasing numbers of dividing Golgi stacks were observed 120 min after BFA washout. BFA-regeneration experiments performed on cells treated with BFA (10 μg mL−1) for only short periods (30–45 min) showed that the formation of ER-Golgi hybrid structures, once initiated by BFA treatment, is an irreversible process, the further incorporation of Golgi membranes into the ER continuing during a subsequent drug washout. Application of the protein kinase A inhibitor H-89, which effectively blocks the reassembly of the Golgi apparatus in mammalian cells, also prevented stack regeneration in BY-2 cells, but only at very high, almost toxic concentrations (>200 μm). Our data suggest that under normal conditions mitosis-related Golgi stack duplication may likely occur via cisternal growth followed by fission.

FIB-SEM tomography of human skin telocytes and their extracellular vesicles.

We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non‐epithelial spaces (stroma) of many organs (see www.telocytes.com). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB‐SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB‐SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon‐like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms. FIB‐SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.

Golgi membrane dynamics after induction of a dominant-negative mutant Sar1 GTPase in tobacco

An inducible system has been established in Nicotiana tabacum plants allowing controlled expression of Sar1-GTP and thus the investigation of protein dynamics after inhibition of endoplasmic reticulum (ER) to Golgi transport. Complete Golgi disassembly and redistribution of Golgi markers into the ER was observed within 18-24h after induction. At the ultrastructural level Sar1-GTP expression led to a decrease in Golgi stack size followed by Golgi fragmentation and accumulation of vesicle remnants. Induction of Sar1-GTP resulted in redistribution of the green fluorescent protein (GFP)-tagged Arabidopsis golgins AtCASP and GC1 (golgin candidate 1, an Arabidopsis golgin 84 isoform) into the ER or cytoplasm, respectively. Additionally, both fusion proteins were observed in punctate structures, which co-located with a yellow fluorescent protein (YFP)-tagged version of Sar1-GTP. The Sar1-GTP-inducible system is compared with constitutive Sar1-GTP expression and brefeldin A treatment, and its potential for the study of the composition of ER exit sites and early cis-Golgi structures is discussed.

Inhibition of Golgi function causes plastid starch accumulation

Little is known about possible interactions between chloroplasts and the Golgi apparatus, although there is increasing evidence for a direct Golgi to chloroplast transport pathway targeting proteins to their destinations within the membranes and stroma of plastids. Here data are presented showing that a blockage of secretion results in a significant increase of starch within plastids. Golgi disassembly promoted either by the secretory inhibitor brefeldin A or through an inducible Sar1-GTP system leads to dramatic starch accumulation in plastids, thus providing evidence for a direct interaction between plastids and Golgi activity. The possibility that starch accumulation is due either to elevated levels of cytosolic sugars because of loss of secretory Golgi activity or even to a blockage of amylase transport from the Golgi to the chloroplast is discussed.

Biogenesis of the plant Golgi apparatus.

It has long been assumed that the individual cisternal stacks that comprise the plant Golgi apparatus multiply by some kind of fission process. However, more recently, it has been demonstrated that the Golgi apparatus can be experimentally disassembled and the reformation process from the ER (endoplasmic reticulum) monitored sequentially using confocal fluorescence and electron microscopy. Some other evidence suggests that Golgi stacks may arise de novo in cells. In the present paper, we review some of the more recent findings on plant Golgi stack biogenesis and propose a new model for their growth de novo from ER exit sites.