Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.

Spatiotemporal dissection of the Golgi apparatus and the ER-Golgi intermediate compartment in budding yeast.

Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis-cisternae, the earliest Golgi sub-compartment, is generated and how the Golgi matures into the trans-Golgi network (TGN). Here, we use high-speed and high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term 'yeast ERGIC'. It occasionally exhibits approach and contact behavior toward the ER exit sites and gradually matures into the cis-Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis- and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins to better understand the Golgi-TGN transition. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the entire cisternal maturation process from the ERGIC to the Golgi and further to the TGN.

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIß activation in gastrointestinal stromal tumor cells.

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase gene, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes Golgi retention of KIT. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT cannot trigger downstream activation. In the Golgi/trans-Golgi network (TGN), KIT activates the PKD2-phosphatidylinositol 4-kinase IIIß (PKD2-PI4KIIIß) pathway through phospholipase Cγ2 (PLCγ2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in the release of KIT from the Golgi/TGN. Our findings show the molecular mechanisms underlying KIT mislocalization and provide evidence for a strategy for inhibition of oncogenic signaling.

The yeast endocytic early/sorting compartment exists as an independent sub-compartment within the trans-Golgi network.

Although budding yeast has been extensively used as a model organism for studying organelle functions and intracellular vesicle trafficking, whether it possesses an independent endocytic early/sorting compartment that sorts endocytic cargos to the endo-lysosomal pathway or the recycling pathway has long been unclear. The structure and properties of the endocytic early/sorting compartment differ significantly between organisms; in plant cells, the trans-Golgi network (TGN) serves this role, whereas in mammalian cells a separate intracellular structure performs this function. The yeast syntaxin homolog Tlg2p, widely localizing to the TGN and endosomal compartments, is presumed to act as a Q-SNARE for endocytic vesicles, but which compartment is the direct target for endocytic vesicles remained unanswered. Here we demonstrate by high-speed and high-resolution 4D imaging of fluorescently labeled endocytic cargos that the Tlg2p-residing compartment within the TGN functions as the early/sorting compartment. After arriving here, endocytic cargos are recycled to the plasma membrane or transported to the yeast Rab5-residing endosomal compartment through the pathway requiring the clathrin adaptors GGAs. Interestingly, Gga2p predominantly localizes at the Tlg2p-residing compartment, and the deletion of GGAs has little effect on another TGN region where Sec7p is present but suppresses dynamics of the Tlg2-residing early/sorting compartment, indicating that the Tlg2p- and Sec7p-residing regions are discrete entities in the mutant. Thus, the Tlg2p-residing region seems to serve as an early/sorting compartment and function independently of the Sec7p-residing region within the TGN.

Super-Resolution Live Imaging of Cargo Traffic Through the Golgi Apparatus in Mammalian Cells.

Super-resolution confocal live imaging microscopy (SCLIM) we developed provides high-speed, high-resolution, three- and four-dimensional, and multicolor simultaneous imaging. Using this technology, we are now able to observe the fine details of various dynamic events going on in living cells, such as membrane traffic and organelle dynamics. The retention using selective hooks (RUSH) system is a powerful tool to control synchronous release of natural cargo proteins of interest from the endoplasmic reticulum in mammalian cells. In this chapter, we describe a method for visualizing secretory cargo traffic within and around the Golgi apparatus in HeLa cells using SCLIM in combination with the RUSH assay.

Dissociation of ERMES clusters plays a key role in attenuating the endoplasmic reticulum stress.

In yeast, ERMES, which mediates phospholipid transport between the ER and mitochondria, forms a limited number of oligomeric clusters at ER-mitochondria contact sites in a cell. Although the number of the ERMES clusters appears to be regulated to maintain proper inter-organelle phospholipid trafficking, its underlying mechanism and physiological relevance remain poorly understood. Here, we show that mitochondrial dynamics control the number of ERMES clusters. Moreover, we find that ER stress causes dissociation of the ERMES clusters independently of Ire1 and Hac1, canonical ER-stress response pathway components, leading to a delay in the phospholipid transport from the ER to mitochondria. Our biochemical and genetic analyses strongly suggest that the impaired phospholipid transport contributes to phospholipid accumulation in the ER, expanding the ER for ER stress attenuation. We thus propose that the ERMES dissociation constitutes an overlooked pathway of the ER stress response that operates in addition to the canonical Ire1/Hac1-dependent pathway.

Quality-controlled ceramide-based GPI-anchored protein sorting into selective ER exit sites.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER. Moreover, we also show that a GPI-AP with a C26 ceramide moiety is monitored by the GPI-glycan remodelase Ted1, which, in turn, is required for receptor-mediated export of GPI-APs. Therefore, our study reveals a quality-control system that ensures lipid-based sorting of GPI-APs into selective ERESs for differential ER export, highlighting the physiological need for this specific export pathway.

The Golgi Apparatus and its Next-Door Neighbors.

The Golgi apparatus represents a central compartment of membrane traffic. Its apparent architecture, however, differs considerably among species, from unstacked and scattered cisternae in the budding yeast Saccharomyces cerevisiae to beautiful ministacks in plants and further to gigantic ribbon structures typically seen in mammals. Considering the well-conserved functions of the Golgi, its fundamental structure must have been optimized despite seemingly different architectures. In addition to the core layers of cisternae, the Golgi is usually accompanied by next-door compartments on its cis and trans sides. The trans-Golgi network (TGN) can be now considered as a compartment independent from the Golgi stack. On the cis side, the intermediate compartment between the ER and the Golgi (ERGIC) has been known in mammalian cells, and its functional equivalent is now suggested for yeast and plant cells. High-resolution live imaging is extremely powerful for elucidating the dynamics of these compartments and has revealed amazing similarities in their behaviors, indicating common mechanisms conserved along the long course of evolution. From these new findings, I would like to propose reconsideration of compartments and suggest a new concept to describe their roles comprehensively around the Golgi and in the post-Golgi trafficking.

The TGN/EE SNARE protein SYP61 and the ubiquitin ligase ATL31 cooperatively regulate plant responses to carbon/nitrogen conditions in Arabidopsis.

Ubiquitination is a post-translational modification involving the reversible attachment of the small protein ubiquitin to a target protein. Ubiquitination is involved in numerous cellular processes, including the membrane trafficking of cargo proteins. However, the ubiquitination of the trafficking machinery components and their involvement in environmental responses are not well understood. Here, we report that the Arabidopsis thaliana trans-Golgi network/early endosome localized SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein SYP61 interacts with the transmembrane ubiquitin ligase ATL31, a key regulator of resistance to disrupted carbon (C)/nitrogen/(N)-nutrient conditions. SYP61 is a key component of membrane trafficking in Arabidopsis. The subcellular localization of ATL31 was disrupted in knockdown mutants of SYP61, and the insensitivity of ATL31-overexpressing plants to high C/low N-stress was repressed in these mutants, suggesting that SYP61 and ATL31 cooperatively function in plant responses to nutrient stress. SYP61 is ubiquitinated in plants, and its ubiquitination level is upregulated under low C/high N-nutrient conditions. These findings provide important insights into the ubiquitin signaling and membrane trafficking machinery in plants.

Assay for dual cargo sorting into endoplasmic reticulum exit sites imaged by 3D Super-resolution Confocal Live Imaging Microscopy (SCLIM).

Understanding how in eukaryotic cells thousands of proteins are sorted from each other through the secretory pathway and delivered to their correct destinations is a central issue of cell biology. We have further investigated in yeast how two distinct types of cargo proteins are sorted into different endoplasmic reticulum (ER) exit sites (ERES) for their differential ER export to the Golgi apparatus. We used an optimized protocol that combines a live cell dual-cargo ER export system with a 3D simultaneous multi-color high-resolution live cell microscopy called Super-resolution Confocal Live Imaging Microscopy (SCLIM). Here, we describe this protocol, which is based on the reversible ER retention of two de novo co-expressed cargos by blocking COPII function upon incubation of the thermo-sensitive COPII allele sec31-1 at restrictive temperature (37°C). ER export is restored by shifting down to permissive temperature (24°C) and progressive incorporation of the two different types of cargos into the fluorescently labelled ERES can be then simultaneously captured at 3D high spatial resolution by SCLIM microscopy. By using this protocol, we have shown that newly synthesized glycosylphosphatidylinositol (GPI)-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized ERES that are distinct from those used by transmembrane secretory proteins. Furthermore, we showed that the chain length of the ceramide present in the ER membrane is critical for this sorting selectivity. Therefore, thanks to the presented method we could obtain the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective ERES.

Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3.

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.

Ceramide chain length-dependent protein sorting into selective endoplasmic reticulum exit sites.

Protein sorting in the secretory pathway is crucial to maintain cellular compartmentalization and homeostasis. In addition to coat-mediated sorting, the role of lipids in driving protein sorting during secretory transport is a longstanding fundamental question that still remains unanswered. Here, we conduct 3D simultaneous multicolor high-resolution live imaging to demonstrate in vivo that newly synthesized glycosylphosphatidylinositol-anchored proteins having a very long chain ceramide lipid moiety are clustered and sorted into specialized endoplasmic reticulum exit sites that are distinct from those used by transmembrane proteins. Furthermore, we show that the chain length of ceramide in the endoplasmic reticulum membrane is critical for this sorting selectivity. Our study provides the first direct in vivo evidence for lipid chain length-based protein cargo sorting into selective export sites of the secretory pathway.

Sec71 separates Golgi stacks in Drosophila S2 cells.

Golgi stacks are the basic structural units of the Golgi. Golgi stacks are separated from each other and scattered in the cytoplasm of Drosophila cells. Here, we report that the ARF-GEF inhibitor Brefeldin A (BFA) induces the formation of BFA bodies, which are aggregates of Golgi stacks, trans-Golgi networks and recycling endosomes. Recycling endosomes are located in the centers of BFA bodies, while Golgi stacks surround them on their trans sides. Live imaging of S2 cells revealed that Golgi stacks repeatedly merged and separated on their trans sides, and BFA caused successive merger by inhibiting separation, forming BFA bodies. S2 cells carrying genome-edited BFA-resistant mutant Sec71M717L did not form BFA bodies at high concentrations of BFA; S2 cells carrying genome-edited BFA-hypersensitive mutant Sec71F713Y produced BFA bodies at low concentrations of BFA. These results indicate that Sec71 is the sole BFA target for BFA body formation and controls Golgi stack separation. Finally, we showed that impairment of Sec71 in fly photoreceptors induces BFA body formation, with accumulation of both apical and basolateral cargoes, resulting in inhibition of polarized transport.

The liverwort oil body is formed by redirection of the secretory pathway.

Eukaryotic cells acquired novel organelles during evolution through mechanisms that remain largely obscure. The existence of the unique oil body compartment is a synapomorphy of liverworts that represents lineage-specific acquisition of this organelle during evolution, although its origin, biogenesis, and physiological function are yet unknown. We find that two paralogous syntaxin-1 homologs in the liverwort Marchantia polymorpha are distinctly targeted to forming cell plates and the oil body, suggesting that these structures share some developmental similarity. Oil body formation is regulated by an ERF/AP2-type transcription factor and loss of the oil body increases M. polymorpha herbivory. These findings highlight a common strategy for the acquisition of organelles with distinct functions in plants, via periodical redirection of the secretory pathway depending on cellular phase transition.

Tricalbins Are Required for Non-vesicular Ceramide Transport at ER-Golgi Contacts and Modulate Lipid Droplet Biogenesis.

Lipid composition varies among organelles, and the distinct lipid composition is important for specific functions of each membrane. Lipid transport between organelles, which is critical for the maintenance of membrane lipid composition, occurs by either vesicular or non-vesicular mechanisms. In yeast, ceramide synthesized in the endoplasmic reticulum (ER) is transported to the Golgi apparatus where inositolphosphorylceramide (IPC) is formed. Here we show that a fraction of Tcb3p, a yeast tricalbin protein, localizes to ER-Golgi contact sites. Tcb3p and their homologs Tcb1p and Tcb2p are required for formation of ER-Golgi contacts and non-vesicular ceramide transport. Absence of Tcb1p, Tcb2p, and Tcb3p increases acylceramide synthesis and subsequent lipid droplet (LD) formation. As LD can sequester excess lipids, we propose that tricalbins act as regulators of ceramide transport at ER-Golgi contact sites to help reduce a potentially toxic accumulation of ceramides.

Formation of Calcified Nodule as a Cause of Early In-Stent Restenosis in Patients Undergoing Dialysis.

Background Dialysis is an independent risk factor for in-stent restenosis (ISR) after stent implantation in coronary arteries. However, the characteristics of ISR in patients undergoing dialysis remain unclear, as there are no histological studies evaluating the causes of this condition. The aim of the present study was to investigate the causes of ISR between patients who are undergoing dialysis and those who are not by evaluating tissues obtained from ISR lesions using directional coronary atherectomy. Methods and Results A total of 29 ISR lesions from 29 patients included in a multicenter directional coronary atherectomy registry of 128 patients were selected for analysis and divided into a dialysis group (n=8) and a nondialysis group (n=21). Histopathological evaluation demonstrated that an in-stent calcified nodule was a major histological characteristic of ISR lesions in the dialysis group and the prevalence of an in-stent calcified nodule was significantly higher in the dialysis group compared with the nondialysis group (75% versus 5%, respectively; P<0.01). On the other hand, the prevalence of an in-stent lipid-rich plaque was significantly lower in the dialysis group compared with the nondialysis group (0% versus 43%, respectively; P=0.03). In all cases with an in-stent calcified nodule, the underlying calcification before stent implantation was moderate to severe. When tissue characteristics were stratified according to duration post-stent implantation, an in-stent calcified nodule in the dialysis group was mainly observed within 1 year after stent implantation. Conclusions In-stent calcified nodules are a common cause of ISR in patients undergoing dialysis and are observed within 1 year after stent implantation, suggesting different causes of ISR between patients undergoing dialysis and those who are not.

Deletion of PIN4 Suppresses the Protein Transport Defects Caused by sec12-4 Mutation in Saccharomyces cerevisiae.

Newly synthesized secretory proteins are released into the lumen of the endoplasmic reticulum (ER). The secretory proteins are surrounded by coat protein complex II (COPII) vesicles, and transported from the ER and reach their destinations through the Golgi apparatus. Sec12p is a guanine nucleotide exchange factor for Sar1p, which initiates COPII vesicle budding from the ER. The activation of Sar1p by Sec12p and the subsequent COPII coat assembly have been well characterized, but the events that take place upstream of Sec12p remain unclear. In this study, we isolated the novel extragenic suppressor of sec12-4, PIN4/MDT1, a cell cycle checkpoint target. A yeast two-hybrid screening was used to identify Pin4/Mdt1p as a binding partner of the casein kinase I isoform Hrr25p, which we have previously identified as a modulator of Sec12p function. Deletion of PIN4 suppressed both defects of temperature-sensitive growth and the partial protein transport observed in sec12-4 mutants. The results of this study suggest that Pin4p provides novel aspects of Sec12p modulations.