Golgi Ribbon Research Articles

Golgi clustering by the deficiency of COPI-SNARE in Drosophila photoreceptors.

A comprehensive study of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the fly genome by RNAi in Drosophila photoreceptors indicated that knockdown of any of the COPI-SNAREs, Syx18, Sec20, and Use1, resulted in the same characteristic phenotypes: Golgi stacks gathering on their trans-side, laterally expanded Golgi cisternae, and a reduced number of discrete Golgi stacks. These Golgi stacks are reminiscent of mammalian Golgi ribbons and Brefeldin A (BFA)-bodies in Drosophila S2 cells. As previously reported, BFA suppresses trans-Golgi network (TGN) fission and Golgi stack separation to form a BFA-body, which is a cluster of Golgi stacks cored by recycling endosomes. We found that the impairing each of COPI-SNAREs results in clustered Golgi stacks similar to BFA-bodies, indicating that COPI-SNAREs have a role to separate clustered Golgi stacks. These results further support the idea that the movement of Golgi stacks and the balance of fusion and fission of the TGN determine the level of clustering and ribbon formation of Golgi stacks within cells.

RNA scaffolds the Golgi ribbon by forming condensates with GM130.

The mammalian Golgi is composed of stacks that are laterally connected into a continuous ribbon-like structure. The integrity and function of the ribbon is disrupted under stress conditions, but the molecular mechanisms remain unclear. Here we show that the ribbon is maintained by biomolecular condensates of RNA and the Golgi matrix protein GM130 (GOLGA2). We identify GM130 as a membrane-bound RNA-binding protein, which directly recruits RNA and associated RNA-binding proteins to the Golgi membrane. Acute degradation of RNA or GM130 in cells disrupts the ribbon. Under stress conditions, RNA dissociates from GM130 and the ribbon is disjointed, but after the cells recover from stress the ribbon is restored. When overexpressed in cells, GM130 forms RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its amino terminus, which binds RNA to induce liquid-liquid phase separation. These co-condensates are sufficient to link purified Golgi membranes, reconstructing lateral linking of stacks into a ribbon-like structure. Together, these studies show that RNA acts as a structural biopolymer that together with GM130 maintains the integrity of the Golgi ribbon.

Upregulation of the secretory pathway Ca2+/Mn2+-ATPase isoform 1 in LPS-stimulated microglia and its involvement in Mn2+-induced Golgi fragmentation.

Microglia play an important protective role in the healthy nervous tissue, being able to react to a variety of stimuli that induce different intracellular cascades for specific tasks. Ca2+ signaling can modulate these pathways, and we recently reported that microglial functions depend on the endoplasmic reticulum as a Ca2+ store, which involves the Ca2+ transporter SERCA2b. Here, we investigated whether microglial functions may also rely on the Golgi, another intracellular Ca2+ store that depends on the secretory pathway Ca2+/Mn2+-transport ATPase isoform 1 (SPCA1). We found upregulation of SPCA1 upon lipopolysaccharide stimulation of microglia BV2 cells and primary microglia, where alterations of the Golgi ribbon were also observed. Silencing and overexpression experiments revealed that SPCA1 affects cell morphology, Golgi apparatus integrity, and phagocytic functions. Since SPCA1 is also an efficient Mn2+ transporter and considering that Mn2+ excess causes manganism in the brain, we addressed the role of microglial SPCA1 in Mn2+ toxicity. Our results revealed a clear effect of Mn2+ excess on the viability and morphology of microglia. Subcellular analysis showed Golgi fragmentation and subsequent alteration of SPCA1 distribution from early stages of toxicity. Removal of Mn2+ by washing improved the culture viability, although it did not effectively reverse Golgi fragmentation. Interestingly, pretreatment with curcumin maintained microglia cultures viable, prevented Mn2+-induced Golgi fragmentation, and preserved SPCA Ca2+-dependent activity, suggesting curcumin as a potential protective agent against Mn2+-induced Golgi alterations in microglia.

The Golgi checkpoint: Golgi unlinking during G2 is necessary for spindle formation and cytokinesis.

Entry into mitosis requires not only correct DNA replication but also extensive cell reorganization, including the separation of the Golgi ribbon into isolated stacks. To understand the significance of pre-mitotic Golgi reorganization, we devised a strategy to first block Golgi segregation, with the consequent G2-arrest, and then force entry into mitosis. We found that the cells forced to enter mitosis with an intact Golgi ribbon showed remarkable cell division defects, including spindle multipolarity and binucleation. The spindle defects were caused by reduced levels at the centrosome of the kinase Aurora-A, a pivotal spindle formation regulator controlled by Golgi segregation. Overexpression of Aurora-A rescued spindle formation, indicating a crucial role of the Golgi-dependent recruitment of Aurora-A at the centrosome. Thus, our results reveal that alterations of the pre-mitotic Golgi segregation in G2 have profound consequences on the fidelity of later mitotic processes and represent potential risk factors for cell transformation and cancer development.

Neural deficits in a mouse model of PACS1 syndrome are corrected with PACS1- or HDAC6-targeting therapy

PACS1 syndrome is a neurodevelopmental disorder (NDD) caused by a recurrent de novo missense mutation in PACS1 (p.Arg203Trp (PACS1R203W)). The mechanism by which PACS1R203W causes PACS1 syndrome is unknown, and no curative treatment is available. Here, we use patient cells and PACS1 syndrome mice to show that PACS1 (or PACS-1) is an HDAC6 effector and that the R203W substitution increases the PACS1/HDAC6 interaction, aberrantly potentiating deacetylase activity. Consequently, PACS1R203W reduces acetylation of α-tubulin and cortactin, causing the Golgi ribbon in hippocampal neurons and patient-derived neural progenitor cells (NPCs) to fragment and overpopulate dendrites, increasing their arborization. The dendrites, however, are beset with varicosities, diminished spine density, and fewer functional synapses, characteristic of NDDs. Treatment of PACS1 syndrome mice or patient NPCs with PACS1- or HDAC6-targeting antisense oligonucleotides, or HDAC6 inhibitors, restores neuronal structure and synaptic transmission in prefrontal cortex, suggesting that targeting PACS1R203W/HDAC6 may be an effective therapy for PACS1 syndrome.

Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

Microtubule-associated septin complexes modulate kinesin and dynein motility with differential specificities

Long-range membrane traffic is guided by microtubule-associated proteins and posttranslational modifications, which collectively comprise a traffic code. The regulatory principles of this code and how it orchestrates the motility of kinesin and dynein motors are largely unknown. Septins are a large family of GTP-binding proteins, which assemble into complexes that associate with microtubules. Using single-molecule invitro motility assays, we tested how the microtubule-associated SEPT2/6/7, SEPT2/6/7/9, and SEPT5/7/11 complexes affect the motilities of the constitutively active kinesins KIF5C and KIF1A and the dynein-dynactin-bicaudal D (DDB) motor complex. We found that microtubule-associated SEPT2/6/7 is a potent inhibitor of DDB and KIF5C, preventing mainly their association with microtubules. SEPT2/6/7 also inhibits KIF1A by obstructing stepping along microtubules. On SEPT2/6/7/9-coated microtubules, KIF1A inhibition is dampened by SEPT9, which alone enhances KIF1A, showing that individual septin subunits determine the regulatory properties of septin complexes. Strikingly, SEPT5/7/11 differs from SEPT2/6/7, in permitting the motility of KIF1A and immobilizing DDB to the microtubule lattice. In hippocampal neurons, filamentous SEPT5 colocalizes with somatodendritic microtubules that underlie Golgi membranes and lack SEPT6. Depletion of SEPT5 disrupts Golgi morphology and polarization of Golgi ribbons into the shaft of somato-proximal dendrites, which is consistent with the tethering of DDB to microtubules by SEPT5/7/11. Collectively, these results suggest that microtubule-associated complexes have differential specificities in the regulation of the motility and positioning of microtubule motors. We posit that septins are an integral part of the microtubule-based code that spatially controls membrane traffic.

The crescent-like Golgi ribbon is shaped by the Ajuba/PRMT5/Aurora-A complex-modified HURP

BackgroundGolgi apparatus (GA) is assembled as a crescent-like ribbon in mammalian cells under immunofluorescence microscope without knowing the shaping mechanisms. It is estimated that roughly 1/5 of the genes encoding kinases or phosphatases in human genome participate in the assembly of Golgi ribbon, reflecting protein modifications play major roles in building Golgi ribbon.MethodsTo explore how Golgi ribbon is shaped as a crescent-like structure under the guidance of protein modifications, we identified a protein complex containing the scaffold proteins Ajuba, two known GA regulators including the protein kinase Aurora-A and the protein arginine methyltransferase PRMT5, and the common substrate of Aurora-A and PRMT5, HURP. Mutual modifications and activation of PRMT5 and Aurora-A in the complex leads to methylation and in turn phosphorylation of HURP, thereby producing HURP p725. The HURP p725 localizes to GA vicinity and its distribution pattern looks like GA morphology. Correlation study of the HURP p725 statuses and GA structure, site-directed mutagenesis and knockdown-rescue experiments were employed to identify the modified HURP as a key regulator assembling GA as a crescent ribbon.ResultsThe cells containing no or extended distribution of HURP p725 have dispersed GA membranes or longer GA. Knockdown of HURP fragmentized GA and HURP wild type could, while its phosphorylation deficiency mutant 725A could not, restore crescent Golgi ribbon in HURP depleted cells, collectively indicating a crescent GA-constructing activity of HURP p725. HURP p725 is transported, by GA membrane-associated ARF1, Dynein and its cargo adaptor Golgin-160, to cell center where HURP p725 forms crescent fibers, binds and stabilizes Golgi assembly factors (GAFs) including TRIP11, GRASP65 and GM130, thereby dictating the formation of crescent Golgi ribbon at nuclear periphery.ConclusionsThe Ajuba/PRMT5/Aurora-A complex integrates the signals of protein methylation and phosphorylation to HURP, and the HURP p725 organizes GA by stabilizing and recruiting GAFs to its crescent-like structure, therefore shaping GA as a crescent ribbon. Therefore, the HURP p725 fiber serves a template to construct GA according to its shape.6goRpNfQL_y2Q765kUtnH_Video

Jaw1/LRMP is associated with the maintenance of Golgi ribbon structure.

Jaw1/LRMP is a membrane protein that is localized to the endoplasmic reticulum and outer nuclear membrane. Previously, we revealed that Jaw1 functions to maintain nuclear shape by interacting with microtubules as a Klarsicht/ANC-1/Syne/homology (KASH) protein. The loss of several KASH proteins causes defects in the position and shape of the Golgi apparatus as well as the nucleus, but the effects of Jaw1 depletion on the Golgi apparatus were poorly understood. Here, we found that siRNA-mediated Jaw1 depletion causes Golgi fragmentation with disordered ribbon structure in the melanoma cell, accompanied by the change in the localization of the Golgi-derived microtubule network. Thus, we suggest that Jaw1 is a novel protein to maintain the Golgi ribbon structure, associated with the microtubule network.

In Vitro Methods to Investigate the Disassembly of the Golgi Ribbon During the G2-M Transition of the Cell Cycle.

The Golgi complex is the central hub of the secretory pathway. In mammalian cells, it is formed by stacks of flattened cisternae organized in a continuous membrane system, the Golgi ribbon, located near the centrosome. During G2, the Golgi ribbon is disassembled into isolated stacks that, at the onset of mitosis, are further fragmented into small tubular-vesicular clusters that disperse throughout the cytoplasm. Here, we describe a set of methods to study the Golgi complex in different phases of thecell cycle, drawing attention to reproducing the mitotic Golgi fragmentation to gain knowledge and acquire the skills to study the mechanisms that regulate mitotic Golgi reorganization as well as its biological significance. The investigations based on these assays have been instrumental in understanding that Golgi disassembly isnot only a consequence of mitosis but is also required for mitotic entry and cell division.

Structural Organization and Function of the Golgi Ribbon During Cell Division.

The Golgi complex has a central role in the secretory traffic. In vertebrate cells it is generally organized in polarized stacks of cisternae that are laterally connected by membranous tubules, forming a structure known as Golgi ribbon. The steady state ribbon arrangement results from a dynamic equilibrium between formation and cleavage of the membrane tubules connecting the stacks. This balance is of great physiological relevance as the unlinking of the ribbon during G2 is required for mitotic entry. A block of this process induces a potent G2 arrest of the cell cycle, indicating that a mitotic “Golgi checkpoint” controls the correct pre-mitotic segregation of the Golgi ribbon. Then, after mitosis onset, the Golgi stacks undergo an extensive disassembly, which is necessary for proper spindle formation. Notably, several Golgi-associated proteins acquire new roles in spindle formation and mitotic progression during mitosis. Here we summarize the current knowledge about the basic principle of the Golgi architecture and its functional relationship with cell division to highlight crucial aspects that need to be addressed to help us understand the physiological significance of the ribbon and the pathological implications of alterations of this organization.

Abstract 2404: Role of TPD52L2 in cell migration and triple negative breast cancer progression

Abstract The Golgi apparatus plays an important role in cell migration and the establishment of epithelial polarity. A dispersed (or fragmented) Golgi ribbon is often seen in stressed cells and in various pathologies, including neurodegeneration or cancer, but whether and how a fragmented Golgi apparatus affect cellular function is poorly defined. Previously our lab has performed and analyzed published RNAi screens to identify genes required to maintain the Golgi ribbon. These candidate genes were filtered for altered expression patterns in breast cancer patients and for their association with survival data. The selected genes reevaluated by RNAi in BT549 breast cancer cells to assess the effect on Golgi fragmentation. One of the most effective hits was TPD52L2, a poorly characterised gene that encodes a membrane-associated protein that is overexpressed in various types of cancer. The aim of this project is to investigate the role of TPD52L2 in cell migration and cell invasion in breast cancer in order to understand the role of Golgi fragmentation in tumorigenesis. In random cell migration assays in dense and sparse seeding conditions, TPD52L2 depleted cells showed an altered actin cytoskeleton and were found to migrate faster in dense seeding without any change in directional persistence. These findings suggest that TPD52L2 not only is required to maintain an intact Golgi structure but also plays an important role in cellular motility and potentially metastasis. Citation Format: Seyedamirabbas Parizadeh, Stephan Geley, Veronika Reiterer-Farhan, Hesso Farhan. Role of TPD52L2 in cell migration and triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2404.

MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane.

The Golgi complex plays an active role in organizing asymmetric microtubule arrays, which are essential for polarized vesicle transport. The coiled-coil protein MTCL1 stabilizes microtubules nucleated from the Golgi membrane. Here, we report an MTCL1 paralog, MTCL2, which preferentially acts on the perinuclear microtubules accumulated around the Golgi. MTCL2 associates with the Golgi membrane through the N-terminal coiled-coil region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown of MTCL2 significantly impaired microtubule accumulation around the Golgi, as well as the compactness of the Golgi ribbon assembly structure. Given that MTCL2 forms parallel oligomers through homo-interaction of the central coiled-coil motifs, our results indicate that MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane. Results of in vitro wound healing assays further suggest that this function of MTCL2 enables integration of the centrosomal and Golgi-associated microtubules on the Golgi membrane, supporting directional migration. Additionally, the results demonstrated the involvement of CLASPs and giantin in mediating the Golgi association of MTCL2.

Structure modeling hints at a granular organization of the Golgi ribbon

BackgroundIn vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this “ribbon” architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses” whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis.ResultsWPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a “breathing” arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions.ConclusionsHypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.

Inhibition of the human secretory pathway Ca2+, Mn2+–ATPase1a by 1,3-thiazole derivatives

The human Golgi/secretory pathway Ca2+,Mn2+-ATPase 1 (hSPCA1) transports Ca2+ and Mn2+ into the Golgi lumen. Studies of the biological functions of hSPCA1 are limited by a lack of selective pharmacological tools for SPCA1 inhibition. The aim of this study was therefore to identify compounds that specifically inhibit hSPCA1 activity. We found that five 1,3-thiazole derivatives exhibited inhibitory action towards the ATP hydrolysis activity of hSPCA1a in a concentration-dependent manner. Among the derivatives tested, compound 1 was the most potent, completely inhibiting hSPCA1a activity with a half-maximal inhibition (IC50) value of 0.8 μM. Compound 1 also partially inhibited the activity of another Ca2+,Mn2+-ATPase (hSPCA2) and Ca2+-ATPase (rSERCA1a), but had no effect on Na+,K+-ATPase or H+,K+-ATPase. Treatment of HeLa cells with compound 1 led to fragmentation of the Golgi ribbon into smaller stacks. In addition, compound 1 mobilized intracellular Ca2+ in HeLa cells that had been pre-treated with thapsigargin. Therefore, based on its selectivity and potency, compound 1 may be a valuable tool with which to further explore the role of SPCA1 in cellular processes.

The life cycle and enigmatic egress of coronaviruses.

There has been considerable recent interest in the life cycle of Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2), the causative agent of the Covid‐19 pandemic. Practically every step in CoV replication—from cell attachment and uptake via genome replication and expression to virion assembly has been considered as a specific event that potentially could be targeted by existing or novel drugs. Interference with cellular egress of progeny viruses could also be adopted as a possible therapeutic strategy; however, the situation is complicated by the fact that there is no broad consensus on how CoVs find their way out of their host cells. The viral nucleocapsid, consisting of the genomic RNA complexed with nucleocapsid proteins obtains a membrane envelope during virus budding into the lumen of the intermediate compartment (IC) at the endoplasmic reticulum (ER)–Golgi interface. From here, several alternative routes for CoV extracellular release have been proposed. Strikingly, recent studies have shown that CoV infection leads to the disassembly of the Golgi ribbon and the mobilization of host cell compartments and protein machineries that are known to promote Golgi‐independent trafficking to the cell surface. Here, we discuss the life cycle of CoVs with a special focus on different possible pathways for virus egress.

Segregation of the membrane cargoes, BACE1 and amyloid precursor protein (APP) throughout the Golgi apparatus.

The intracellular trafficking of β‐site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid‐β production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans‐Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aβ production in non‐neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high‐resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis‐Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP‐1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.

Monitoring Intra-Golgi Transport with Acute Spatiotemporal Control of a Synthetic Cargo.

We hereby describe a method to image cargo trafficking from the cis- to the trans-face of the Golgi apparatus. Briefly, we combine nocodazole treatment that breaks down the Golgi ribbon, temperature blocks that slow down cargo transport, and a drug-controlled aggregation system that controls the size of the cargo and its retention at different stages of the secretory pathway. Using this method, we first position the cargo within the cis-face of the Golgi. When traffic resumes upon temperature block release, kinetics of transport can be assessed by confocal microscopy through colocalization of the cargo with cis- and trans-Golgi markers. This method allows for testing various modes of intra-Golgi transports and can be adapted to investigate other steps of the secretory pathway.

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system.

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. They have recently been found to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, making it extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed cell lines with conditional KO (CKO) of either Rab1 (Rab1A and Rab1B) or Rab5 (Rab5A, Rab5B and Rab5C) by using the auxin-inducible protein degradation system. Rab1 CKO and Rab5 CKO led to eventual cell death from 18 h and 48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and endoplasmic reticulum (ER)-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress

The Golgi complex is an essential organelle of the eukaryotic exocytic pathway. A subfamily of Golgi matrix proteins, called GRASPs, is central in stress-induced unconventional secretion, Golgi dynamics during mitosis/apoptosis, and Golgi ribbon formation. The Golgi ribbon is vertebrate-specific and correlates with the appearance of two GRASP paralogues and two Golgins (GM130/Golgin45), which form specific GRASP-Golgin pairs. The molecular details of their appearance only in Metazoans are unknown. Moreover, despite new functionalities supported by GRASP paralogy, little is known about their structural and evolutionary differences. Here, we used ancestor sequence reconstruction and biophysical/biochemical approaches to assess the evolution of GRASPs structure/dynamics, fibrillation, and how they started anchoring their Golgin partners. Our data showed that a GRASP ancestor anchored Golgins before gorasp gene duplication in Metazoans. After gene duplication, variations within the GRASP binding pocket determined which paralogue would recruit which Golgin. These interactions are responsible for their specific Golgi location and Golgi ribbon appearance. We also suggest that GRASPs have a long-standing capacity to form supramolecular structures, affecting their participation in stress-induced processes.