GRASP55 regulates intra‐Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis‐trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI‐based retrograde transport vesicles, thus concentrating them in the trans‐Golgi. In genome‐edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis‐Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.     

NPC1 regulates the distribution of phosphatidylinositol 4‐kinases at Golgi and lysosomal membranes

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann‐Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4‐phosphate (PtdIns4P) countertransport cycle between Golgi‐endoplasmic reticulum (ER), as well as lysosome‐ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4‐kinases—PI4KIIα and PI4KIIIβ—which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.     

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

The fidelity of Golgi glycosylation is, in part, ensured by compartmentalization of enzymes within the stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterization of the clientele of GOLPH3 and its paralogue GOLPH3L is lacking. GOLPH3’s role is of particular interest as it is frequently amplified in several solid tumor types. Here, we apply two orthogonal proteomic methods to identify GOLPH3+3L clients and find that they act in diverse glycosylation pathways or have other roles in the Golgi. Binding studies, bioinformatics, and a Golgi retention assay show that GOLPH3+3L bind the cytoplasmic tails of their clients through membrane-proximal positively charged residues. Furthermore, deletion of GOLPH3+3L causes multiple defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.     

Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases

Gangliosides, complex glycosphingolipids containing sialic acids, are synthesized in the endoplasmic reticulum and in the Golgi complex. These neobiosynthesized gangliosides move via vesicular transport to the plasma membrane, becoming components of the external leaflet. Gangliosides can undergo endocytosis followed by recycling to the cell surface or sorting to the Golgi complex or lysosomes for remodeling and catabolism. Recently, glycosphingolipid catabolic enzymes (glycohydrolases) have been found to be associated with the plasma membrane, where they display activity on the membrane components. In this work, we demonstrated that ecto-ganglioside glycosyltransferases may catalyze ganglioside synthesis outside the Golgi compartment, particularly at the cell surface. Specifically, we report the first direct evidence of expression and activity of CMP-NeuAc:GM3 sialyltransferase (Sial-T2) at the cell surface of epithelial and melanoma cells, with membrane-integrated ecto-Sial-T2 being able to sialylate endogenously synthesized GM3 ganglioside as well as exogenously incorporated substrate. Interestingly, we also showed that ecto-Sial-T2 was able to synthesize GD3 ganglioside at the cell surface using the endogenously synthesized cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) available at the extracellular milieu. In addition, the expression of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase (GalNAc-T) was also detected at the cell surface of epithelial cells, whose catalytic activity was only observed after feeding the cells with exogenous GM3 substrate. Thus, the relative interplay between the plasma membrane-associated glycosyltransferase and glycohydrolase activities, even when acting on a common substrate, emerges as a potential level of regulation of the local glycosphingolipid composition in response to different external and internal stimuli.     

GOLPH3 keeps the Golgi residents at home

In this issue of JCB, Welch et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202106115) show that GOLPH3 mediates the sorting of numerous Golgi proteins into recycling COPI transport vesicles. This explains how many resident proteins are retained at the Golgi and reveals a key role for GOLPH3 in maintaining Golgi homeostasis.

The Golgi apparatus lies at the heart of the secretory pathway, where its major functions are the posttranslational modification of cargo proteins and lipids, particularly at the level of glycosylation, and the sorting of cargo to its correct onward destination. The Golgi is composed of stacked membrane compartments called cisternae, which contain numerous resident enzymes that act on the cargo as it passes through the organelle, from the entry or cis side to the exit or trans side. Each resident enzyme has its own distribution within the Golgi stack, resulting in the sequential modification of the secretory cargo as it moves through the Golgi.Various mechanisms exist to ensure that Golgi residents are retained within the Golgi despite the huge flux of protein and lipid through this organelle (1). Major players are COPI vesicles, which recycle Golgi residents from later to earlier cisternae, at the same time as the cisternae are thought to slowly migrate across the stack, as on a conveyor belt, progressively changing composition in a process referred to as cisternal maturation (2). Unlike the Golgi resident enzymes, which enter recycling vesicles, cargo is thought to remain within the maturing cisternae as it moves through the Golgi. Certain Golgi enzymes can bind directly to the COPI coat, explaining their inclusion in COPI vesicles (3), but for other enzymes and resident proteins, their retention mechanism is less obvious.Previous studies on the peripheral Golgi membrane protein GOLPH3 and its paralogue GOLPH3L (herein I will refer to both proteins as GOLPH3) indicated it can bind to certain Golgi enzymes and to the COPI coat, thereby acting as an adaptor to mediate sorting of these enzymes into COPI vesicles (4, 5). This was first shown for the yeast orthologue Vps74p (6, 7) and has also been demonstrated for the Drosophila version of the protein (8), consistent with a conserved function in Golgi enzyme retention. However, the extent to which GOLPH3 might participate in retention of different Golgi enzymes and other resident proteins, and its importance relative to other methods of protein retention in the Golgi, has remained unclear. Indeed, a recent study suggested that GOLPH3 selectively mediates the retention of enzymes involved in glycosphingolipid synthesis, consistent with a fairly selective role in retaining only a subset of resident Golgi enzymes (9). It should also be noted that GOLPH3 has been implicated in other functions, namely budding of exocytic vesicles from the Golgi, the DNA damage response, and mechanistic target of rapamycin signaling (10).In their current paper, Welch et al. used a combination of approaches to reassess the role of GOLPH3 at the Golgi (11). Using proteomics, they could identify numerous GOLPH3 binding partners, which included COPI, as expected, and a large number of other Golgi residents, including numerous Golgi enzymes and other membrane proteins. The ability of GOLPH3 to retain enzymes at the Golgi was confirmed using microscopy and an innovative flow cytometry–based assay to quantify surface versus Golgi abundance. The large number of possible interactors suggested that GOLPH3 could mediate the Golgi retention of many proteins. To further assess this possibility, the authors took advantage of previous observations showing that Golgi enzymes may be misrouted to the lysosome and degraded upon their failure to be retained in the Golgi (6, 7, 9). Using mass spectrometry, they could show that numerous Golgi resident proteins were depleted in GOLPH3 knockout cells, many of which were also found in the GOLPH3 interactome. This included many enzymes involved in glycosylation, consistent with GOLPH3 playing an important role in maintaining Golgi-dependent glycosylation of proteins and lipids. This was supported by lectin analysis, which showed marked changes in a broad range of glycans in the GOLPH3 knockout cells.The large number of GOLPH3 clients raises the question as to how it can recognize so many proteins. Previous work has shown binding to the cytoplasmic tails of Golgi enzymes and an interaction motif has been described for Vps74p and more recently for GOLPH3 (6, 9). However, bioinformatics analysis of the many GOLPH3 clients combined with mutational analysis, as performed in the current study, revealed the lack of a consensus sequence for GOLPH3 binding, with the common feature being a strong net positive charge combined with short cytoplasmic tail length. This would result in a high positive charge proximal to the membrane, which likely allows interaction with an acidic patch on the surface of GOLPH3. This mode of binding could mediate selective retention of many Golgi residents, while allowing for the forward trafficking of cargo proteins that have longer, less charged, or folded cytoplasmic domains.GOLPH3 is an oncogene associated with many types of cancer (12). Several mechanisms have been proposed to account for the oncogenic properties of GOLPH3, but most compelling is that changes in glycosylation are responsible. It was recently shown that GOLPH3-dependent changes in glycosphingolipids affects cell growth by altering mitogenic signaling (9). Changes in glycosylation of surface receptors has also been reported, which can affect surface abundance and hence signaling (13). The new results from Welch et al. suggest that glycosylation of many proteins and lipids may be relevant in cancer and that potentially a broad range of downstream targets contribute to oncogenesis. Such targets could influence processes beyond signaling, including cell adhesion and migration, that are known to be sensitive to changes in the surface glycome and which have been reported in previous studies on GOLPH3 (12).The study by Welch et al. indicates a major role for GOLPH3 in Golgi protein retention (Fig. 1). Clearly though, other retention mechanisms exist, including direct binding to COPI, and transmembrane domain length is also important, where the short transmembrane domain of resident proteins favors partitioning into recycling COPI vesicles and Golgi cisternal membranes of a similar thickness (1). Additional COPI adaptors are also likely, with TM9SF2 recently identified as a likely candidate, being present in Golgi vesicles and able to bind certain Golgi enzymes (1). It is possible that different resident proteins use different adaptors, or that a combination of retention mechanisms act in conjunction for certain residents, providing robustness to the retention process. However, any redundancy would seem incomplete given the strong phenotype seen upon loss of GOLPH3. GOLPH3 is localized toward the trans side of the Golgi, so it is possible that other adaptors, such as TM9SF2 and possibly others, might act earlier in the Golgi, or that direct coat binding is more important within the early Golgi. Hence different residents may be more likely to use different retention mechanisms depending on their location in the Golgi. Because GOLPH3 acts late in the Golgi and can bind many clients, we may think of it as a gatekeeper to prevent loss of numerous Golgi residents from the organelle.Open in a separate windowFigure 1.GOLPH3 plays a major role in Golgi protein retention. Golgi resident proteins, including many glycosylation enzymes, depicted by lollipops, are sorted into recycling COPI vesicles to maintain retention in the Golgi in the face of onward cisternal maturation and secretory cargo transport. Different enzymes are depicted by different lollipop shapes and colors, with GOLPH3 clients indicated by horizontal ovals. Enzymes retained by other mechanisms are depicted by lollipops with circles (transmembrane domain length), squares or vertical ovals (binding to other COPI adaptors, indicated in turquoise and purple), or hexagons (direct binding to the COPI coat). GOLPH3, which is more abundant toward the trans side of the Golgi, has many clients.With regard to possible future studies, although we have a good idea of how GOLPH3 recognizes its clients, detailed structural analysis will prove informative in elucidating how it can bind so many proteins. Similarly, identification of additional adaptors linking Golgi residents to the COPI coat will be important to generate a more comprehensive view of Golgi protein retention. Finally, in the context of disease, further analysis of the glycoproteins and glycolipids whose levels are altered because of changes in GOLPH3 expression, of which there are likely to be many, should provide significant new insights into the mechanisms underlying GOLPH3-mediated tumorigenesis.     

Golgi Phosphoprotein 3 Triggers Signal-mediated Incorporation of Glycosyltransferases into Coatomer-coated (COPI) Vesicles

Newly synthesized membrane and secreted proteins undergo a series of posttranslational modifications in the Golgi apparatus, including attachment of carbohydrate moieties. The final structure of so-formed glycans is determined by the order of execution of the different glycosylation steps, which seems intimately related to the spatial distribution of glycosyltransferases and glycosyl hydrolases within the Golgi apparatus. How cells achieve an accurate localization of these enzymes is not completely understood but might involve dynamic processes such as coatomer-coated (COPI) vesicle-mediated trafficking. In yeast, this transport is likely to be regulated by vacuolar protein sorting 74 (Vps74p), a peripheral Golgi protein able to interact with COPI coat as well as with a binding motif present in the cytosolic tails of some mannosyltransferases. Recently, Golgi phosphoprotein 3 (GOLPH3), the mammalian homolog of Vps74, has been shown to control the Golgi localization of core 2 N-acetylglucosamine-transferase 1. Here, we highlight a role of GOLPH3 in the spatial localization of α-2,6-sialyltransferase 1. We show, for the first time, that GOLPH3 supports incorporation of both core 2 N-acetylglucosamine-transferase 1 and α-2,6-sialyltransferase 1 into COPI vesicles. Depletion of GOLPH3 altered the subcellular localization of these enzymes. In contrast, galactosyltransferase, an enzyme that does not interact with GOLPH3, was neither incorporated into COPI vesicles nor was dependent on GOLPH3 for proper localization.     

PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking

Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of PtdIns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases.     

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non‐plasma membrane PI(4,5)P₂, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P₂ phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over‐activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P₂ phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.     

The Golgi microtubules regulate single cell durotaxis

Current understandings on cell motility and directionality rely heavily on accumulated investigations of the adhesion–actin cytoskeleton–actomyosin contractility cycles, while microtubules have been understudied in this context. Durotaxis, the ability of cells to migrate up gradients of substrate stiffness, plays a critical part in development and disease. Here, we identify the pivotal role of Golgi microtubules in durotactic migration of single cells. Using high‐throughput analysis of microtubule plus ends/focal adhesion interactions, we uncover that these non‐centrosomal microtubules actively impart leading edge focal adhesion (FA) dynamics. Furthermore, we designed a new system where islands of higher stiffness were patterned within RGD peptide coated polyacrylamide gels. We revealed that the positioning of the Golgi apparatus is responsive to external mechanical cues and that the Golgi–nucleus axis aligns with the stiffness gradient in durotaxis. Together, our work unveils the cytoskeletal underpinning for single cell durotaxis. We propose a model in which the Golgi–nucleus axis serves both as a compass and as a steering wheel for durotactic migration, dictating cell directionality through the interaction between non‐centrosomal microtubules and the FA dynamics.     

The needs of a synapse—How local organelles serve synaptic proteostasis

Synaptic function crucially relies on the constant supply and removal of neuronal membranes. The morphological complexity of neurons poses a significant challenge for neuronal protein transport since the machineries for protein synthesis and degradation are mainly localized in the cell soma. In response to this unique challenge, local micro‐secretory systems have evolved that are adapted to the requirements of neuronal membrane protein proteostasis. However, our knowledge of how neuronal proteins are synthesized, trafficked to membranes, and eventually replaced and degraded remains scarce. Here, we review recent insights into membrane trafficking at synaptic sites and into the contribution of local organelles and micro‐secretory pathways to synaptic function. We describe the role of endoplasmic reticulum specializations in neurons, Golgi‐related organelles, and protein complexes like retromer in the synthesis and trafficking of synaptic transmembrane proteins. We discuss the contribution of autophagy and of proteasome‐mediated and endo‐lysosomal degradation to presynaptic proteostasis and synaptic function, as well as nondegradative roles of autophagosomes and lysosomes in signaling and synapse remodeling. We conclude that the complexity of neuronal cyto‐architecture necessitates long‐distance protein transport that combines degradation with signaling functions.     

Golgi Phosphoprotein 3 Determines Cell Binding Properties under Dynamic Flow by Controlling Golgi Localization of Core 2 N-Acetylglucosaminyltransferase 1

Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLe^x), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR⁹ sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT^1–16-Leu^17–32-Gly^33–42-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA⁹) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLe^x associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1.     

Glycohydrolases β-hexosaminidase and β-galactosidase are associated with lipid microdomains of Jurkat T-lymphocytes

Growing evidence suggests the presence of active lysosomal enzymes in extra-lysosomal compartments, such as the plasma membrane. Although in the past little attention was paid to glycohydrolases acting on cellular compartments different from lysosomes, there is now increasing interest on plasma membrane-associated glycohydrolases because they should be involved, together with glycosyltransferases, in glycosphingolipids oligosaccharide modification processes regulating cell-to-cell and/or cell-environment interactions in both physiological and pathological conditions. Starting from the previous evidence of the presence of β-hexosaminidase and β-galactosidase at the plasma membrane of cultured fibroblasts, we here investigated the association of these glycohydrolases with lipid microdomains of Jurkat T-lymphocytes. Monosialoganglioside GM3 represents the major glycosphingolipid constituent of T-cell plasma membrane and its amount largely increases after T-cell stimulation. β-hexosaminidase and β-galactosidase cleave specific β-linked terminal residues from a wide range of glycoconjugates and in particular are involved in the stepwise degradation of GM1 to GM3 ganglioside. Here we demonstrated that fully processed plasma membrane-associated β-hexosaminidase and β-galactosidase co-distribute with the lipid microdomain markers and co-immunoprecipitate with the signalling protein lck in Jurkat T-cell. Furthermore, Jurkat cell stimulation up-regulates the expression and activity of lysosomal β-hexosaminidase and β-galactosidase and increases their targeting to lipid microdomains. The non-random distribution of plasma membrane-associated β-hexosaminidase and β-galactosidase and their localization within lipid microdomains, suggest a role of these enzymes in the local reorganization of glycosphingolipid-based signalling units.     

Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.     

GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster

The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. Here, we characterize the Drosophila melanogaster homologue of human GOLPH3 during cell division. We show that GOLPH3 accumulates at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. We demonstrate that the molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, we show that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results we propose that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, our studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis.     

Sac1–Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.     

Glycyrrhizin regulates rat TMJOA progression by inhibiting the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway

To investigate the role of glycyrrhizin on the progression of temporomandibular joint osteoarthritis (TMJOA) and the underlying mechanism by regulation of the high‐mobility group box 1 (HMGB1) receptor for advanced glycation end products (RAGE)/toll‐like receptor 4 (TLR4)‐nuclear factor kappa B (NF‐κB)/protein kinase B (AKT) pathway. After a rat model of TMJOA was built by intra‐articular injection of monosodium iodoacetate, glycyrrhizin was intragastrically administered at low concentration (20 mg/kg) or high concentration (50 mg/kg). Micro‐computed tomography, histological and immunohistochemical analysis were used to reveal the progression of TMJOA. Rat TMJ chondrocytes and disc cells were cultured in inflammatory condition with different doses of glycyrrhizin. Western blot was used to evaluate the effect of glycyrrhizin on the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway. Administration of glycyrrhizin alleviated cartilage degeneration, lowered the levels of inflammatory and catabolic mediators and reduced the production of HMGB1, RAGE and TLR4 in TMJOA animal model. Increased production of RAGE and TLR4, and activated intracellular NF‐κB and/or AKT signalling pathways in chondrocytes and disc cells were found in inflammatory condition. Upon activation, matrix metalloprotease‐3 and interleukin‐6 were upregulated. Glycyrrhizin inhibited not only HMGB1 release but also RAGE and TLR4 in inflammatory condition. Glycyrrhizin alleviated the pathological changes of TMJOA by regulating the HMGB1‐RAGE/TLR4‐NF‐kB/AKT signalling pathway. This study revealed the potential of glycyrrhizin as a novel therapeutic drug to suppress TMJ cartilage degradation.     

USP42 protects ZNRF3/RNF43 from R‐spondin‐dependent clearance and inhibits Wnt signalling

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R‐spondin induces auto‐ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R‐spondin by protecting ZNRF3/RNF43 from ubiquitin‐dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R‐spondin‐LGR4‐ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R‐spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.     

Study of GOLPH3: a Potential Stress-Inducible Protein from Golgi Apparatus

Although the Golgi apparatus has been studied extensively for over 100 years, the complex structure-function relationships have yet to be elucidated. It is well known that the Golgi complex plays an important role in the transport, processing, sorting, and targeting of numerous proteins and lipids destined for secretion, plasma membrane, and lysosomes. Increasing evidence suggests that the Golgi apparatus is a sensor and common downstream effector of stress signals in cell death pathways. It undergoes disassembly and fragmentation in several neurological disorders. Recent studies indicate that Golgi phosphoprotein 3 (GOLPH3 also known as GPP34/GMx33/MIDAS), a peripheral membrane protein of trans-Golgi network, represents an exciting new class of oncoproteins involved in cell signal transduction and is potentially mobilized by stress. In this review, we focus on the importance of GOLPH3 in vesicular trafficking, Golgi architecture maintenance, receptor sorting, protein glycosylation, and further discuss its potential in signal sensing in stress response.     

Balancing the length of the distal tip by septins is key for stability and signalling function of primary cilia

Primary cilia are antenna‐like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady‐state to promote signalling is largely unknown. Here, we define that mammalian primary cilia axonemes are formed by proximal segment (PS) and distal segment (DS) delineated by tubulin polyglutamylation‐rich and ‐poor regions, respectively. The analysis of proximal/distal segmentation indicated that perturbations leading to cilia over‐elongation influenced PS or DS length with a different impact on cilia behaviour. We identified septins as novel repressors of DS growth. We show that septins control the localisation of MKS3 and CEP290 required for a functional transition zone (TZ), and the cilia tip accumulation of the microtubule‐capping kinesin KIF7, a cilia‐growth inhibitor. Live‐cell imaging and analysis of sonic‐hedgehog (SHH) signalling activation established that DS over‐extension increased cilia ectocytosis events and decreased SHH activation. Our data underlines the importance of understanding cilia segmentation for length control and cilia‐dependent signalling.     

Cellular and molecular biology of glycosphingolipid glycosylation

Brain tissue is characterized by its high glycosphingolipid content, particularly those containing sialic acid (gangliosides). As a result of this observation, brain tissue was a focus for studies leading to the characterization of the enzymes participating in ganglioside biosynthesis, and their participation in driving the compositional changes that occur in glycolipid expression during brain development. Later on, this focus shifted to the study of cellular aspects of the synthesis, which lead to the identification of the site of synthesis in the neuronal soma and their axonal transport toward the periphery. In this review article, we will focus in subcellular aspects of the biosynthesis of glycosphingolipid oligosaccharides, particularly the mechanisms underlying the trafficking of glycosphingolipid glycosyltransferases from the endoplasmic reticulum to the Golgi, those that promote their retention in the Golgi and those that participate in their topological organization as part of the complex membrane bound machinery for the synthesis of glycosphingolipids.