Essential role of the conserved oligomeric Golgi complex in .

The Golgi is an essential eukaryotic organelle and a major place for protein sorting and glycosylation. Among apicomplexan parasites, retains the most developed Golgi structure and produces many glycosylated factors necessary for parasite survival. Despite its importance, Golgi function received little attention in the past.

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients.

A missense mouse model for Smith-McCort dysplasia shows bone resorption defects and altered protein glycosylation.

Smith McCort (SMC) dysplasia is a rare, autosomal recessive, osteochondrodysplasia that can be caused by pathogenic variants in either or genes. These genes codes for proteins that are located at the Golgi apparatus and have a role in intracellular vesicle trafficking. We generated mice that carry a disease-causing variant, c.

Tethered platelet capture provides a mechanism for restricting circulating platelet activation to the wound site.

Background: Puncture wounding is a longstanding challenge to human health for which understanding is limited, in part, by a lack of detailed morphological data on how the circulating platelet capture to the vessel matrix leads to sustained, self-limiting platelet accumulation.

Objectives: The objective of this study was to produce a paradigm for self-limiting thrombus growth in a mouse jugular vein model.

Methods: Data mining of advanced electron microscopy images was performed from authors' laboratories.

Rapid COG Depletion in Mammalian Cell by Auxin-Inducible Degradation System.

Conserved oligomeric Golgi (COG) complex orchestrates intra-Golgi retrograde trafficking and glycosylation of macromolecules, but the detailed mechanism of COG action is unknown. Previous studies employed prolonged protein knockout and knockdown approaches which may potentially generate off-target and indirect mutant phenotypes. To achieve a fast depletion of COG subunits in human cells, the auxin-inducible degradation system was employed.

High-Pressure Freezing Followed by Freeze Substitution: An Optimal Electron Microscope Technique to Study Golgi Apparatus Organization and Membrane Trafficking.

A major goal of structural biologists is to preserve samples as close to their living state as possible. High-pressure freezing (HPF) is a state-of-art technique that freezes the samples at high pressure (~2100 bar) and low temperature (-196 °C) within milliseconds. This ultrarapid fixation enables simultaneous immobilization of all cellular components and preserves the samples in a near-native state.

Acute COG complex inactivation unveiled its immediate impact on Golgi and illuminated the nature of intra-Golgi recycling vesicles.

Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins.

Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases.

Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities.

Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates.

Primary hemostasis results in a platelet-rich thrombus that has long been assumed to form a solid plug. Unexpectedly, our 3-dimensional (3D) electron microscopy of mouse jugular vein puncture wounds revealed that the resulting thrombi were structured about localized, nucleated platelet aggregates, pedestals and columns, that produced a vaulted thrombus capped by extravascular platelet adherence. Pedestal and column surfaces were lined by procoagulant platelets.

Dense cellular segmentation for EM using 2D-3D neural network ensembles.

Biologists who use electron microscopy (EM) images to build nanoscale 3D models of whole cells and their organelles have historically been limited to small numbers of cells and cellular features due to constraints in imaging and analysis. This has been a major factor limiting insight into the complex variability of cellular environments. Modern EM can produce gigavoxel image volumes containing large numbers of cells, but accurate manual segmentation of image features is slow and limits the creation of cell models.

Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis.

The canalicular system (CS) has been defined as: an inward, invaginated membrane connector that supports entry into and exit from the platelet; a static structure stable during platelet isolation; and the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3.

3D ultrastructural analysis of α-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

Background: State-of-the-art 3-dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure.

Objectives: Our goal was to determine the 3D organization of α-granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships.

Defects in COG-Mediated Golgi Trafficking Alter Endo-Lysosomal System in Human Cells.

The conserved oligomeric complex (COG) is a multi-subunit vesicle tethering complex that functions in retrograde trafficking at the Golgi. We have previously demonstrated that the formation of enlarged endo-lysosomal structures (EELSs) is one of the major glycosylation-independent phenotypes of cells depleted for individual COG complex subunits. Here, we characterize the EELSs in HEK293T cells using microscopy and biochemical approaches.

SNARE-dependent membrane fusion initiates α-granule matrix decondensation in mouse platelets.

Platelet α-granule cargo release is fundamental to both hemostasis and thrombosis. Granule matrix hydration is a key regulated step in this process, yet its mechanism is poorly understood. In endothelial cells, there is evidence for 2 modes of cargo release: a jack-in-the-box mechanism of hydration-dependent protein phase transitions and an actin-driven granule constriction/extrusion mechanism.

Alterations in platelet secretion differentially affect thrombosis and hemostasis.

We genetically manipulated the major platelet vesicle-associated membrane proteins (VAMP2, VAMP3, and VAMP8) to create mice with varying degrees of disrupted platelet secretion. As previously shown, loss of VAMP8 reduced granule secretion, and this defect was exacerbated by further deletion of VAMP2 and VAMP3. VAMP238 platelets also had reduced VAMP7.

Membrane detachment is not essential for COG complex function.

COG is a multisubunit vesicle tethering complex in the Golgi. We demonstrate that both COG subcomplexes can be permanently attached to Golgi membranes and that major COG functions do not require cycling between the membrane and cytosol.

Golgi proteins in circulating human platelets are distributed across non-stacked, scattered structures.

Platelets are small, anucleate cell fragments that are central to hemostasis, thrombosis, and inflammation. They are derived from megakaryocytes from which they inherit their organelles. As platelets can synthesize proteins and contain many of the enzymes of the secretory pathway, one might expect all mature human platelets to contain a stacked Golgi apparatus, the central organelle of the secretory pathway.

Cog5-Cog7 crystal structure reveals interactions essential for the function of a multisubunit tethering complex.

The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation.

Fluorescent microscopy as a tool to elucidate dysfunction and mislocalization of Golgi glycosyltransferases in COG complex depleted mammalian cells.

Staining of molecules such as proteins and glycoconjugates allows for an analysis of their localization within the cell and provides insight into their functional status. Glycosyltransferases, a class of enzymes which are responsible for glycosylating host proteins, are mostly localized to the Golgi apparatus, and their localization is maintained in part by a protein vesicular tethering complex, the conserved oligomeric Golgi (COG) complex. Here we detail a combination of fluorescent lectin and immuno-staining in cells depleted of COG complex subunits to examine the status of Golgi glycosyltransferases.

COG6 interacts with a subset of the Golgi SNAREs and is important for the Golgi complex integrity.

Vesicular tethers and SNAREs are two key protein components that govern docking and fusion of intracellular membrane carriers in eukaryotic cells. The conserved oligomeric Golgi (COG) complex has been specifically implicated in the tethering of retrograde intra-Golgi vesicles. Using yeast two-hybrid and co-immunoprecipitation approaches, we show that the COG6 subunit of the COG complex is capable of interacting with a subset of Golgi SNAREs, namely STX5, STX6, GS27 and SNAP29.

The lipoprotein lipase (LPL) S447X gain of function variant involves increased mRNA translation.

Objective: A common gain-of-function LPL variant, LPLS447X, has favorable clinical features and involves a C→G base change at nucleotide 1595 of the LPL cDNA, along with a haplotype, which includes other non-coding SNPs. The mechanism for the LPL gain-in-function is not clear. LPL translation is regulated by epinephrine by an RNA-protein complex, consisting of PKA subunits and an A kinase anchoring protein (AKAP), which targets the 3'UTR.

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery.

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized.

The translational regulation of lipoprotein lipase by epinephrine involves an RNA binding complex including the catalytic subunit of protein kinase A.

The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively. Catecholamines stimulate adipocyte lipolysis through reversible phosphorylation of hormone-sensitive lipase, and simultaneously inhibit LPL activity. However, LPL regulation is complex and previous studies have described translational regulation of LPL in response to catecholamines because of an RNA-binding protein that interacts with the 3'-untranslated region of LPL mRNA.

Nuclear UDP-glucuronosyltransferases: identification of UGT2B7 and UGT1A6 in human liver nuclear membranes.

We have demonstrated the subcellular localization of the human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A6, in endoplasmic reticulum (ER) and nuclear membrane from human hepatocytes and cell lines, by in situ immunostaining and Western blot. Double immunostaining for UGT2B7 and calnexin, an ER resident protein, showed that UGT2B7 was equally present in ER and nuclear membrane whereas calnexin was present almost exclusively in ER. Immunogold labeling of HK293 cells expressing UGT2B7 established the presence of UGT2B7 in both nuclear membranes.