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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 a-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. Statistical analysis reveals that the a-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets. [ABSTRACT FROM AUTHOR]

Smith McCort (SMC) dysplasia is a rare, autosomal recessive, osteochondrodysplasia that can be caused by pathogenic variants in either RAB33B or DYM 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 Rab33b disease-causing variant, c.136A>C (p.Lys46Gln), which is identical to that of members from a consanguineous family diagnosed with SMC. In male mice at 4 months of age, the Rab33b variant caused a mild increase in trabecular bone thickness in the spine and femur and in femoral mid-shaft cortical thickness with a concomitant reduction of the femoral medullary area, suggesting a bone resorption defect. In spite of the increase in trabecular and cortical thickness, bone histomorphometry showed a 4-fold increase in osteoclast parameters in homozygous Rab33b mice suggesting a putative impairment in osteoclast function, while dynamic parameters of bone formation were similar in mutant versus control mice. Femur biomechanical tests showed an increased in yield load and a progressive elevation, from WT to heterozygote to homozygous mutants, of bone intrinsic properties. These findings suggest an overall impact on bone material properties which may be caused by disturbed protein glycosylation in cells contributing to skeletal formation, supported by the altered and variable pattern of lectin staining in murine and human tissue cultured cells and in liver and bone murine tissues. The mouse model only reproduced some of the features of the human disease and was sex-specific, manifesting in male but not female mice. Our data reveal a potential novel role of RAB33B in osteoclast function and protein glycosylation and their dysregulation in SMC and lay the foundation for future studies. [ABSTRACT FROM AUTHOR]

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. v‐SNAREs (GS15, GS28) and v‐tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t‐SNAREs (STX5, YKT6), t‐tethers (GM130, p115), and most of Rab proteins remained Golgi‐associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi‐based vesicular coats—COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra‐Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells. [ABSTRACT FROM AUTHOR]

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. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient's fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs. [ABSTRACT FROM AUTHOR]

The canalicular system (CS) has been defined as: 1) an inward, invaginated membrane connector that supports entry into and exit from the platelet; 2) a static structure stable during platelet isolation; and 3) 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. Qualitative and quantitative analysis of whole platelet reconstructions, obtained from immediately fixed or washed platelets fixed post-washing, indicated that CS, even in the presence of activation inhibitors, reorganized during platelet isolation to generate a more interconnected network. Further, CS redistribution into the PM at different times, post-activation, appeared to account for only about half the PM expansion seen in thrombin-activated platelets, in vitro, suggesting that CS reorganization is not sufficient to serve as a dominant membrane reservoir for activated platelets. In sum, our analysis highlights the need to revisit past assumptions about the platelet CS to better understand how this membrane system contributes to platelet function. [ABSTRACT FROM AUTHOR]

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. By thin section electron microscopy, stacked membranes resembling the stacked Golgi compartment in megakaryocytes and other nucleated cells can be detected in both proplatelets and platelets. However, the incidence of such structures is low and whether each and every platelet contains such a structure remains an open question. By single-label, immunofluorescence staining, Golgi glycosyltransferases are found within each platelet and map to scattered structures. Whether these structures are positive for marker proteins from multiple Golgi subcompartments remains unknown. Here, we have applied state-of-the-art techniques to probe the organization state of the Golgi apparatus in resting human platelets. By the whole cell volume technique of serial-block-face scanning electron microscopy (SBF-SEM), we failed to observe stacked, Golgi-like structures in any of the 65 platelets scored. When antibodies directed against Golgi proteins were tested against HeLa cells, labeling was restricted to an elongated juxtanuclear ribbon characteristic of a stacked Golgi apparatus. By multi-label immunofluorescence microscopy, we found that each and every resting human platelet was positive for cis, trans, and trans Golgi network (TGN) proteins. However, in each case, the proteins were found in small puncta scattered about the platelet. At the resolution of deconvolved, widefield fluorescence microscopy, these proteins had limited tendency to map adjacent to one another. When the results of 3D structured illumination microscopy (3D SIM), a super resolution technique, were scored quantitatively, the Golgi marker proteins failed to map together indicating at the protein level considerable degeneration of the platelet Golgi apparatus relative to the layered stack as seen in the megakaryocyte. In conclusion, we suggest that these results have important implications for organelle structure/function relationships in the mature platelet and the extent to which Golgi apparatus organization is maintained in platelets. Our results suggest that Golgi proteins in circulating platelets are present within a series of scattered, separated structures. As separate elements, selective sets of Golgi enzymes or sugar nucleotides could be secreted during platelet activation. The establishment of the functional importance, if any, of these scattered structures in sequential protein modification in circulating platelets will require further research. [ABSTRACT FROM PUBLISHER]

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 glycosylate. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1-4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG's eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5-Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5-Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylate. [ABSTRACT FROM AUTHOR]

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. Enzymatic assays with UGT2B7 substrates confirmed the presence of functional UGT2B7 protein in ER, whole nuclei, and both outer and inner nuclear membranes. This study has identified, for the first time, the presence of UGT2B7 and UGT1A6 in the nucleus and of UGT2B7 in the inner and outer nuclear membranes. This localization may play an important functional role within nuclei: protection from toxic compounds and/or control of steady-state concentrations of nuclear receptor ligands. [Copyright &y& Elsevier]

Adipose lipoprotein lipase (LPL) plays an important role in regulating plasma triglycerides and lipid metabolism. We have previously demonstrated that PKC α depletion using specific antisense oligomers inhibits LPL activity in 3T3 F442A adipocytes. This inhibition of LPL activity was identified as translational and mediated by the interaction of cytoplasmic RNA binding proteins with sequences on the 3′UTR of LPL mRNA. Using in vitro translation experiments we have now identified the minimum essential region on the 3′UTR of LPL mRNA required for the inhibition of translation. These data have been confirmed by RNase protection analysis using cytoplasmic proteins isolated from the adipocytes treated with PKC α antisense oligomers and LPL 3′UTR transcript (LPL 3′UTR 1512-1640). Using a LPL mRNA affinity column we have separated and identified the components involved in this interaction to be, the catalytic and the regulatory subunits of PKA (Cα and RII β), and AKAP 121. The data indicated that the RNA inhibitory complex consisted of the same RNA binding proteins which have been identified by us previously as mediators of LPL translational inhibition by epinephrine. We therefore examined whether PKC α depletion was activating PKA. PKC depletion by prolonged TPA treatment (16h) or PKC α antisense oligomers increased PKA activity in 3T3 F442A adipocytes, and this activation was comparable to PKA activation following 2h of epinephrine treatment. [ABSTRACT FROM AUTHOR]

Abstract: 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. Methods: To examine LPL translation of the LPLS447X variant, in vitro translation of LPL mRNA constructs was studied in the presence of cytoplasmic extracts from 3T3-F442A adipocytes treated with/without epinephrine. Results: When the C→G base change at nucleotide 1595 was introduced, LPL mRNA was less susceptible to inhibition by the adipocyte extract. Similarly, a lessened susceptibility to translation inhibition occurred when the complete haplotype was constructed in the full-length 3.6kb LPL mRNA, when an irrelevant coding sequence was introduced into the LPL mRNA construct, and in response to the use of components of the RNA binding complex (PKA C and R subunits, and KH region of AKAP149). Conclusion: These studies suggest that the LPLS447X gain of function may be due to the base change in the LPL mRNA resulting in a decreased susceptibility to translational inhibition. [Copyright &y& Elsevier]

Importance: The Golgi is an essential eukaryotic organelle and a major place for protein sorting and glycosylation. Among apicomplexan parasites, Toxoplasma gondii 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. In the current study, we identified and characterized the conserved oligomeric Golgi complex and its novel partners critical for protein transport in T. gondii tachyzoites. Our results suggest that T. gondii broadened the role of the conserved elements and reinvented the missing components of the trafficking machinery to accommodate the specific needs of the opportunistic parasite T. gondii ., Competing Interests: The authors declare no conflict of interest.

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. This method of protein regulation allows a very fast and efficient depletion of COG subunits, which provides the ability to accumulate COG complex dependent (CCD) vesicles and investigate initial cellular defects associated with the acute depletion of COG complex subunits. This protocol is applicable to other vesicle tethering complexes and can be utilized to investigate anterograde and retrograde intracellular membrane trafficking pathways., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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. This facilitates the study of dynamic processes in Golgi apparatus organization and membrane trafficking. The work in our laboratory shows that high-pressure freezing followed by freeze substitution (FS), the introduction of organic solvents at low temperature prior to plastic embedding, can better preserve the structure of Golgi apparatus and Golgi-associated vesicles. Here, we present a protocol for freezing monolayer cell cultures on sapphire disks followed by freeze substitution. We were able to use this protocol to successfully study Golgi organization and membrane trafficking in HeLa cells. The protocol gives decidedly better preservation of Golgi apparatus and associated vesicles than conventional chemically fixed preparation and as a plastic embedded preparation can be readily extended to 3D electron microscopy imaging through sequential block face-scanning electron microscopy. The 3D imaging of a multi-micron thick organelle such as the Golgi apparatus located near the cell nucleus is greatly facilitated relative to hydrated sample imaging techniques such as cryo-electron microscopy., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Methods: We used serial block face-scanning electron microscopy images to render the 3D ultrastructure of α-granules, dense granules, mitochondria, canalicular system, and plasma membrane for 30 human platelets, 10 each from 3 donors. α-Granule compositional data were assessed by sequential, serial section cryo-immunogold electron microscopy and by immunofluorescence (structured illumination microscopy)., Results and Conclusions: α-Granule number correlated linearly with platelet size, while dense granule and mitochondria number had little correlation with platelet size. For all subcellular compartments, individual organelle parameters varied considerably and organelle volume fraction had little correlation with platelet size. Three-dimensional data from 30 platelets indicated only limited spatial intermixing of the different organelle classes. Interestingly, almost 70% of α-granules came within ≤35 nm of each other, a distance associated in other cell systems with protein-mediated contact sites. Size and shape analysis of the 1488 α-granules analyzed revealed no more variation than that expected for a Gaussian distribution. Protein distribution data indicated that all α-granules likely contained the same major set of proteins, albeit at varying amounts and varying distribution within the granule matrix., Competing Interests: The authors report nothing to disclose., (© 2019 University of Arkansas for Medical sciences. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

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. Our analysis revealed that the EELSs are highly acidic and that vATPase-dependent acidification is essential for the maintenance of this enlarged compartment. The EELSs are accessible to both trans -Golgi enzymes and endocytic cargo. Moreover, the EELSs specifically accumulate endolysosomal proteins Lamp2, CD63, Rab7, Rab9, Rab39, Vamp7, and STX8 on their surface. The EELSs are distinct from lysosomes and do not accumulate active Cathepsin B. Retention using selective hooks (RUSH) experiments revealed that biosynthetic cargo mCherry-Lamp1 reaches the EELSs much faster as compared to both receptor-mediated and soluble endocytic cargo, indicating TGN origin of the EELSs. In support to this hypothesis, EELSs are enriched with TGN specific lipid PI4P. Additionally, analysis of COG4/VPS54 double KO cells revealed that the activity of the GARP tethering complex is necessary for EELSs' accumulation, indicating that protein mistargeting and the imbalance of Golgi-endosome membrane flow leads to the formation of EELSs in COG-deficient cells. The EELSs are likely to serve as a degradative storage hybrid organelle for mistargeted Golgi enzymes and underglycosylated glycoconjugates. To our knowledge this is the first report of the formation of an enlarged hybrid endosomal compartment in a response to malfunction of the intra-Golgi trafficking machinery.

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. The third alternative considered is a prefusion, channel-mediated granule swelling, analogous to the membrane "ballooning" seen in procoagulant platelets. Using thrombin-stimulated platelets from a set of secretion-deficient, soluble N -ethylmaleimide factor attachment protein receptor (SNARE) mutant mice and various ultrastructural approaches, we tested predictions of these mechanisms to distinguish which best explains the α-granule release process. We found that the granule decondensation/hydration required for cargo expulsion was (1) blocked in fusion-protein-deficient platelets; (2) characterized by a fusion-dependent transition in granule size in contrast to a preswollen intermediate; (3) determined spatially with α-granules located close to the plasma membrane (PM) decondensing more readily; (4) propagated from the site of granule fusion; and (5) traced, in 3-dimensional space, to individual granule fusion events at the PM or less commonly at the canalicular system. In sum, the properties of α-granule decondensation/matrix hydration strongly indicate that α-granule cargo expulsion is likely by a jack-in-the-box mechanism rather than by gradual channel-regulated water influx or by a granule-constriction mechanism. These experiments, in providing a structural and mechanistic basis for cargo expulsion, should be informative in understanding the α-granule release reaction in the context of hemostasis and thrombosis.

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.

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. The combination of these techniques allows for a detailed characterization of the changes in function and localization of Golgi glycosyltransferases with respect to transient COG subunit depletion.

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. In this study, we identified several protein components of an LPL RNA binding complex. Using an LPL RNA affinity column, we identified two of the RNA-binding proteins as the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), and A kinase anchoring protein (AKAP) 121/149, one of the PKA anchoring proteins, which has known RNA binding activity. To determine whether the C subunit was involved in LPL translation inhibition, the C subunit was depleted from the cytoplasmic extract of epinephrine-stimulated adipocytes by immunoprecipitation. This resulted in the loss of LPL translation inhibition activity of the extract, along with decreased RNA binding activity in a gel shift assay. To demonstrate the importance of the AKAPs, inhibition of PKA-AKAP binding with a peptide competitor (HT31) prevented epinephrine-mediated inhibition of LPL translation. C subunit kinase activity was necessary for LPL RNA binding and translation inhibition, suggesting that the phosphorylation of AKAP121/149 or other proteins was an important part of RNA binding complex formation. The hormonal activation of PKA results in the reversible phosphorylation of hormone-sensitive lipase, which is the primary mediator of adipocyte lipolysis. These studies demonstrate a dual role for PKA to simultaneously inhibit LPL-mediated lipogenesis through inhibition of LPL translation.