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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.

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.

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.

Background Serglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function. Objective To understand how SRGN affects platelet granule packaging, cargo release, and extra-platelet microenvironments. Methods Platelets and megakaryocytes from SRGN-/- mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding. Results Metabolic, 35 SO4 labeling identified SRGN as a major sulfated macromolecule in megakaryocytes. SRGN colocalized with α-granule markers (platelet factor 4 [PF4], von Willebrand factor [VWF], and P-selectin), but its deletion did not affect α-granule morphology or number. Platelet α-granule composition was altered, with a reduction in basic proteins (pI ≥8; e.g., PF4, SDF-1, angiogenin) and constitutive release of PF4 from SRGN-/- megakaryocytes. P-Selectin, VWF, and fibrinogen were unaffected. Serotonin (5-HT) uptake and β-hexosaminidase (HEXB) were slightly elevated. Thrombin-induced exocytosis of PF4 from platelets was defective; however, release of RANTES/CCL5 was normal and osteopontin secretion was more rapid. Release of 5-HT and HEXB (from dense granules and lysosomes, respectively) were unaffected. Ultrastructural studies showed distinct morphologies in activated platelets. The α-granule lumen of SRGN-/- platelet had a grainy staining pattern, whereas that of wild-type granules had only fibrous material remaining. α-Granule swelling and decondensation were reduced in SRGN-/- platelets. Upon stimulation of platelets, a SRGN/PF4 complex was released in a time- and agonist-dependent manner. Shedding of GPVI from SRGN-/- platelets was modestly enhanced. Shedding of GP1b was unaffected. Conclusion The polyanionic proteoglycan SRGN influences α-granule packaging, cargo release, and shedding of platelet membrane proteins.

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.

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.

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.

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.

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. Furthermore, early steps in thrombus assembly were sensitive to P2Y12 inhibition and late steps to thrombin inhibition. Based on these results, we propose a Cap and Build, puncture wound paradigm that should have translational implications for bleeding control and hemostasis., Rhee, Pokrovskaya et al. utilize 3D electron microscopy of mouse jugular vein puncture wounds to reveal thrombi structured around localized, nucleated platelet aggregates that produced a vaulted thrombus capped by extravascular platelet adherence. As a result, the authors propose a “Cap and Build” paradigm of primary hemostasis.

Mice and mouse platelets are major experimental models for hemostasis and thrombosis; however, important physiological data from this model has received little to no quantitative, 3D ultrastructural analysis. We used state-of-the-art, serial block imaging scanning electron microscopy (SBF-SEM, nominal Z-step size was 35 nm) to image resting platelets from C57BL/6 mice. α-Granules were identified morphologically and rendered in 3D space. The quantitative analysis revealed that mouse α-granules typically had a variable, elongated, rod shape, different from the round/ovoid shape of human α--granules. This variation in length was confirmed qualitatively by higher-resolution, focused ion beam (FIB) SEM at a nominal 5 nm Z-step size. The unexpected α-granule shape raises novel questions regarding α-granule biogenesis and dynamics. Does the variation arise at the level of the megakaryocyte and α-granule biogenesis or from differences in α-granule dynamics and organelle fusion/fission events within circulating platelets? Further quantitative analysis revealed that the two major organelles in circulating platelets, α-granules and mitochondria, displayed a stronger linear relationship between organelle number/volume and platelet size, i.e., a scaling in number and volume to platelet size, than found in human platelets suggestive of a tighter mechanistic regulation of their inclusion during platelet biogenesis. In conclusion, the overall spatial arrangement of organelles within mouse platelets was similar to that of resting human platelets, with mouse α-granules clustered closely together with little space for interdigitation of other organelles.

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. Segmentation algorithms based on convolutional neural networks can process large volumes quickly, but achieving EM task accuracy goals often challenges current techniques. Here, we define dense cellular segmentation as a multiclass semantic segmentation task for modeling cells and large numbers of their organelles, and give an example in human blood platelets. We present an algorithm using novel hybrid 2D–3D segmentation networks to produce dense cellular segmentations with accuracy levels that outperform baseline methods and approach those of human annotators. To our knowledge, this work represents the first published approach to automating the creation of cell models with this level of structural detail.

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. VAMP2Δ3Δ8-/- platelets also had reduced VAMP7. Loss of VAMP2 and VAMP3 (VAMP2Δ3Δ) had a minimal impact on secretion when VAMP7 and VAMP8 were present. Integrin αIIbβ3 activation and aggregation were not affected, although spreading was reduced in VAMP2Δ3Δ8-/- platelets. Using these mice as tools, we asked how much secretion is needed for proper thrombosis and hemostasis in vivo. VAMP2Δ3Δ mice showed no deficiency, whereas VAMP8-/- mice had attenuated formation of occlusive thrombi upon FeCl3-induced arterial injury but no excessive bleeding upon tail transection. VAMP2Δ3Δ8-/- mice bled profusely and failed to form occlusive thrombi. Plasma-coagulation factors were normal in all of the strains, but phosphatidylserine exposure was reduced in VAMP2Δ3Δ and VAMP2Δ3Δ8-/- platelets. From our data, an ∼40% to 50% reduction in platelet secretion in vitro (dense and α granule) correlated with reduced occlusive thrombosis but no compromise in hemostasis. At a >50% reduction, thrombosis and hemostasis were defective in vivo. Our studies are the first systematic manipulation of platelet exocytic machinery to demonstrate a quantitative linkage between in vitro platelet secretion and hemostasis and thrombosis in vivo. The animals described will be invaluable tools for future investigations into how platelet secretion affects other vascular processes.

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., The conserved oligomeric Golgi (COG) complex is a vesicle tether of the “complexes associated with tethering containing helical rods” family, which functions on the cytoplasmic side of Golgi. It is currently unknown whether COG function, or function of any multisubunit vesicular tether, depends on cycling between the membrane and cytosol. Therefore, we permanently anchored key subunits of COG subcomplexes (COG4, COG7, and COG8) to Golgi membranes using transmembrane protein TMEM115 (TMEM-COG). All TMEM-COG subunits tested were Golgi localized, integrated into the COG complex, and stabilized membrane association of endogenous subunits. Interestingly, TMEM-COG4 and TMEM-COG7 equally rescued COG function in organization of Golgi markers, glycosylation, and abundance of COG-sensitive proteins. In contrast, TMEM-COG8 was not as effective, indicating that N-terminal attachment of COG8 interfered with overall COG structure and function, and none of the TMEM-COG subunits rescued the abnormal Golgi architecture caused by COG knockout. Collectively, these data indicate that both subcomplexes of the COG complex can perform most of COG function when permanently attached to membranes and that the cytosolic pool of COG is not completely essential to COG function.

Vascular damage presents in many forms and varying geometries. Nevertheless, the platelet response to endothelial damage to the blood vessel wall, be it through a prick or a full puncture wound, is thought to be staged by a qualitatively similar temporal variance in signaling agonists. For example, endothelial damage in the microvasculature is thought to be initially dominated by thrombin and later by platelet released ADP and thromboxane. The same temporal sequence in signaling has been proposed to exist in a profusely bleeding puncture wound 1. If so, platelet morphology, a gold standard of platelet activation state, could provide a strong readout of temporally distinct signaling effects. Platelet morphology has long been considered to be a reliable indicator of a strong agonist such as thrombin acting through PAR receptors that produces a rounded, pseudopod extending, degranulated, highly adhesive platelet versus weaker agonists such as ADP or thromboxane acting through P2Y 12 receptors to produce a less adhesive, somewhat rounded platelet. A testable prediction of existing hemostasis models is that temporal staging of signaling leads to temporal differences in platelet morphology within the forming/remodeling thrombus. Such hypothesized temporal differences in signaling are clinically significant as they form the basis for hypothesizing phenotypically distinct outcomes for direct acting anti-coagulants (DOACs) affecting thrombin versus anti-platelet drugs affecting P2Y 12, ADP receptors. Advances in imaging, e.g., wide area transmission electron microscopy (WA-TEM), make possible the local determination of platelet activation state with high precision 2. Taking a mouse jugular vein puncture wound model 1,2, we found that all morphologically recognized platelet activation states were present early, 1 min post puncture, with loosely bound discoid shaped platelets being the most peripherally located. For bleeding, early-stage puncture wound, these loosely adherent, low activation state platelets were located on both intravascular and extravascular thrombus aggregates. Once the puncture wound is closed, loosely adherent platelets were only found on the intravascular surfaces of the thrombus. We propose that this result is most consistent with a platelet conversion model in which new loosely adherent platelets rapidly convert to tightly packed platelets. As the thrombus remodels, 5 and 20 min post-puncture, the thrombus continued to accumulate platelets both intravascularly and extravascularly. Peripheral, discoid shaped platelets provided a source for intravascular thrombus growth. However, any subsequent extravascular thrombus growth must be due to platelet migration. Significantly, we found that cangrelor, a direct acting P2Y 12 inhibitor, stalled thrombus formation/remodeling at an early stage (Figure 1A,C,E see also ref 1,2). By WA-TEM, the accumulation of discoid-shaped, loosely adherent platelets appeared to be enhanced in a cangrelor treated 5 min thrombus (Figure 1E,F). We suggest that P2Y 12 receptors must act early in thrombus formation with the conversion of discoid to more activated platelets being most affected. In contrast, a 5-min post puncture dabigatran (DOAC) treated showed deformed architecture with inhibition of the accumulation of discoid shaped platelets/rounded loosely adherent platelets being most affected (Figure 1D,F, see also ref 2). Accumulation of degranulated platelets appeared to be lessened in both cangrelor and dabigatran treated thrombi. We propose that the simplest explanation of these results is that multiple signaling pathways act in parallel with select activation states being more dependent on one pathway than another. Clinically, our results suggest that P2Y 12 inhibitors can affect thrombus formation at early time points in addition to the late time points projected by current models. 1. Tomaiuolo M., Matzko C.N., Posentud-Fuentes I., Weisel J.W., Brass L.F. & Stalker T.J. Interrelationships between structure and function during the hemostatic response to injury. Proc Natl Acad Sci USA. 116. 2243-2252 (2019). 2. Rhee, Pokrovskaya I.D.,BallK., LingK., VedanapartiY., CohenJ., CruzD., ZhaoO.S., AronovaM.A., ZhangG., Kamykowski J.A., LeapmanR.D., & StorrieB. Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates. Commun. Biol., accepted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Vascular damage comes in many forms with the puncture wound likely the longest known to humans. Experimentally, vascular damage has typically been visualized in mouse models in which there is little bleeding. Under these conditions, damage is limited mostly to the endothelial layer lining the vessel. Visualization has varied from light to scanning electron microscopy. Interpretation has been dominated by intravital microscopy outcomes in which an initial layer of p-selection-exposed, i.e., α-granule secretion-positive, platelets is deposited in association with the damaged vessel wall and extended by the accumulation of a less activated outer layer of platelets. These results have given rise to a Core and Shell model of platelet-rich thrombus formation [Tomaiuolo et al., 2017]. Recently, new mouse models have been presented in which the vessel is punctured to create a 300 to 600 micron wound hole [Tomoiuolo et al., 2019]. Bleeding is now profuse. The puncture wound results have been interpreted within a Core and Shell model. However, two important aspects of the experimental data [Tomoiuolo et al., 2019] suggest that the existing model may not explain the actual results. First, p-selectin expression as a marker for α-granule secretion and platelet activation was present in limited areas towards the periphery of the resulting thrombus, not as well-defined Core. Second, the hemostatic thrombus when viewed at early stages, ex vivo, showed a pebbly distribution of platelet aggregates suggestive of nucleated platelet accumulation rather than the smooth layers that would follow from a Core and Shell model. We hypothesize that nucleated accumulation of platelet aggregates within the puncture hole could provide pedestals upon which localized accumulation of platelets form the infrastructure of a vaulted thrombus whose extravascular capping leads to bleeding cessation. Methods: To test the proposed hypothesis, we visualized the interior and overall structure of the forming puncture wound thrombus in full 3D at sub-platelet level resolution. To achieve this end, we took our proven serial block face scanning electron microscopy (SBF-SEM) protocols for visualizing platelet organelles in 3D [Pokrovskaya et al., 2018] and adapted them to the visualization of forming thrombi over 1000s of image. To localize samples for electron microscopy, we used in vivo antibody labeling [Tomaiuolo et al., 2019]. This approach had the added advantage of enabling correlative light microscopy mapping overall p-selectin, a marker of platelet secretion, and fibrin distributions against 3D, platelet resolution, thrombus morphology. Results: We found that a 1 min thrombus, pre-bleeding cessation, was structured about the localized accumulation of pedestal-like platelet aggregates along the sides of the puncture hole, extended and spaced along the extravascular adventitia. Subsequent pedestal extension formed a "pontoon" bridge that "capped" extravascularly the puncture hole. At 5 min, full bleeding cessation, we found that forming platelet thrombus had a Swiss cheese-like interior of vaults that were continuous with the intravascular vessel lumen and framed by columns of platelets, presumed pedestal extensions. The thrombus was sealed on the extravascular side by a platelet "cap" (Figure). As expected after bleeding cessation, red blood cells accumulated on the intravascular side of the cap. Formation of a tightly sealed cap was dependent on α-granule secretion as indicated by the effect of knockout of VAMP-8, the primary SNARE protein involved in a-granule release. Based upon morphology, vaults within the forming thrombus were lined with apparent procoagulant platelets providing a potential protected surface for coagulation factor activation. Conclusions: We conclude that bleeding cessation in a true puncture wound occurs from the extravascular side of the thrombus rather than through the formation of a platelet plug that fills the hole. We propose an alternative model of bleeding cessation in which localized platelet aggregates are the starting pedestal upon which all subsequent steps in puncture thrombus formation build, i.e., "Cap and Build". The extent to which properties differ among systems remains an open question. Tomaiuolo et al. 2017. Intervent. Cardiol. Clin. 6: 1-12. Pokrovskaya et al. 2018. Blood Adv. 2: 2947-2958 Tomaiuolo et al. 2019. Proc. Natl. Acad. Sci. USA 116:2 243-2252 Figure Disclosures No relevant conflicts of interest to declare.

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.

Anucleate platelets are produced by fragmentation of megakaryocytes. Platelets circulate in the blood stream for a finite period: Upon vessel injury, they are activated to participate in hemostasis; upon senescence, unused platelets are cleared. Platelet hypofunction leads to bleeding. Conversely, pathogenic platelet activation leads to occlusive events that precipitate strokes and heart attacks. Recently, we and others have shown that autophagy occurs in platelets and is important for platelet production and normal functions including hemostasis and thrombosis. Due to the unique properties of platelets, such as their lack of nuclei and their propensity for activation, methods for studying platelet autophagy must be specifically tailored. Here, we describe useful methods for examining autophagy in both human and mouse platelets.

UNLABELLED ESSENTIALS: How platelets organize their α-granule cargo and use their canalicular system remains controversial. Past structural studies were limited due to small sampling volumes or decreased resolution. Our analyses revealed homogeneous granules and a closed canalicular system that opened on activation. Understanding how platelets alter their membranes during activation and secretion elucidates hemostasis. SUMMARY BACKGROUND Platelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their α-granules. Alpha-granules may be biochemically and structurally heterogeneous; however, other studies suggest that they may be more homogeneous with the observed variation reflecting granule dynamics rather than fundamental differences. OBJECTIVES Our aim was to address how the structural organization of α-granules supports their dynamics. METHODS To preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image nearly entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM). RESULTS AND CONCLUSIONS In resting platelets, we observed a morphologically homogeneous α-granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations (i.e., macro-homogeneity). In resting platelets, the incidence of tubular granule extensions was low, ~4%, but this increased by > 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially decondensing α-granules and the canalicular system remained as separate membrane domains. Decondensing α-granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased by about three-fold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet α-granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for the timed release of α-granule proteins.

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.

The conserved oligomeric Golgi (COG) complex controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle trafficking within the Golgi. Human COG defects lead to severe multisystemic diseases known as COG-congenital disorders of glycosylation (COG-CDG). To gain better understanding of COG-CDGs, we compared COG knockout cells with cells deficient to 2 key enzymes, Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase and uridine diphosphate-glucose 4-epimerase (GALE), which contribute to proper N- and O-glycosylation. While all knockout cells share similar defects in glycosylation, these defects only account for a small fraction of observed COG knockout phenotypes. Glycosylation deficiencies were not associated with the fragmented Golgi, abnormal endolysosomes, defective sorting and secretion or delayed retrograde trafficking, indicating that these phenotypes are probably not due to hypoglycosylation, but to other specific interactions or roles of the COG complex. Importantly, these COG deficiency specific phenotypes were also apparent in COG7-CDG patient fibroblasts, proving the human disease relevance of our CRISPR knockout findings. The knowledge gained from this study has important implications, both for understanding the physiological role of COG complex in Golgi homeostasis in eukaryotic cells, and for better understanding human diseases associated with COG/Golgi impairment.

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50–100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.

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. 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 glycosylation.

Introduction: Platelet recruitment to generate a thrombus is key to bleeding cessation. That recruitment is dependent on a series of platelet activation processes that include adhesion to the exposed vessel matrix, platelet-platelet adhesion and platelet granule release. How platelet activation is patterned to generate a thrombus has previously been studied by intravital light microscopy, two-photon microscopy and scanning electron microscopy at resolutions insufficient to infer platelet activation at the level of the individual platelet. Here, we present a collaborative effort to stratify spatially the extent of platelet activation at the cellular level in a mouse jugular vein puncture model. We used wide-area transmission electron microscopy (WA-TEM) and serial block face scanning electron microscopy (SBF-SEM) at a resolution of 3 to 100 nm across whole thrombi to determine activation state of individual platelets. Our results, indicate a pattern of platelet stratification within the puncture wound that varies in a time-dependent manner with distinct structural stages in the formation of the thrombus. Methods: Jugular vein thrombi from C57BL/6 mice were collected 1, 5, or 20 min after a 300 µm needle puncture and prepared for EM imaging. For WA-TEM, hundreds of overlapping 3 nm resolution images were acquired using a gondimeter stage. The images sets were aligned using NIH Fiji software to create a single high-res, thrombus-wide image. Individual platelets were stratified into morphologically defined activation states (1: no activation, 2: decreased granule number, 3: no visible granules left, 4: hollow inside). The spatial distribution of platelet stratification was analyzed using iVision software. For SBF-SEM, 100-nm XY-resolution SEM images were collected every 200 nm and 20 nm XY-resolution images every 20 µm. Semi-automated stratification of platelet activation state in individual slices of the thrombus were combined into a 3-D representation using Amira software. Volumetric distributions of platelets with respect to the puncture hole and the vascular wall were quantified. Results: Thrombus Formation Stage 1 (anchor and extend) -- One min post puncture, platelets were anchored in clumps along the exposed vessel wall. Near the damaged vessel wall was a peripheral layer of activated or degranulated platelets (states 3 and 4) covered by additional layers of less-activated platelets (state 1 and 2). Short cylindrical ingrowths extended into the 300 µm hole. Unexpectedly, large aggregates of platelets with a mixture of activation states (states 1 - 4) were found extending from these anchor points into the hole and vertically into the intravascular space. Aggregate surface layers were composed mostly of degranulated platelets (states 3 and 4). Less than 40% of neighboring platelets were of the same activation state as their neighbor. Surprisingly, Stage 2 (cap and erect) -- At 5 min after injury, the puncture hole was capped. ~70% of platelets neighboring degranulated platelets (3 and 4) formed visible aggregates within the thrombus. These aggregates were found along the exposed vessel wall and encasing vertical platelet aggregate towers containing a mixture of platelets in different states (1 - 4). Towers were typically separated by large cavities. SBF-SEM images, a machine-based, unbiased sampling of the underlying platelet distribution, revealed that ~10% of the platelet volume in the puncture hole of the thrombus and the intravascular towers contained largely degranulated platelets, similar to the data from WA-TEM. Stage 3 (infill and remodeling) - At 20 min post-puncture, the thrombus was filled with a mixture of platelets of varying activation states, which surrounded central, vertical aggregates (towers) of degranulated platelets seen at 5 min. Only minor cavity space was apparent. The intravascular surface of the thrombus was covered with an ~10 platelet-thick layer of loosely packed, variably activated platelets (states 1 - 4). Conclusions: Our results demonstrate dynamic spatial patterns of platelet activation within a forming puncture-wound thrombus. Such patterns yield insights into thrombus formation and suggest the need to reference platelets defects and anti-thrombotics drugs against new models. Figure Disclosures No relevant conflicts of interest to declare.

The conserved oligomeric Golgi (COG) complex is a peripheral membrane protein complex which orchestrates tethering of intra-Golgi vesicles. We found that COG1-4 (lobe A) and 5–8 (lobe B) protein assemblies are present as independent sub-complexes on cell membranes. Super-resolution microscopy demonstrates that COG sub-complexes are spatially separated on the Golgi with lobe A preferential localization on Golgi stacks and the presence of lobe B on vesicle-like structures, where it physically interacts with v-SNARE GS15. The localization and specific interaction of the COG sub-complexes with the components of vesicle tethering/fusion machinery suggests their different roles in the vesicle tethering cycle. We propose and test a novel model that employs association/disassociation of COG sub-complexes as a mechanism that directs vesicle tethering at Golgi membranes. We demonstrate that defective COG assembly or restriction of tethering complex disassembly by a covalent COG1-COG8 linkage is inhibitory to COG complex activity, supporting the model.

The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this study we have used the CRISPR approach to generate HEK293T knock-out (KO) cell lines missing individual COG subunits. These cell lines were characterized for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. We found that all KO cell lines were uniformly deficient in cis/medial-Golgi glycosylation and each had nearly abolished binding of Cholera toxin. In addition, all cell lines showed defects in Golgi morphology, retrograde trafficking and sorting, sialylation and fucosylation, but severities varied according to the affected subunit. Lobe A and Cog6 subunit KOs displayed a more severely distorted Golgi structure, while Cog2, 3, 4, 5, and 7 knock outs had the most hypo glycosylated form of Lamp2. These results led us to conclude that every subunit is essential for COG complex function in Golgi trafficking, though to varying extents. We believe that this study and further analyses of these cells will help further elucidate the roles of individual COG subunits and bring a greater understanding to the class of MTCs as a whole.

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. Interaction with SNAREs is accomplished via the universal SNARE-binding motif of COG6. Overexpression of COG6, or its depletion from cells, disrupts the integrity of the Golgi complex. Importantly, COG6 protein lacking the SNARE-binding domain is deficient in Golgi binding, and is not capable of inducing Golgi complex fragmentation when overexpressed. These results indicate that COG6-SNARE interactions are important for both COG6 localization and Golgi integrity.

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.

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane-associated protein complex that functions in a retrograde intra-Golgi trafficking through associations with coiled-coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex-interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15-positive COG complex-dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP-tagged and endogenous COG complex subunits associated with inclusions in a serovar-independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v-SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid-cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi-derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.

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. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.

We interrogated in 3D-space the arrangement of alpha-granules and their proteins within 30 human platelets in order to address two central structure/function questions. First, are alpha-granules based on their size, shape and protein content a single population or not, and second, what implications could organelle frequency and organization within the resting platelet have for platelet biogenesis and membrane fusion events in activated platelets? We used serial block face scanning electron microscopy (SBF-SEM) to render in full platelet volume the ultrastructure of alpha-granules, dense granules, mitochondria, canalicular system (CS), and plasma membrane (PM) in 30 platelets, 10 each from 3 donors. Size and shape were measured for 1488 a-granules. Compositional data were assessed for multiple proteins over hundreds of granules by 3D-structured illumination microscopy (SIM) and serial section cryo-immunogold electron microscopy. Data analysis led to 3 conclusions: 1) Based on size, shape and protein composition, there was one major population of alpha-granules in resting human platelets, 2) Alpha-granules clustered tightly together while dense granules were more peripherally located and distal from one another suggesting a spatial arrangement that in the one case supports compound granule fusion and in the other case rapid fusion with the PM, and 3) Based on the weak relationship between platelet size and organelle number and volume, we inferred that platelet biogenesis is likely relatively imprecise with alpha-granule inclusion being more precisely metered than that of other organelles,. These results provide a strong, informative baseline for a-granule structural properties and suggest a spatial clustering of organelles within the resting platelet that may be functionally significant during platelet activation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Toxin trafficking studies provide valuable information about endogenous pathways of intracellular transport. Subtilase cytotoxin (SubAB) is transported in a retrograde manner through the endosome to the Golgi and then to the endoplasmic reticulum (ER), where it specifically cleaves the ER chaperone BiP/GRP78 (Binding immunoglobin protein/Glucose-Regulated Protein of 78 kDa). To identify the SubAB Golgi trafficking route, we have used siRNA-mediated silencing and immunofluorescence microscopy in HeLa and Vero cells. Knockdown (KD) of subunits of the conserved oligomeric Golgi (COG) complex significantly delays SubAB cytotoxicity and blocks SubAB trafficking to the cis Golgi. Depletion of Rab6 and beta-COP proteins causes a similar delay in SubAB-mediated GRP78 cleavage and did not augment the trafficking block observed in COG KD cells, indicating that all three Golgi factors operate on the same 'fast' retrograde trafficking pathway. SubAB trafficking is completely blocked in cells deficient in the Golgi SNARE Syntaxin 5 and does not require the activity of endosomal sorting nexins SNX1 and SNX2. Surprisingly, depletion of Golgi tethers p115 and golgin-84 that regulates two previously described coat protein I (COPI) vesicle-mediated pathways did not interfere with SubAB trafficking, indicating that SubAB is exploiting a novel COG/Rab6/COPI-dependent retrograde trafficking pathway.

Adipose LPL (lipoprotein lipase) plays an important role in regulating plasma triacylglycerols and lipid metabolism. We have previously demonstrated that PKCalpha (protein kinase Calpha) depletion inhibits LPL translation in 3T3-F442A adipocytes. Using in vitro translation experiments, the minimum essential region on the 3'UTR (3'-untranslated region) of LPL mRNA required for the inhibition of translation was identified as the proximal 39 nt. These results were confirmed by RNase protection analysis using cytoplasmic proteins isolated from the adipocytes treated with PKCalpha antisense oligomers and the LPL 3'UTR transcript (LPL 3'UTR nt: 1512-1640). The protein components involved in this RNA-binding interaction from PKCalpha depletion were passed through an affinity column containing a sequence of the LPL 3'UTR and, after Western blotting, the RNA-binding proteins were identified as the catalytic and the regulatory subunits of PKA (protein kinase A), Calpha and RIIbeta, and AKAP (A-kinase-anchoring protein) 121. This RNA inhibitory complex consisted of the same RNA-binding proteins that have been identified previously as mediators of LPL translational inhibition by PKA activation, suggesting that PKCalpha depletion inhibits LPL translation through PKA activation. In additional experiments, PKC depletion by prolonged PMA treatment or PKCalpha antisense oligomers resulted in an increase in PKA activity in 3T3-F442A adipocytes, comparable with PKA activation with adrenaline (epinephrine) treatment. These results demonstrate that LPL translational inhibition occurs through an RNA-binding complex involving PKA subunits and AKAP121, and this complex can be activated either through traditional PKA activation methods or through the depletion of PKCalpha.