Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

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. Statistical analysis reveals that the α-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.

COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.

A Rab33b 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 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.

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis.

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury. Abnormalities in this process lead to either excessive bleeding or uncontrolled thrombosis/insufficient antithrombotic activity, which translates into vessel occlusion and its sequelae. The FeCl3-induced carotid injury model is a valuable tool in probing how thrombosis initiates and progresses in vivo. This model involves endothelial damage/denudation and subsequent clot formation at the injured site. It provides a highly sensitive, quantitative assay to monitor vascular damage and clot formation in response to different degrees of vascular damage. Once optimized, this standard technique can be used to study the molecular mechanisms underlying thrombosis, as well as the ultrastructural changes in platelets in a growing thrombus. This assay is also useful to study the efficacy of antithrombotic and antiplatelet agents. This article explains how to initiate and monitor FeCl3-induced arterial thrombosis and how to collect samples for analysis by electron microscopy.

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. Results: Wide-area transmission electron mcrographs revealed initial platelet capture to the exposed adventitia resulted in localized patches of degranulated, procoagulant-like platelets. Platelet activation to a procoagulant state was sensitive to dabigatran, a direct-acting PAR receptor inhibitor, but not to cangrelor, a P2Y12 receptor inhibitor. Subsequent thrombus growth was sensitive to both cangrelor and dabigatran and sustained by the capture of discoid platelet strings first to collagen-anchored platelets and later to loosely adherent peripheral platelets. Spatial examination indicated that staged platelet activation resulted in a discoid platelet tethering zone that was pushed progressively outward as platelets converted from one activation state to another. As thrombus growth slowed, discoid platelet recruitment became rare and loosely adherent intravascular platelets failed to convert to tightly adherent platelets. Conclusions: In summary, the data support a model that we term Capture and Activate, in which the initial high platelet activation is directly linked to the exposed adventitia, all subsequent tethering of discoid platelets is to loosely adherent platelets that convert to tightly adherent platelets, and self-limiting, intravascular platelet activation over time is the result of decreased signaling intensity.

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. 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.

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. 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.

Platelet α-granule cargo packaging and release are affected by the luminal proteoglycan, serglycin.

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 ß-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.

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. 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.

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

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.

Structural analysis of resting mouse platelets by 3D-EM reveals an unexpected variation in α-granule shape.

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.

Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport.

Rab6, the most abundant Golgi associated small GTPase, consists of 2 equally common isoforms, Rab6A and Rab6A', that differ in 3 amino acids and localize to trans Golgi cisternae. The two isoforms are largely redundant in function and hence are often referred to generically as Rab6. Rab6 loss-of-function inhibits retrograde Golgi trafficking, induces an increase in Golgi cisternal number in HeLa cells and delays the cell surface appearance of the anterograde cargo protein, VSVG. We hypothesized that these effects are linked and might be explained by a cisternal-specific delay in cargo transport. In pulse chase experiments using a deconvolved, confocal line scanning approach to score the distribution of the tsO45 mutant of VSVG protein in Rab6 depleted cells, we found that anterograde transport at 32 °C, permissive conditions, through the Golgi apparatus was locally delayed, almost tenfold, between medial and trans Golgi cisterna. Cis to medial transport was nearly normal as was trans Golgi to TGN transport. TGN exit was unaffected by Rab6 depletion. These effects were the same with either of two siRNAs. Similar intra-Golgi transport delays were seen at 37 °C with RUSH VSVG or a RUSH GPI-anchored construct using a biotin pulse to release the marker proteins from the ER. Using 3D-SIM, a super resolution approach, we found that RUSH VSVG transport was delayed pre-trans Golgi. These visual approaches suggest a selective slowing of anterograde transport relative to 3 different marker proteins downstream of the trans Golgi. Using a biochemical approach, we found that the onset of VSVG endoglycosidase H resistance in Rab6 depleted cells was delayed. Depletion of neither Rab6A or Rab6A' isoforms alone had any effect on anterograde transport through the Golgi suggesting that Rab6A and Rab6A' act coordinately. Delayed cargo transport conditions correlate strongly with a proliferation of Golgi cisternae observed in earlier electron microscopy. Our results strongly indicate that Rab6 is selectively required for rapid anterograde transport from the medial to trans Golgi. We suggest that the observed correlation with localized cisternal proliferation fits best with a cisternal progression model of Golgi function.

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. 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.

Epistatic Analysis of the Contribution of Rabs and Kifs to CATCHR Family Dependent Golgi Organization.

Multisubunit members of the CATCHR family: COG and NRZ complexes, mediate intra-Golgi and Golgi to ER vesicle tethering, respectively. We systematically addressed the genetic and functional interrelationships between Rabs, Kifs, and the retrograde CATCHR family proteins: COG3 and ZW10, which are necessary to maintain the organization of the Golgi complex. We scored the ability of siRNAs targeting 19 Golgi-associated Rab proteins and all 44 human Kifs, microtubule-dependent motor proteins, to suppress CATCHR-dependent Golgi fragmentation in an epistatic fluorescent microscopy-based assay. We found that co-depletion of Rab6A, Rab6A', Rab27A, Rab39A and two minus-end Kifs, namely KIFC3 and KIF25, suppressed both COG3- and ZW10-depletion-induced Golgi fragmentation. ZW10-dependent Golgi fragmentation was suppressed selectively by a separate set of Rabs: Rab11A, Rab33B and the little characterized Rab29. 10 Kifs were identified as hits in ZW10-depletion-induced Golgi fragmentation, and, in contrast to the double suppressive Kifs, these were predominantly plus-end motors. No Rabs or Kifs selectively suppressed COG3-depletion-induced Golgi fragmentation. Protein-protein interaction network analysis indicated putative direct and indirect links between suppressive Rabs and tether function. Validation of the suppressive hits by EM confirmed a restored organization of the Golgi cisternal stack. Based on these outcomes, we propose a three-way competitive model of Golgi organization in which Rabs, Kifs and tethers modulate sequentially the balance between Golgi-derived vesicle formation, consumption, and off-Golgi transport.

Autophagy in Platelets.

Anucleate platelets are produced by fragmentation of megakaryocytes. Platelets circulate in the bloodstream 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.

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. 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.

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. 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 αIIbß3 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.

Rab proteins as major determinants of the Golgi complex structure.

GTP-ases of the Rab family (about 70 in human) are key regulators of intracellular transport and membrane trafficking in eukaryotic cells. Remarkably, almost one third associate with membranes of the Golgi complex and TGN (trans-Golgi network). Through interactions with a variety of effectors that include molecular motors, tethering complexes, scaffolding proteins and lipid kinases, they play an important role in maintaining Golgi architecture.

GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells.

GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome.

Cellubrevin/vesicle-associated membrane protein-3-mediated endocytosis and trafficking regulate platelet functions.

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbß3, αvß3), and purinergic receptors (P2Y1, P2Y12), and thus normal platelet function. However, the molecular machinery required and possible trafficking routes are still ill-defined. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing, soluble N-ethylmaleimide factor attachment protein receptor (v-SNARE) called cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) in platelet function. Although not required for normal platelet exocytosis or hemostasis, VAMP-3-/- mice had less platelet-associated Fg, indicating a defect in Fg uptake/storage. Other granule markers were unaffected. Direct experiments, both in vitro and in vivo, showed that loss of VAMP-3 led to a robust defect in uptake/storage of Fg in platelets and cultured megakaryocytes. Uptake of the fluid-phase marker, dextran, was only modestly affected. Time-dependent uptake and endocytic trafficking of Fg and dextran were followed using 3-dimensional-structured illumination microscopy. Dextran uptake was rapid compared with Fg, but both cargoes progressed through Rab4+, Rab11+, and von Willebrand factor (VWF)+ compartments in wild-type platelets in a time-dependent manner. In VAMP-3-/- platelets, the 2 cargoes showed limited colocalization with Rab4, Rab11, or VWF. Loss of VAMP-3 also affected some acute platelet functions, causing enhanced spreading on Fg and fibronectin and faster clot retraction compared with wild-type. In addition, the rate of Janus kinase 2 phosphorylation, initiated through the thrombopoietin receptor (TPOR/Mpl) activation, was affected in VAMP-3-/- platelets. Collectively, our studies show that platelets are capable of a range of endocytosis steps, with VAMP-3 being pivotal in these processes.