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.

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.

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.

Defective platelet autocrine signaling in HPS.

In this issue of Blood, Meng et al and Sharda et al use the Hermansky-Pudlak syndrome (HPS) as a model to show that adenosine 5'-diphosphate (ADP) released by dense granules serves as an autocrine signal to potentiate platelet release of α-granule and lysosome cargo and protein disulfide isomerase (PDI), all of which serve to stabilize thrombus formation.

Autophagy is induced upon platelet activation and is essential for hemostasis and thrombosis.

Autophagy is important for maintaining cellular homeostasis, and thus its deficiency is implicated in a broad spectrum of human diseases. Its role in platelet function has only recently been examined. Our biochemical and imaging studies demonstrate that the core autophagy machinery exists in platelets, and that autophagy is constitutively active in resting platelets. Moreover, autophagy is induced upon platelet activation, as indicated by agonist-induced loss of the autophagy marker LC3II. Additional experiments, using inhibitors of platelet activation, proteases, and lysosomal acidification, as well as platelets from knockout mouse strains, show that agonist-induced LC3II loss is a consequence of platelet signaling cascades and requires proteases, acidic compartments, and membrane fusion. To assess the physiological role of platelet autophagy, we generated a mouse strain with a megakaryocyte- and platelet-specific deletion of Atg7, an enzyme required for LC3II production. Ex vivo analysis of platelets from these mice shows modest defects in aggregation and granule cargo packaging. Although these mice have normal platelet numbers and size distributions, they exhibit a robust bleeding diathesis in the tail-bleeding assay and a prolonged occlusion time in the FeCl3-induced carotid injury model. Our results demonstrate that autophagy occurs in platelets and is important for hemostasis and thrombosis.

The cellular basis of platelet secretion: Emerging structure/function relationships.

Platelet activation has long been known to be accompanied by secretion from at least three types of compartments. These include dense granules, the major source of small molecules; α-granules, the major protein storage organelle; and lysosomes, the site of acid hydrolase storage. Despite ~60 years of research, there are still many unanswered questions about the cell biology of platelet secretion: for example, how are these secretory organelles organized to support cargo release and what are the key routes of cargo release, granule to plasma membrane or granule to canalicular system. Moreover, in recent years, increasing evidence points to the platelet being organized for secretion of the contents from other organelles, namely the dense tubular system (endoplasmic reticulum) and the Golgi apparatus. Conceivably, protein secretion is a widespread property of the platelet and its organelles. In this review, we concentrate on the cell biology of the α-granule and its structure/function relationships. We both review the literature and discuss the wide array of 3-dimensional, high-resolution structural approaches that have emerged in the last few years. These have begun to reveal new and unanticipated outcomes and some of these are discussed. We are hopeful that the next several years will bring rapid advances to this field that will resolve past controversies and be clinically relevant.

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

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

Distinct sets of Rab6 effectors contribute to ZW10--and COG-dependent Golgi homeostasis.

The organization of the Golgi apparatus is determined in part by the interaction of Rab proteins and their diverse array of effectors. Here, we used multiple approaches to identify and characterize a small subset of effectors that mimicked the effects of Rab6 on Golgi ribbon organization. In a visual-based, candidate protein screen, we found that the individual depletion of any of three Rab6 effectors, myosin IIA (MyoIIA), Kif20A and Bicaudal D (BicD), was sufficient to suppress Golgi ribbon fragmentation/dispersal coupled to retrograde tether proteins in a manner paralleling Rab6. MyoIIA and Kif20A depletions were pathway selective and suppressed ZW10-dependent Golgi ribbon fragmentation/dispersal only whereas BicD depletion, like Rab6, suppressed both ZW10- and COG-dependent Golgi ribbon fragmentation. The MyoIIA effects could be produced in short-term assays by the reversible myosin inhibitor, blebbistatin. At the electron microscope level, the effects of BicD-depletion mimicked many of those of Rab6-depletion: longer and more continuous Golgi cisternae and a pronounced accumulation of coated vesicles. Functionally, BicD-depleted cells were inhibited in transport of newly synthesized VSV-G protein to the cell surface. In summary, our results indicate small, partially overlapping subsets of Rab6 effectors are differentially important to two tether-dependent pathways essential to Golgi organization and function.

Electron tomography reveals Rab6 is essential to the trafficking of trans-Golgi clathrin and COPI-coated vesicles and the maintenance of Golgi cisternal number.

We have shown previously that Rab6, a small, trans-Golgi-localized GTPase, acts upstream of the conserved oligomeric Golgi complex (COG) and ZW10/RINT1 retrograde tether complexes to maintain Golgi homeostasis. In this article, we present evidence from the unbiased and high-resolution approach of electron microscopy and electron tomography that Rab6 is essential to the trans-Golgi trafficking of two morphological classes of coated vesicles; the larger corresponds to clathrin-coated vesicles and the smaller to coat protein I (COPI)-coated vesicles. On the basis of the site of coated vesicle accumulation, cisternal dilation and the normal kinetics of cargo transport from the endoplasmic reticulum (ER) to Golgi followed by delayed Golgi to cell surface transport, we suggest that Golgi function in cargo transport is preferentially inhibited at the trans-Golgi/trans-Golgi network (TGN). The >50% increase in Golgi cisternae number in Rab6-depleted HeLa cells that we observed may well be coupled to the trans-Golgi accumulation of COPI-coated vesicles; depletion of the individual Rab6 effector, myosin IIA, produced an accumulation of uncoated vesicles with if anything a decrease in cisternal number. These results are the first evidence for a Rab6-dependent protein machine affecting Golgi-proximal, coated vesicle accumulation and probably transport at the trans-Golgi and the first example of concomitant cisternal proliferation and increased Golgi stack organization under inhibited transport conditions.

Live-cell assays to identify regulators of ER-to-Golgi trafficking.

We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (ß1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.

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.

Quantitative immunofluorescence mapping reveals little functional coclustering of proteins within platelet α-granules.

Platelets are small anucleate blood cells that aggregate to seal leaks at sites of vascular injury and are important in the pathology of atherosclerosis, acute coronary syndromes, rheumatoid arthritis, cancer, and the regulation of angiogenesis. In all cases, platelet aggregation requires release of stored proteins from α-granules. However, how proteins with potentially antagonistic functions are packaged within α-granules is controversial. One possibility is the packaging of functional agonists and antagonists into different α-granule populations. By quantitative immunofluorescence colocalization, we found that pair-wise comparisons of 15 angiogenic-relevant α-granule proteins displayed little, if any, pattern of functional coclustering. Rather, the data suggested a Gaussian distribution indicative of stochastic protein delivery to individual granules. The apparent physiologic paradox raised by these data may be explained through alternate mechanisms, such as differential content release through incomplete granule fusion or dampened and balanced regulatory networks brought about by the corelease of antagonistic factors.

Are Rab proteins the link between Golgi organization and membrane trafficking?

The fundamental separation of Golgi function between subcompartments termed cisternae is conserved across all eukaryotes. Likewise, Rab proteins, small GTPases of the Ras superfamily, are putative common coordinators of Golgi organization and protein transport. However, despite sequence conservation, e.g., Rab6 and Ypt6 are conserved proteins between humans and yeast, the fundamental organization of the organelle can vary profoundly. In the yeast Saccharomyces cerevisiae, the Golgi cisternae are physically separated from one another, while in mammalian cells, the cisternae are stacked one upon the other. Moreover, in mammalian cells, many Golgi stacks are typically linked together to generate a ribbon structure. Do evolutionarily conserved Rab proteins regulate secretory membrane trafficking and diverse Golgi organization in a common manner? In mammalian cells, some Golgi-associated Rab proteins function in coordination of protein transport and maintenance of Golgi organization. These include Rab6, Rab33B, Rab1, Rab2, Rab18, and Rab43. In yeast, these include Ypt1, Ypt32, and Ypt6. Here, based on evidence from both yeast and mammalian cells, we speculate on the essential role of Rab proteins in Golgi organization and protein transport.

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.

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.

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.

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.

Rab33b and Rab6 are functionally overlapping regulators of Golgi homeostasis and trafficking.

We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra-Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra-Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex- or Zeste White 10 (ZW10)-depletion induced disruption of the Golgi ribbon in HeLa cells. Moreover, efficient GTP-restricted Rab6 induced relocation of Golgi enzymes to the endoplasmic reticulum (ER) was Rab33b-dependent, but not vice versa, suggesting that the two Rabs act sequentially in an intra-Golgi Rab cascade. In support of this hypothesis, we found that overexpression of GTP-Rab33b induced the dissociation of Rab6 from Golgi membranes in vivo. In addition, the transport of Shiga-like toxin B fragment (SLTB) from the trans to cis Golgi and ER required Rab33b. Surprisingly, depletion of Rab33b had little, if any, immediate effect on cell growth and multiplication. Furthermore, anterograde trafficking of tsO45G protein through the Golgi apparatus was normal. We suggest that the Rab33b/Rab6 regulated intra-Golgi retrograde trafficking pathway must coexist with other Golgi trafficking pathways. In conclusion, we provide the first evidence that Rab33b and Rab6 act to coordinate a major intra-Golgi retrograde trafficking pathway. This coordination may have parallels with Rab conversion/cascade events that regulate endosome, phagosome and exocytic processes.