Author Correction: FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new insight into the three-dimensional architecture of the Golgi complex: Characterization of unusual structures in epididymal principal cells.

Principal epididymal cells have one of the largest and more developed Golgi complex of mammalian cells. In the present study, we have used this cell as model for the study of the three-dimensional architecture of the Golgi complex of highly secretory and endocytic cells. Electron tomography demonstrated the presence in this cell type of some unknown or very unusual Golgi structures such as branched cisternae, pocket-like cisternal invaginations or tubular connections. In addition, we have used this methodology and immunoelectron microscopy to analyze the close relationship between this organelle and both the endoplasmic reticulum and microtubules, and to describe in detail how these elements interact with compact and non-compact regions of the ribbon.

FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42.

The Rho-family small GTPase Cdc42 localizes at plasma membrane and Golgi complex and aside from protrusion and migration operates in vesicle trafficking, endo- and exocytosis as well as establishment and/or maintenance of cell polarity. The formin family members FMNL2 and -3 are actin assembly factors established to regulate cell edge protrusion during migration and invasion. Here we report these formins to additionally accumulate and function at the Golgi apparatus. As opposed to lamellipodia, Golgi targeting of these proteins required both their N-terminal myristoylation and the interaction with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore, absence of these proteins led to enlargement of endosomes as well as defective maturation and/or sorting into late endosomes and lysosomes. In line with Cdc42 - recently established to regulate anterograde transport through the Golgi by cargo sorting and carrier formation - FMNL2/3 depletion also affected anterograde trafficking of VSV-G from the Golgi to the plasma membrane. Our data thus link FMNL2/3 formins to actin assembly-dependent functions of Cdc42 in anterograde transport through the Golgi apparatus.

Golgi apparatus dis- and reorganizations studied with the aid of 2-deoxy-D-glucose and visualized by 3D-electron tomography.

We studied Golgi apparatus disorganizations and reorganizations in human HepG2 hepatoblastoma cells by using the nonmetabolizable glucose analogue 2-deoxy-D-glucose (2DG) and analyzing the changes in Golgi stack architectures by 3D-electron tomography. Golgi stacks remodel in response to 2DG-treatment and are replaced by tubulo-glomerular Golgi bodies, from which mini-Golgi stacks emerge again after removal of 2DG. The Golgi stack changes correlate with the measured ATP-values. Our findings indicate that the classic Golgi stack architecture is impeded, while cells are under the influence of 2DG at constantly low ATP-levels, but the Golgi apparatus is maintained in forms of the Golgi bodies and Golgi stacks can be rebuilt as soon as 2DG is removed. The 3D-electron microscopic results highlight connecting regions that interlink membrane compartments in all phases of Golgi stack reorganizations and show that the compact Golgi bodies mainly consist of continuous intertwined tubules. Connections and continuities point to possible new transport pathways that could substitute for other modes of traffic. The changing architectures visualized in this work reflect Golgi stack dynamics that may be essential for basic cell physiologic and pathologic processes and help to learn, how cells respond to conditions of stress.

2-Deoxy-D-glucose treatment changes the Golgi apparatus architecture without blocking synthesis of complex lipids.

The classic Golgi apparatus organization, an arrangement of highly ordered cisternal stacks with tubular-vesicular membrane specializations on both sides, is the functional image of a continuous flow of contents and membranes with input, metabolization, and output in a dynamic steady state. In response to treatment with 2-deoxy-D-glucose (2-DG), which lowers the cellular ATP level by about 70% within minutes, this organization is rapidly replaced by tubular-glomerular membrane convolutes described as Golgi networks and bodies. 2-DG is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis, which has become attractive in the context of therapeutic approaches for several kinds of tumors specifically targeting glycolysis in cancer. With the question of whether the functions of the Golgi apparatus in lipid synthesis would be influenced by the 2-DG-induced Golgi apparatus reorganization, we focused on lipid metabolism within the Golgi bodies. For this, we applied a fluorophore-labeled short-chain ceramide (BODIPY-Cer) in various combinations with 2-DG treatment to HepG2 cell cultures and followed uptake, enrichment and metabolization to higher ordered lipids. The cellular ATP status in each experiment was controlled with a bioluminescence assay, and the response of the Golgi apparatus was tracked by immunostaining of the trans-Golgi network protein TGN46. For electron microscopy, the fluorescent BODIPY-Cer signals were converted into electron-dense precipitates by photooxidation of diaminobenzidine (DAB); DAB precipitates labeled trans-Golgi areas in control cultures but also compartments at the periphery of the Golgi bodies formed in response to 2-DG treatment, thus indicating that concentration of ceramide takes place in spite of the Golgi apparatus reorganization. Lipid analyses by thin-layer chromatography (TLC) performed in parallel showed that BODIPY-Cer is not only concentrated in compartments of the 2-DG-induced Golgi bodies but is partly metabolized to BODIPY-sphingomyelin. Both, uptake and condensation of BODIPY-Cer and its conversion to complex lipids indicate that functions of the Golgi apparatus in the cellular lipid metabolism persist although the classic Golgi apparatus organization is abolished.

Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae.

The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression-maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.

Viewing Golgi structure and function from a different perspective--insights from electron tomography.

Historically, ultrastructural investigations, which have focused on elucidating the biological idiosyncrasies of the Golgi apparatus, have tended towards oversimplified or fallacious hypotheses when postulating how the Golgi apparatus reorganizes itself both structurally and functionally to fulfill the plethora of cellular processes underpinned by this complex organelle. Key questions are still unanswered with regard to how changes in Golgi architecture correlate so reproducibly to changes in its functional priorities under different physiological conditions or experimental perturbations. This fact alone serves to highlight how the technical limitations associated with conventional two-dimensional imaging approaches employed in the past failed to adequately capture the extraordinary complexity of the Golgi's three-dimensional (3D) structure-now a hallmark of this challenging organelle. Consequently, this has hampered progress towards developing a clear understanding of how changes in its structure and function typically occur in parallel. In this chapter, we highlight but a few of the significant new insights regarding variations in the Golgi's structure-function relationships that have been afforded over recent years through advanced electron microscopic techniques for 3D image reconstruction, commonly referred to as electron tomography.

High-density lipoprotein endocytosis in endothelial cells.

AIM: To describe the way stations of high-density lipoprotein (HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescence microscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type I mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrin-coated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

Monitoring of platelet activation in platelet concentrates using transmission electron microscopy.

OBJECTIVE: The quality of platelet concentrates (PC) is important for the in vivo recovery of thrombostasis in patients suffering from bleeding disorders and in tumor patients after chemotherapy. In this respect, activated platelets (PLT) cannot display their full functionality in the recipient and even can cause adverse effects. Therefore, we developed a transmission electron microscopy (TEM) method for quality assessment of PC. METHODS: Score values taken from panorama TEM images describe the progress of PLT activation. To exemplify this method, i) 19 apheresis PC isolated with the Baxter Amicus system (BA) were compared with 14 PC obtained from pooled buffy coats (BC). ii) The score values of 33 PC derived from BA as well from BC were compared with flow-cytometric CD62P determinations by cross correlation. iii) Changes in the score value profiles during storage of a single pathogen-reduced BA PC were monitored over a period of 7 days. RESULTS: The TEM evaluation described allows for demonstrating particular PLT activation stages. i) Significant differences between the percentages of the score values 0, 1 and 2 could be demonstrated in both processing groups. No significant differences were found comparing these two groups. ii) A weak correlation could be shown when comparing the percentages of score values 2 plus 3 with the percentage of CD62P-positive PLT. iii) The pathogen reduction affected slightly the score profiles during storage due to an increase of dead PLT. CONCLUSION: Our investigations demonstrate the unique detailed quality information of PC obtained by the TEM method. This method can be performed in every routine electron microscopy laboratory.

Electron microscopy of endocytic pathways.

Detailed insight into the fine structure and 3D-architecture of the complex and dynamic compartments of the endocytic system is essential for a morpho-functional analysis of retrograde traffic from the cell surface to different intracellular destinations. Here, we describe a cytochemical approach for electron microscopic exploration of endocytic pathways with the use of wheat germ agglutinin (WGA) in combination with either conventional chemical fixation or ultrafast physical fixation of the cells by high pressure-freezing. Horseradish peroxidase-labeled WGA endocytozed by human hepatoma cells for various periods of time served as a marker. Its intracellular routes were visualized by means of diaminobenzidine oxidation either done conventionally after chemical fixation or in living cells prior to physical fixation. The latter protocol permits the combination of peroxidase-catalyzed cytochemistry with high pressure-freezing (HPF), which is state of the art for ultrastructural studies of complex and dynamic organelles at high spatial and temporal resolutions. The technique yields distinct cytochemical reactions and excellently preserved fine structures well qualified for detailed electron microscopic and 3D-studies of the complex endocytic architectures.

Photooxidation technology for correlative light and electron microscopy.

Correlative microscopic approaches combine the advantages of both light and electron microscopy. Here we show a correlative approach that uses the photooxidation capacity of fluorescent dyes. Through illumination with high energetic light, the chromogen diaminobenzidine is oxidized and stable deposits are formed at the sites of the former fluorescent signals, which after osmification are then visible in the electron microscope. The potential of the method is illustrated by tracing the endocytic pathway of three different ligands: the lipid ceramide, high density lipoproteins, and the lectin wheat germ agglutinin. The ligands were labeled either with BODIPY or Alexa dyes. Following cell surface binding, uptake, and time-dependent intracellular progression, the route taken by these molecules together with the organelles that have been visited is characterized. Correlative microscopic data are recorded at various levels. First, by fluorescence and phase contrast illumination with the light microscope, followed by the analysis of semithin sections after photooxidation, and finally of thin sections at the ultrastructural level.

Human endothelial progenitor cells internalize high-density lipoprotein.

Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular pathway and accumulated prominently in all parts of the Golgi apparatus and in lipid droplets. Subsequently, also the RER and mitochondria were involved. These studies demonstrated the different intracellular pathway of HDL-derived bodipy-cholesterol and HDL-derived bodipy-cholesteryl oleate by EPCs, with concomitant.

Electron tomography of fusiform vesicles and their organization in urothelial cells.

The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 µm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4-15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.

Structural organization of the presynaptic density at identified synapses in the locust central nervous system.

In a synaptic active zone, vesicles aggregate around a densely staining structure called the presynaptic density. We focus on its three-dimensional architecture and a major molecular component in the locust. We used electron tomography to study the presynaptic density in synapses made in the brain by identified second-order neuron of the ocelli. Here, vesicles close to the active zone are organized in two rows on either side of the presynaptic density, a level of organization not previously reported in insect central synapses. The row of vesicles that is closest to the density's base includes vesicles docked with the presynaptic membrane and thus presumably ready for release, whereas the outer row of vesicles does not include any that are docked. We show that a locust ortholog of the Drosophila protein Bruchpilot is localized to the presynaptic density, both in the ocellar pathway and compound eye visual neurons. An antibody recognizing the C-terminus of the Bruchpilot ortholog selectively labels filamentous extensions of the presynaptic density that reach out toward vesicles. Previous studies on Bruchpilot have focused on its role in neuromuscular junctions in Drosophila, and our study shows it is also a major functional component of presynaptic densities in the central nervous system of an evolutionarily distant insect. Our study thus reveals Bruchpilot executes similar functions in synapses that can sustain transmission of small graded potentials as well as those relaying large, spike-evoked signals.

Combined light and electron microscopy using diaminobenzidine photooxidation to monitor trafficking of lipids derived from lipoprotein particles.

Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.

Urothelial plaque formation in post-Golgi compartments.

Urothelial plaques are specialized membrane domains in urothelial superficial (umbrella) cells, composed of highly ordered uroplakin particles. We investigated membrane compartments involved in the formation of urothelial plaques in mouse umbrella cells. The Golgi apparatus did not contain uroplakins organized into plaques. In the post-Golgi region, three distinct membrane compartments containing uroplakins were characterized: i) Small rounded vesicles, located close to the Golgi apparatus, were labelled weakly with anti-uroplakin antibodies and they possessed no plaques; we termed them "uroplakin-positive transporting vesicles" (UPTVs). ii) Spherical-to-flattened vesicles, termed "immature fusiform vesicles" (iFVs), were uroplakin-positive in their central regions and contained small urothelial plaques. iii) Flattened "mature fusiform vesicles" (mFVs) contained large plaques, which were densely labelled with anti-uroplakin antibodies. Endoytotic marker horseradish peroxidase was not found in these post-Golgi compartments. We propose a detailed model of de novo urothelial plaque formation in post-Golgi compartments: UPTVs carrying individual 16-nm particles detach from the Golgi apparatus and subsequently fuse into iFV. Concentration of 16-nm particles into plaques and removal of uroplakin-negative membranes takes place in iFVs. With additional fusions and buddings, iFVs mature into mFVs, each carrying two urothelial plaques toward the apical surface of the umbrella cell.

The ceramide-enriched trans-Golgi compartments reorganize together with other parts of the Golgi apparatus in response to ATP-depletion.

In this study, the ceramide-enriched trans-Golgi compartments representing sites of synthesis of sphingomyelin and higher organized lipids were visualized in control and ATP-depleted hepatoma and endothelial cells using internalization of BODIPY-ceramide and the diaminobenzidine photooxidation method for combined light-electron microscopical exploration. Metabolic stress induced by lowering the cellular ATP-levels leads to reorganizations of the Golgi apparatus and the appearance of tubulo-glomerular bodies and networks. The results obtained with three different protocols, in which BODIPY-ceramide either was applied prior to, concomitantly with, or after ATP-depletion, revealed that the ceramide-enriched compartments reorganize together with other parts of the Golgi apparatus under these conditions. They were found closely associated with and integrated in the tubulo-glomerular bodies formed in response to ATP-depletion. This is in line with the changes of the staining patterns obtained with the Helix pomatia lectin and the GM130 and TGN46 immuno-reactions occurring in response to ATP-depletion and is confirmed by 3D electron tomography. The 3D reconstructions underlined the glomerular character of the reorganized Golgi apparatus and demonstrated continuities of ceramide positive and negative parts. Most interestingly, BODIPY-ceramide becomes concentrated in compartments of the tubulo-glomerular Golgi bodies, even though the reorganization took place before BODIPY-ceramide administration. This indicates maintained functionalities although the regular Golgi stack organization is abolished; the results provide novel insights into Golgi structure-function relationships, which might be relevant for cells affected by metabolic stress.

High-pressure freezing combined with in vivo-DAB-cytochemistry: a novel approach for studies of endocytic compartments.

Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.