Ultrathin Cryosections Research Articles

An optimized protocol for immuno-electron microscopy of endogenous LC3

ABSTRACT MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions. Abbreviations: BafA1: bafilomycin A1; BSA: bovine serum albumin; BSA-c: acetylated BSA; BSA5: BSA conjugated to 5-nm gold particles; CLEM: correlative light-electron microscopy; EGFP: enhanced green fluorescent protein; EM: electron microscopy; FBS: fetal bovine serum; FSG: fish skin gelatin; GA: glutaraldehyde; IF: immunofluorescence; LAMP1: lysosomal associated membrane protein 1; LC3s: LC3 proteins; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ON: overnight; PAG: protein A-conjugated gold particles; PAG1-3: PAG5, PAG10, PAG15, protein A conjugated to 1-3-, 5-, 10-, or 15-nm gold particles; PB: Sorensen’s phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RT: room temperature.

Cryo-ultramicrotomy and Mass Spectrometry Imaging Analysis of Nudibranch Microstructures.

In this paper, we investigate the presence of latrunculin A in the outer rim of a nudibranch Chromodoris kuiteri and show that by combining ultrathin cryosection methods with MALDI MSI we can achieve improved lateral (x and y) resolution and very high resolution in the z dimension by virtue of the ultrathin 200 nm thin cryosections. We also demonstrate that a post ionization laser increases sensitivity. Recent advances in MALDI source design have improved the lateral resolution (x and y) and sensitivity during MSI. Taken together, very high z resolution, from ultrathin sections, and improved lateral (x and y) resolution will allow for subcellular molecular imaging with the potential for subcellular 3D volume reconstruction.

Characterization of different oligomeric forms of CRISP2 in the perinuclear theca versus the fibrous tail structures of boar spermatozoa†

Mammalian sperm carry a variety of highly condensed insoluble protein structures such as the perinuclear theca, the fibrous sheath and the outer dense fibers, which are essential to sperm function. We studied the role of cysteine rich secretory protein 2 (CRISP2); a known inducer of non-pathological protein amyloids, in pig sperm with a variety of techniques. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High-resolution localization by immunogold labeling electron microscopy of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. Interestingly, we found that under native, non-reducing conditions CRISP2 formed oligomers both in the tail and the head but with different molecular weights and different biochemical properties. The tail oligomers were insensitive to reducing conditions but nearly complete dissociated into monomers under 8M urea treatment, while the head 250kDa CRISP2 positive oligomer completely dissociated into CRISP2 monomers under reducing conditions. The head specific dissociation of CRISP2 oligomer is likely a result of the reduction of various sulfhydryl groups in the cysteine rich domain of this protein. The sperm head CRISP2 shared typical solubilization characteristics with other perinuclear theca proteins as was shown with sequential detergent and salt treatments. Thus, CRISP2 is likely to participate in the formation of functional protein complexes in both the sperm tail and sperm head, but with differing oligomeric organization and biochemical properties. Future studies will be devoted to the understand the role of CRISP2 in sperm protein complexes formation and how this contributes to the fertilization processes.

Altered nuclear architecture in blood cells from Huntington's disease patients.

Cell nuclear architecture has been explored in cancer and laminopathies but not in neurodegenerative disorders. Huntington's disease (HD) is a neurodegenerative disorder that leads to neuronal death. Chromosome-wide changes in gene expression have been reported in HD, not only in the brain but also in peripheral blood cells, but whether this translates into nuclear and chromosome architecture alterations has not yet been studied. We investigate nuclear structure and chromosome organization in HD blood cells using fluorescence in situ hybridization in ultrathin cryosections (cryoFISH), coupled with machine learning image analysis to evaluate size, distribution, and morphology of nuclei and chromosomes. Four chromosomes were analyzed based on up- or downregulation of gene expression in HD. We show that blood cells from HD patients display increased nuclear size and filamentary shape, increased size of gene-rich chromosome 19, decreased filamentary shape of gene-rich chromosome 22, and a more radially centralized position for chromosome 19, whereas chromosomes 4 and 5 do not show detectable differences. We identify gross changes in nuclear architecture and chromosome organization associated with HD in blood. This adds a new layer of information onto disrupting mechanisms in HD and increases the potential of using blood to survey HD.

Comparison of Two Transmission Electron Microscopy Methods to Visualize Drug-Induced Alterations of Gram-Negative Bacterial Morphology.

In this study, we optimized and compared different transmission electron microscopy (TEM) methods to visualize changes to Gram-negative bacterial morphology induced by treatment with a robenidine analogue (NCL195) and colistin combination. Aldehyde-fixed bacterial cells (untreated, treated with colistin or NCL195 + colistin) were prepared using conventional TEM methods and compared with ultrathin Tokuyasu cryo-sections. The results of this study indicate superiority of ultrathin cryo-sections in visualizing the membrane ultrastructure of Escherichia coli and Pseudomonas aeruginosa, with a clear delineation of the outer and inner membrane as well as the peptidoglycan layer. We suggest that the use of ultrathin cryo-sectioning can be used to better visualize and understand drug interaction mechanisms on the bacterial cell membrane.

Various Nucleolar Stress Inducers Result in Highly Distinct Changes in Water, Dry Mass and Elemental Content in Cancerous Cell Compartments: Investigation Using a Nano-Analytical Approach.

Rationale: Numerous chemotherapeutic drugs that affect ribosome biogenesis in the nucleolus induce nucleolar stress. Improving our understanding of the effects of these drugs will require uncovering and comparing their impact on several biophysical parameters of the major cell compartments. Here, we quantified the water content and dry mass of cancerous cells treated with CX-5461, DRB or DAM to calculate macromolecular crowding and the volume occupied by free water, as well as elemental content.Methods: HeLa-H2B-GFP cells were treated with CX-5461, DRB or DAM. Water content and dry mass were measured in numerous regions of interest of ultrathin cryo-sections by quantitative scanning transmission electron microscope dark-field imaging and the elements quantified by energy dispersive X-ray spectrometry. The data were used to calculate macromolecular crowding and the volume occupied by free water in all cell compartments of control and treated cells. Hydrophobic and unfolded proteins were revealed by 8-Anilinonaphtalene-1-sulfonic acid (ANS) staining and imaging by two-photon microscopy. Immunolabeling of UBF, pNBS1 and pNF-κB was carried out and the images acquired with a confocal microscope for 3D imaging to address whether the localization of these proteins changes in treated cells.Results: Treatment with CX-5461, DRB or DAM induced completely different changes in macromolecular crowding and elemental content. Macromolecular crowding and elemental content were much higher in CX-5461-treated, moderately higher in DRB-treated, and much lower in DAM-treated cells than control cells. None of the drugs alone induced nucleolar ANS staining but it was induced by heat-shock of control cells and cells previously treated with DAM. UBF and pNBS1 were systematically co-localized in the nucleolus of CX-5461- and DAM-treated cells. pNF-κB only localized to the nucleolar caps of pre-apoptotic DAM-treated cells.Conclusion: We directly quantified water and ion content in cell compartments using cryo-correlative electron microscopy. We show that different chemotherapeutic nucleolar stress inducers result in distinctive, thus far-unrecognized changes in macromolecular crowding and elemental content which are known to modify cell metabolism. Moreover we were able to correlate these changes to the sensitivity of treated cells to heat-shock and the behavior of nucleolar pNBS1 and pNF-κB.

Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy.

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue.

The extended, dynamic mitochondrial reticulum in skeletal muscle and the creatine kinase (CK)/phosphocreatine (PCr) shuttle are working hand in hand for optimal energy provision.

The existence of a mitochondrial reticulum or network was postulated already in 1986 (Kirkwood et al. 1986) and the effect of endurance training on the fine structure of this very reticulum was documented in the following year (Kirkwood et al. 1987). A decade later the concept of mitochondrial networks in skeletal muscle definitely gained support by improved techniques, using a detailed ultra-structural analysis of ultrathin cryo-sections of human muscle by electron microscopy (Ogata and Yamasaki 1997). Now, in their letter on ‘‘Mitochondrial reticulum for cellular energy distribution in muscle’’ the authors Glancy et al. with Balaban (Nature 523:617–620, 2015) presented very elegant and technically brilliant work corroborating the existence of a mitochondrial reticulum network inside skeletal muscle fibers. In addition, these authors provide new evidence that this mitochondrial reticulum network provides a conductive pathway for energy distribution and helps to minimize diffusion distances for metabolites such as ATP. The authors propose that mitochondrial membrane potential conduction via this mitochondrial reticulum is the dominant pathway for energy distribution in skeletal muscle and that the phospho-creatine (PCr)/kinase creatine kinase (CK) shuttle (Bessman and Geiger 1981; Wallimann et al. 1992) would not be critical for normal muscle function. In their discussion it is stated, however, that CK would probably still provide a significant evolutionary advantage, justifying the retention or development of the system (Glancy et al. 2015). As a matter of fact, evolutionarily the phosphagen kinase system dates back several hundred millions of years to early metazoan CK (Uda et al. 2012) or even bacteria with arginine kinase (AK) (Suzuki et al. 2013). To support the interpretation of their data, the authors argue that the skeletal muscle phenotypes of CK knock-out or creatine(Cr)-deficiency mice are small with nearly normal muscle function and that only modest adaptations are seen in these transgenic animals. Unfortunately, both statements are incorrect. First, at a closer look, the phenotype of the CK double knock-out mouse is physiologically relevant in terms of muscle force and relaxation (Steeghs et al. 1997), and CK injection into CK-deficient skeletal muscle fibers restores contractile function and calcium handling (Dahlstedt et al. 2003) that are both distinctly disturbed in the CK double knock-outs lacking expression of cytosolic muscle MM-CK as well as mitochondrial mtCK (Steeghs et al. 1997; 1998). Even skeletal muscles of mice that are deficient only in cytosolic MM-CK clearly lack muscle burst activity (van Deursen et al. 1993) and down-regulated expression of MM-CK in skeletal muscle of transgenic mice leads to a correspondingly lowered ability to perform muscle contractile burst activity that closely correlates with the level of MM-CK expression (Van Deursen et al. 1994). Second, the adaptation changes observed in skeletal muscle due to abrogation of the CK system are astonishing, e.g. the mitochondrial content in fast-type glycolytic muscles (normally devoid of intra-myofibrillar mitochondria) of these transgenic animals is highly up-regulated and the appearance of these transformed muscles lacking CK rather resemble oxidative insect endurance flight muscles with a very high mitochondrial content and a vast propensity of intra-myofibrillar mitochondria (van Deursen et al. 1993, see Fig. 2; and Steeghs et al. 1997, see Fig. 4) with a corresponding mitochondrial network. Most relevant with respect to muscle pathology is the observed formation & Theo Wallimann theo.wallimann@cell.biol.ethz.ch

Imaging Drosophila brain by combining cryo-soft X-ray microscopy of thick vitreous sections and cryo-electron microscopy of ultrathin vitreous sections.

Cryo-soft X-ray microscopy is an emerging imaging tool complementary to cryo-electron microscopy, allowing to image frozen hydrated specimens ten to hundred times thicker, but at lower resolution. We describe how the method, so far restricted to isolated small cells or cell monolayers, can be extended to large cells and tissue. We image the synapses of the Kenyon cells in frozen hydrated Drosophila brains combining cryo-soft X-ray microscopy of thick vitreous sections, and cryo-electron microscopy of ultrathin vitreous sections. We show how to obtain frozen hydrated sections of thicknesses ranging from 40 nm up to 2.5 μm, by tuning the sectioning speed of the cryo-microtome. A fluorescent stereo-microscope mounted on the cryo-microtome allowed us to target the regions of interest after GFP-labeling of synapses. Thick cryo-sections were imaged by cryo-soft X-ray microscopy at a resolution better than 25 nm, while ultrathin cryo-sections of the same regions were explored in parallel at the nanometre level of resolution by cryo-electron microscopy.

Targeted nano analysis of water and ions in the nucleus using cryo-correlative microscopy.

The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.

Therapeutic Efficacy of Stable Analogues of Vasoactive Intestinal Peptide against Pathogens

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide recently identified as a potential antimicrobial peptide. To overcome the metabolic limitations of VIP, we modified the native peptide sequence and generated two stable synthetic analogues (VIP51 and VIP51(6-30)) with better antimicrobial profiles. Herein we investigate the effects of both VIP analogues on cell viability, membrane integrity, and ultrastructure of various bacterial strains and Leishmania species. We found that the two VIP derivatives kill various non-pathogenic and pathogenic Gram-positive and Gram-negative bacteria as well as the parasite Leishmania major through a mechanism that depends on the interaction with certain components of the microbial surface, the formation of pores, and the disruption of the surface membrane. The cytotoxicity of the VIP derivatives is specific for pathogens, because they do not affect the viability of mammalian cells. Docking simulations indicate that the chemical changes made in the analogues are critical to increase their antimicrobial activities. Consequently, we found that the native VIP is less potent as an antibacterial and fails as a leishmanicidal. Noteworthy from a therapeutic point of view is that treatment with both derivatives increases the survival and reduces bacterial load and inflammation in mice with polymicrobial sepsis. Moreover, treatment with VIP51(6-30) is very effective at reducing lesion size and parasite burden in a model of cutaneous leishmaniasis. These results indicate that the VIP analogues emerge as attractive alternatives for treating drug-resistant infectious diseases and provide key insights into a rational design of novel agents against these pathogens.

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis

In this article the tools, techniques, and instruments appropriate for quantitative measurements of intracellular elemental content using the technique known as electron probe microanalysis (EPMA) are described. Intramitochondrial calcium is a particular focus because of the critical role that mitochondrial calcium overload plays in neurodegenerative diseases. The method is based on the analysis of X-rays generated in an electron microscope (EM) by interaction of an electron beam with the specimen. In order to maintain the native distribution of diffusible elements in electron microscopy specimens, EPMA requires "cryofixation" of tissue followed by the preparation of ultrathin cryosections. Rapid freezing of cultured cells or organotypic slice cultures is carried out by plunge freezing in liquid ethane or by slam freezing against a cold metal block, respectively. Cryosections nominally 80 nm thick are cut dry with a diamond knife at ca. -160 °C, mounted on carbon/pioloform-coated copper grids, and cryotransferred into a cryo-EM using a specialized cryospecimen holder. After visual survey and location mapping at ≤-160 °C and low electron dose, frozen-hydrated cryosections are freeze-dried at -100 °C for ~30 min. Organelle-level images of dried cryosections are recorded, also at low dose, by means of a slow-scan CCD camera and subcellular regions of interest selected for analysis. X-rays emitted from ROIs by a stationary, focused, high-intensity electron probe are collected by an energy-dispersive X-ray (EDX) spectrometer, processed by associated electronics, and presented as an X-ray spectrum, that is, a plot of X-ray intensity vs. energy. Additional software facilitates: 1) identification of elemental components by their "characteristic" peak energies and fingerprint; and 2) quantitative analysis by extraction of peak areas/background. This paper concludes with two examples that illustrate typical EPMA applications, one in which mitochondrial calcium analysis provided critical insight into mechanisms of excitotoxic injury and another that revealed the basis of ischemia resistance.

Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes

Superoxide dismutases (SODs) are metalloenzymes that belong to the essential antioxidant enzyme systems of virtually all oxygen-respiring organisms. SODs catalyze the dismutation of highly reactive superoxide radicals into hydrogen peroxide and molecular oxygen. For the subcellular localization of the manganese superoxide dismutase (SOD2) in eukaryotic cells, a dual mitochondrial localization and peroxisomal localization were proposed in the literature. However, our own observation from immunofluorescence preparations of human and mouse tissues suggested that SOD2 serves as an excellent marker protein for mitochondria but never co-localized with peroxisomes. To clarify whether our observations were correct, we have carefully reinvestigated the subcellular localization of SOD2 using sensitive double-immunofluorescence methods on frozen and paraffin sections as well as in cell culture preparations. In addition, ultrastructural analyses were performed with post-embedding immunoelectron microscopy on LR White sections as well as labeling of ultrathin cryosections with various immunogold techniques. In all morphological experiments, the SOD2 localization was compared to one of the catalase, a typical marker protein for peroxisomes, solely localized in these organelles. Moreover, biochemical subcellular fractions of mouse liver was used to isolate enriched organelles and highly purified peroxisomal fractions for Western blot analyses of the exact subcellular distributions of SOD2 and catalase. All results with the various methodologies, tissues, and cell types used revealed that catalase and SOD2 were always confined to distinct and separate subcellular compartments. SOD2 was unequivocally in mitochondria, but never present in peroxisomes. Furthermore, our results are supported by accumulating database information on organelle proteomes that also indicate that SOD2 is a pure mitochondrial protein.

Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach.

The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5%, respectively. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83% (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128-462mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.

Supramolecular Architecture of Rhodopsin in Native Photoreceptors Revealed by Cryo-Electron Tomography

Vision begins with the absorption of photons by rhodopsin, the visual pigment in photoreceptors. Rhodopsin belongs to the family of G protein-coupled receptors (GPCRs). Some family members form functional dimers or oligomers. Whether rhodopsin forms oligomers and whether these oligomers are functionally relevant is controversial 1-3. We study rhodopsin organization in vitreous ultrathin cryo-sections of intact, dark-adapted photoreceptors by cryo-electron tomography. This technique ascertains that rhodopsin is preserved in a close-to-native state. Briefly: retina is fixed by high-pressure freezing, ultra-thin sectioned and visualized by cryo-electron tomography. In the reconstructed and processed tomograms the organization of rhodopsin molecules becomes visible. We identify three levels of hierarchical supramolecular organization. Rhodopsin forms dimers; the dimers form rows; and rows come in special pairs like rail tracks. Rows are aligned parallel to the disk incisure. A row is comprised of at least 10 dimers and is about 50 nm in length. The distance between rows within a track is 5 nm; and tracks are separated by 15 nm. We propose that rhodopsin tracks provide a template that organizes the spatio-temporal interaction of preassembled signalling components on the disk surface. Aligned rows of immobile rhodopsin renders photoreceptors highly dichroic and might provide the structural basis for detection of polarized light. We envisage that rhodopsin-like type A GPCRs, which are highly homologous, also entertain a supramolecular organization.References1 Chabre, M., Cone, R. & Saibil, H. R. Biophysics - is rhodopsin dimeric in native rods? Nature 426, 30-31 (2003).2 Chabre, M. & le Maire, M. Monomeric G-protein-coupled receptor as a functional unit. Biochemistry 44, 9395-94O3 (2005).3 Fotiadis, D. et al. Atomic-force microscopy: rhodopsin dimers in native disc membranes. Nature 421, 127-128 (2003).

Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Tu1133 Ultrastructural Localization of Claudin-3 in the Rat Esophageal Stratified Epithelium Under Normal Conditions and Chronic Reflux Esophagitis

Background and Aim The role of the mucosal barrier on the esophagus has not been fully clarified. Tight junction (TJ) and Claudin are considered as one of the mucosal barrier of the esophagus. We have studied the protein expression of Claudin which composed of TJ using experimental model of the rat reflux esophagitis(RE). We suggested that Claudin-3 was one of the key TJ barrier proteins and alterations in Claudin-3 proteins might change TJ structures in the rat RE model (Asaoka D et al. J Gastroenterol 2005, Oguro M et al. J Gastroenterol 2011). However, the detailed structural change of TJ has not been investigated. The aim of this study was to explore the relationship between the morphological TJ structure and Claudin-3 protein in the rat esophageal epithelium in the control and RE model using electron microscopy. Materials and Methods Chronic acid RE model was prepared in male Wister rats by the method (Asaoka D et al. J Gastroenterol 2005), and Sham operation was implemented to the controls. Two weeks after the operation, the rats were sacrificed. After their esophagus was removed and fixed, the following studies were performed; 1) electron microscopy observation 2) freeze fracture electron microscopy observation 3) immunoelectron microscopy observation with anti-Claudin 3 antibody. Especially, we observed the samples in detail using the modified grid-mapped freeze fracture technique instead of the conventional freeze fracture method to prevent the occurrence of the fragmentation and specify the location of the fracture plane. Briefly, a gold finder grid was bonded to the replicated tissue by using 2% polycarbonate dissolved in dichloroethane. The polycarbonatestabilized samples were immersed in PBS and removed from the gold specimen disk. The samples were then stained with DAPI and mapped by using the confocal microscope. Results We observed TJ-related structures in the granular layer and spinous layer in the esophageal epithelia of both groups of the rats using transmission electron microscopy. However, no typical strands were observed by freeze fracture electron microscopy. The specimen wasn't stained with Claudin-3 in both the Sham and RE model rats by immunoelectron microscopy of ultrathin cryosections. Conclusion Claudin-3 protein wasn't involved in the composition of TJ structure. This result indicates that claudin-3 protein may play the role in the maintenance of the mucosal barrier in the different area from where TJ complex exists.

Close Association of Aquaporin-2 Internalization with Caveolin-1

Aquaporin 2 (AQP2) is a membrane water channel protein that traffics between the intracellular membrane compartment and the plasma membrane in a vasopressin-dependent manner in the renal collecting duct cell to control the amount of water reabsorption. We examined the relation between AQP2 internalization from the plasma membrane and caveolin-1, which is a major protein in membrane microdomain caveolae, in Mardin-Darby canine kidney cells expressing human AQP2 (MDCK-hAQP2 cells). Double-immunofluorescence microscopy showed that AQP2 is colocalized with caveolin-1 in the apical plasma membrane by stimulating the intracellular signaling cascade of vasopressin with forskolin. After washing forskolin, both AQP2 and caveolin-1 were internalized to early endosomes and then separately went back to their individual compartments, which are subapical compartments and the apical membrane, respectively.Double-immunogold electron microscopy in ultrathin cryosections confirmed the colocalization of AQP2 with caveolin-1 at caveolar structures on the apical plasma membrane of forskolin-treated cells and the colocalization within the same intracellular vesicles after washing forskolin. A co-immunoprecipitation experiment showed the close interaction between AQP2 and caveolin-1 in forskolin-treated cells and in cells after washing forskolin. These results suggest that a caveolin-1-dependent and possibly caveolar-dependent pathway is a candidate for AQP2 internalization in MDCK cells.