Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area.

Arabidopsis PLETHORA transcription factors control phyllotaxis.

The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1].

A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth.

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities.

VIS2FIX: a high-speed fixation method for immuno-electron microscopy.

Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h.

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing.

CHAP is a newly identified Z-disc protein essential for heart and skeletal muscle function.

In recent years, the perception of Z-disc function has changed from a passive anchor for myofilaments that allows transmission of force, to a dynamic multicomplex structure, capable of sensing and transducing extracellular signals. Here, we describe a new Z-disc protein, which we named CHAP (cytoskeletal heart-enriched actin-associated protein), expressed in differentiating heart and skeletal muscle in vitro and in vivo. Interestingly, in addition to its sarcomeric localization, CHAP was also able to translocate to the nucleus.

Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers, by TEM and FIB-SEM.

Aclar, a copolymer film with properties very similar to those of tissue culture plastic, is a versatile substrate to grow cells for light (including fluorescence) and electron microscopic applications in combination with both chemical fixation and cryoimmobilization. In this paper, we describe complete procedures to perform correlative light and electron microscopy using Aclar as substrate for the culture of cell monolayers to be finally embedded in plastic. First, we developed straightforward, efficient and flexible ways to mark the surface of the Aclar to create substrates to locate cells first at the light microscopy and then the electron microscopy level.

Spiral coating of the endothelial caveolar membranes as revealed by electron tomography and template matching.

Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3-D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high-pressure frozen, freeze-substituted and epon-embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises--as confirmed by 3-D immunolabeling for caveolin of 'intact' cells--bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi-caveolar bodies.

Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.

Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type-specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation.

Focused ion beam-scanning electron microscope: exploring large volumes of atherosclerotic tissue.

Atherogenesis is a pathological condition in which changes in the ultrastructure and in the localization of proteins occur within the vasculature during all stages of the disease. To gain insight in those changes, high-resolution imaging is necessary. Some of these changes will only be present in a small number of cells, positioned in a 'sea' of non-affected cells.

Cell division ring, a new cell division protein and vertical inheritance of a bacterial organelle in anammox planctomycetes.

Anammox bacteria are members of the phylum Planctomycetes that oxidize ammonium anaerobically and produce a significant part of the atmosphere's dinitrogen gas. They contain a unique bacterial organelle, the anammoxosome, which is the locus of anammox catabolism. While studying anammox cell and anammoxosome division with transmission electron microscopy including electron tomography, we observed a cell division ring in the outermost compartment of dividing anammox cells.

Golgi-associated cPLA2alpha regulates endothelial cell-cell junction integrity by controlling the trafficking of transmembrane junction proteins.

In endothelial cells specifically, cPLA2alpha translocates from the cytoplasm to the Golgi complex in response to cell confluence. Considering the link between confluence and cell-cell junction formation, and the emerging role of cPLA2alpha in intracellular trafficking, we tested whether Golgi-associated cPLA2alpha is involved in the trafficking of junction proteins. Here, we show that the redistribution of cPLA2alpha from the cytoplasm to the Golgi correlates with adherens junction maturation and occurs before tight junction formation.

Sol-gel transitions and liquid crystal phase transitions in concentrated aqueous suspensions of colloidal gibbsite platelets.

In this paper, we present a comprehensive study of the sol-gel transitions and liquid crystal phase transitions in aqueous suspensions of positively charged colloidal gibbsite platelets at pH 4-5 over a wide range of particle concentrations (50-600 g/L) and salt concentrations (10(-4)-10(-1) M NaCl). A detailed sol-gel diagram was established by oscillatory rheological experiments. These demonstrate the presence of kinetically arrested states both at high and at low salt concentrations, enclosing a sol region.

Septal pore complex morphology in the Agaricomycotina (Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales.

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina.

Membrane contact sites between apicoplast and ER in Toxoplasma gondii revealed by electron tomography.

Toxoplasma gondii is an obligate intracellular parasite from the phylum Apicomplexa. A hallmark of these protozoans is the presence of a unique apical complex of organelles that includes the apicoplast, a plastid acquired by secondary endosymbiosis. The apicoplast is indispensible for parasite viability.

Quantitative structural analysis of binary nanocrystal superlattices by electron tomography.

Binary nanocrystal superlattices, that is, ordered structures of two sorts of nanocolloids, hold promise for a series of functional materials with novel collective properties. Here we show that based on electron tomography a comprehensive, quantitative, three-dimensional characterization of these systems down to the single nanocrystal level can be achieved, which is key in understanding the emerging materials properties. On four binary lattices composed of PbSe, CdSe, and Au nanocrystals, we illustrate that ambiguous interpretations based on two-dimensional transmission electron microscopy can be prevented, nanocrystal sizes and superlattice parameters accurately determined, individual crystallographic point and plane defects studied, and the order/disorder at the top and bottom surfaces imaged.

Induced membrane domains as visualized by electron tomography and template matching.

Membranes play a crucial role in many cellular processes, and it is therefore not surprising that many electron tomographic studies in life sciences concern membranous structures. While these tomographic studies provide many new insights into membrane connections and continuities in three dimensions, they are mostly limited to a macro-morphological level. In this paper, we demonstrate that by combining electron tomography and three-dimensional template matching we are able to investigate membrane morphology at a new level: membrane domains in three dimensions.

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging.

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB-SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons.

Tannic acid-mediated osmium impregnation after freeze-substitution: a strategy to enhance membrane contrast for electron tomography.

In this technical note we report a tannic acid-mediated osmium impregnation method that, applied after freeze-substitution, increases membrane contrast in cells for transmission electron microscopy and tomography studies. The general staining that is achieved allows visualization of organelles, plasma membrane and associated specializations (e.g.

Cryo-electron tomography of mouse hepatitis virus: Insights into the structure of the coronavirion.

Coronaviruses are enveloped viruses containing the largest reported RNA genomes. As a result of their pleomorphic nature, our structural insight into the coronavirion is still rudimentary, and it is based mainly on 2D electron microscopy. Here we report the 3D virion structure of coronaviruses obtained by cryo-electron tomography.