Gold-substituted Silver-intensified Peroxidase Immunolabeling for FIB-SEM Imaging.

Modern electron microscopy offers a wide variety of tools to investigate the ultrastructural organization of cells and tissues and to accurately pinpoint intracellular localizations of macromolecules of interest. New volumetric electron microscopy techniques and new instrumentation provide unique opportunities for high-throughput analysis of comparatively large volumes of tissue and their complete reconstitution in three-dimensional (3D) electron microscopy. However, due to a variety of technical issues such as the limited penetration of label into the tissue, low antigen preservation, substantial electron density of secondary detection reagents, and many others, the adaptation of immuno-detection techniques for use with such 3D imaging methods as focused ion beam-scanning electron microscopy (FIB-SEM) has been challenging. Here, we describe a sample preparation method for 3D FIB-SEM, which results in an optimal preservation and staining of ultrastructural details at a resolution necessary for tracing immunolabeled neuronal structures and detailed reconstruction of synapses. This technique is applicable to neuronal and non-neuronal cells, tissues, and a wide variety of antigens.

Prolonged cigarette smoke exposure alters mitochondrial structure and function in airway epithelial cells.

BACKGROUND: Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis. METHODS: We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls. RESULTS: We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1ß. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1. CONCLUSION: The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

Surface characteristics of nanoparticles determine their intracellular fate in and processing by human blood-brain barrier endothelial cells in vitro.

A polarized layer of endothelial cells that comprises the blood-brain barrier (BBB) precludes access of systemically administered medicines to brain tissue. Consequently, there is a need for drug delivery vehicles that mediate transendothelial transport of such medicines. Endothelial cells use a variety of endocytotic pathways for the internalization of exogenous materials, including clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis. The different modes of endocytosis result in the delivery of endocytosed material to distinctive intracellular compartments and therewith correlated differential processing. To obtain insight into the properties of drug delivery vehicles that direct their intracellular processing in brain endothelial cells, we investigated the intracellular processing of fixed-size nanoparticles in an in vitro BBB model as a function of distinct nanoparticle surface modifications. Caveolar endocytosis, adsorptive-mediated endocytosis, and receptor-mediated endocytosis were promoted by the use of uncoated 500-nm particles, attachment of the cationic polymer polyethyleneimine (PEI), and attachment of prion proteins, respectively. We demonstrate that surface modifications of nanoparticles, including charge and protein ligands, affect their mode of internalization by brain endothelial cells and thereby their subcellular fate and transcytotic potential.

Formation of E-cadherin/beta-catenin-based adherens junctions in hepatocytes requires serine-10 in p27(Kip1).

The adhesion between epithelial cells at adherens junctions is regulated by signaling pathways that mediate the intracellular trafficking and assembly of its core components. Insight into the molecular mechanisms of this is necessary to understand how adherens junctions contribute to the functional organization of epithelial tissues. Here, we demonstrate that in human hepatic HepG2 cells, oncostatin M-p42/44 mitogen-activated protein kinase signaling stimulates the phosphorylation of p27(Kip1) on Ser-10 and promotes cell-cell adhesion. The overexpression of wild-type p27 or a phospho-mimetic p27S10D mutant in HepG2 cells induces a hyper-adhesive phenotype. In contrast, the overexpression of a nonphosphorylatable p27S10A mutant prevents the mobilization of E-cadherin and beta-catenin at the cell surface, reduces basal cell-cell adhesion strength, and prevents the stimulatory effect of oncostatin M on cell-cell adhesion. As part of the underlying molecular mechanism, it is shown that in p27S10A-expressing cells beta-catenin interacts with p27 and is prevented from interacting with E-cadherin. The intracellular retention of E-cadherin and beta-catenin is also observed in hepatocytes from p27S10A knockin mice that express the p27S10A mutant instead of wild-type p27. Together, these data suggest that the formation of adherens junctions in hepatocytes requires Ser-10 in p27.

Cell polarity development and protein trafficking in hepatocytes lacking E-cadherin/beta-catenin-based adherens junctions.

Using a mutant hepatocyte cell line in which E-cadherin and beta-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical-basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical-basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin-based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin-based adherens junctions. Together, our data show that E-cadherin/beta-catenin-based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/beta-catenin-based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.

Adhesion receptors mediate efficient non-viral gene delivery.

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties. Here we demonstrate that transfection of epithelial cells with lipoplexes is almost exclusively mediated by the beta1 integrin cell surface receptor. More important, we show that in general, adhesion receptors can be exploited by lipoplexes to gain access to cells, including difficult-to-transfect primary neural stem cells and suspension cells, thereby leading to productive transfection. We propose that adhesion receptors serve as "natural" receptors for lipoplexes. As no natural cellular receptors for lipoplexes have previously been identified, our results are an important step forward in understanding the mechanisms of non-viral gene delivery. Moreover, the finding that adhesion receptors mediate efficient non-viral gene delivery paves the way for the optimization of (standard) transfection procedures as well as ex vivo gene therapy protocols using non-viral vectors.

The dynamic developmental localization of the full-length corticotropin-releasing factor receptor type 2 in rat cerebellum.

Corticotropin releasing factor receptor 2 (CRF-R2) is strongly expressed in the cerebellum and plays an important role in the development of the cerebellar circuitry, particularly in the development of the dendritic trees and afferent input to Purkinje cells. However, the mechanisms responsible for the distribution and stabilization of CRF-R2 in the cerebellum are not well understood. Here, we provide the first detailed analysis of the cellular localization of the full-length form of CRF-R2 in rat cerebellum during early postnatal development. We document unique and developmentally regulated subcellular distributions of CRF-R2 in cerebellar cell types, e.g. granule cells after postnatal day 15. The presence of one or both receptor isoforms in the same cell may provide a molecular basis for distinct developmental processes. The full-length form of CRF-R2 may be involved in the regulation of the first stage of dendritic growth and at later stages in the controlling of the structural arrangement of immature cerebellar circuits and in the autoregulatory pathway of the cerebellum.

Rho kinase, myosin-II, and p42/44 MAPK control extracellular matrix-mediated apical bile canalicular lumen morphogenesis in HepG2 cells.

The molecular mechanisms that regulate multicellular architecture and the development of extended apical bile canalicular lumens in hepatocytes are poorly understood. Here, we show that hepatic HepG2 cells cultured on glass coverslips first develop intercellular apical lumens typically formed by a pair of cells. Prolonged cell culture results in extensive organizational changes, including cell clustering, multilayering, and apical lumen morphogenesis. The latter includes the development of large acinar structures and subsequent elongated canalicular lumens that span multiple cells. These morphological changes closely resemble the early organizational pattern during development, regeneration, and neoplasia of the liver and are rapidly induced when cells are cultured on predeposited extracellular matrix (ECM). Inhibition of Rho kinase or its target myosin-II ATPase in cells cultured on glass coverslips mimics the morphogenic response to ECM. Consistently, stimulation of Rho kinase and subsequent myosin-II ATPase activity by lipoxygenase-controlled eicosatetranoic acid metabolism inhibits ECM-mediated cell multilayering and apical lumen morphogenesis but not initial apical lumen formation. Furthermore, apical lumen remodeling but not cell multilayering requires basal p42/44 MAPK activity. Together, the data suggest a role for hepatocyte-derived ECM in the spatial organization of hepatocytes and apical lumen morphogenesis and identify Rho kinase, myosin-II, and MAPK as potentially important players in different aspects of bile canalicular lumen morphogenesis.

CRF and urocortin differentially modulate GluRdelta2 expression and distribution in parallel fiber-Purkinje cell synapses.

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density. However, only UCN treatment results in increased delta2 protein level per Purkinje cell, implying the existence of posttranscriptional regulation of GluRdelta2 mRNA. CRF, in contrast, reduces the number of delta2-positive dendritic shafts per cell, implying that the increase of GluRdelta2 in remaining synapses may be mainly due to its retargeting. We further observed different patterns of GluRdelta2 distribution in the zone of postsynaptic density upon CRF and UCN treatment. CRF treatment results in a clustered distribution of GluRdelta2 along the postsynaptic density, whereas UCN treatment provides a linear distribution.

Cilia in the head of hornets: form and function.

In the head of the Oriental hornet, beneath the cuticle, there are plaques of hair cells. These are distributed throughout the upper front part of the head; to wit: in the region of the vertex (i.e., around and behind the ocelli), in the genae around and behind the compound eyes (the ommatidia), and in the region of the forehead or frons. These hair cells are arranged with their thin whip-like part (i.e., cilia) directed outward and morphologically fall into three distinct groups: type (a) thin elongated cilia connected to each other alongside by side-links; type (b) thin elongated cilia of which two or more interconnect at their distal ends via a delicate nerve fiber bearing a knob at its center; and type (c) shorter and thicker cilia that roughly resemble a triangular thorn and are also interconnected by a thin thread, which, however, bears a ball rather then a knob at its center. The knob in the one case and the ball in the other vary in their diameter, but in both instances the interconnecting elements, be they nerve fibers or threads, are seemingly multidirectional. Beneath the frons, in the region of the coronal suture, the hair cells (cilial plaques) are inwardly directed and bear a large trachea at their center. Presumably, the "weighted" cilial cells that are directed toward the exterior of the body aid the hornet in navigation and gravity determination whereas the inwardly directed ciliary cells may possibly serve in acoustic communication. Another element worthy of mention within the hair cells are yellow granules (yg). These yg's originate from the whip-like portion of the ciliary cells that are distributed beneath the frons plate, and also in the yellow stripes of the gastral cuticle. Conceivably, these yellow granules, in both cases, may play a role in the absorption and storage of solar energy. In summary, ciliary structures are involved in the hornet in gravity sensing, in acoustical communication and in light sensing, i.e., with some similarity with what happens in vertebrates in the inner ear and in the photoreceptor.

Ontogenesis of peripheral electromagnetic receptors in hornets.

This article traces the ontogenesis of peripheral electromagnetic receptors (PER) in the cuticle of the Oriental hornet (Vespa orientalis). In the abdominal cuticle of adult hornets, the PERs are densely distributed throughout, but there are even more than 30 at the margins of the segments. These organelles develop as a network in the hornet cuticle immediately upon its completion. Briefly, from each basic cell of a PER grows a bulge towards the exterior, that is, towards the illuminated region of the cuticle. This bulge develops rapidly and as it grows it starts to push out and lift up the various layers of the cuticle, the while pressing them together. By a spiraling movement, the bulge insinuates itself between the layers, whereupon it dissolves and punctures its way through all the layers of the hypocuticle, via the endocuticle up to the exocuticle. The only cuticular layer that remains intact is the epicuticle, but even that undergoes change, assuming the shape of a smooth surface with a depression at its center. The indented part in the epicuticle is circular, approximately 2.5 microm in diameter and enables the entry of radiation (illumination) from the outside into the PER, which is located half-way down the cuticle, with the distance from the exterior to the base of the PER being approximately 25 microm. The numerous lamellae of the cuticle run parallel to one another, but in the region of the bulge they are either perpendicular or directed upwards. This ontogeny of the PERs lends the cuticle a sandwich-like shape, being radically perforated by the PERs bulges, yet covered at the top by the epicuticle and at the bottom by basal cells. The PERs also extend shoots into the cuticular layer and these further perforate the cuticle but also interlink the various PERs. From all the above, it is clear that the cuticle forms first and only subsequently does the network of PERs develop and interpenetrate its various layers.

The postnatal developmental expression pattern of urocortin in the rat olivocerebellar system.

Urocortin belongs to the family of corticotropin-releasing factor (CRF)-like peptides, which play an important role in sensorimotor coordination. CRF induces locomotor activity, and urocortin has an inhibitory effect. Here, we document the regional and subcellular localization of urocortin in the developing rat cerebellum to compare it with CRF. During the first postnatal week, urocortin immunoreactivity (UCN-ir), within the white matter and cerebellar cortex, was strongest in vermal lobules I, II, IX, and X, closely followed by lobules IV, V, and VIII; lobules VI and VII showed the weakest labeling. Cortical immunoreactivity was in the form of puncta that encircled Purkinje cell somata. By postnatal day (PD) 12, UCN-ir had increased appreciably in all lobules. In Purkinje cells, labeling was spread throughout their somata and proximal dendrites. By PD 15, labeling in lobules I-IV appeared to wane, yet still prevailed in the central and posterior lobules. This anterior-to-posterior gradient persisted through to adulthood. The study shows that urocortin and CRF have similar regional distribution profiles during development, suggesting synergistic roles within the vestibulocerebellum. The onset of the adult distributional pattern of urocortin at the stage when rats are capable of fluent walking patterns further strengthens the correlation between CRF-like peptides and postural control. An important difference between urocortin and CRF is the localization of urocortin, and not CRF, within Purkinje cells, implying that urocortin probably has an additional role in modulating the signals emanating from the cerebellar cortex to the deep cerebellar nuclei.

AFM study of microstructures on the cornea of the compound eye and ocelli of the hornet Vespa orientalis (Insecta, Hymenoptera).

The present study describes a novel structure occurring on the cornea in the compound eye and the ocellus of the Oriental hornet. This description is based on observations carried out via scanning electron microscopy (SEM) and primarily via atomic force microscopy (AFM). We report herein that the vespan cornea is densely covered with cupola-shaped protrusions, which in the compound eye have bases about 0.2 microm in diameter and an average height of 0.03 microm while upon the ocelli their diameter is rather variable, with some measuring 0.2-0.3 microm, and a length of submicrons to microns. Interspersed among the brief, cupola-shaped structures there are winding, longer structures whose height appears uniform at around 0.03 microm. It appears that the structures are formed from the epicuticular layer of the cornea and we conjecture that in both the compound eye and in the ocelli they serve to enhance the absorption of light, as well as to reduce light reflection.