Dense Vesicles Research Articles

Synaptic relationships between proopiomelanocortin- and ghrelin-containing neurons in the rat arcuate nucleus

Both proopiomelanocortin (POMC) and ghrelin peptides are implicated in the feeding regulation. The synaptic relationships between POMC- and ghrelin-containing neurons in the hypothalamic arcuate nucleus were studied using double-immunostaining methods at the light and electron microscope levels. Many POMC-like immunoreactive axon terminals were found to be apposed to ghrelin-like immunoreactive neurons and also to make synapses with ghrelin-like immunoreactive neuronal perikarya and dendritic processes. Most of the synapses were symmetrical in shape. A small number of synapses made by ghrelin-like immunoreactive axon terminals on POMC-like immunoreactive neurons were also identified. Both the POMC- and ghrelin-like immunoreactive neurons were found to contain large dense granular vesicles. These data suggest that the POMC-producing neurons are modulated via synaptic communication with ghrelin-containing neurons. Moreover, ghrelin-containing neurons may also have a feedback effect on POMC-containing neurons through direct synaptic contacts.

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.

Loss of the Atp2c1 Secretory Pathway Ca2+-ATPase (SPCA1) in Mice Causes Golgi Stress, Apoptosis, and Midgestational Death in Homozygous Embryos and Squamous Cell Tumors in Adult Heterozygotes

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.

Overexpression of the autoantigen IA-2 puts beta cells into a pre-apoptotic state: autoantigen-induced, but non-autoimmune-mediated, tissue destruction

IA-2 is a major autoantigen in type 1 diabetes and autoantibodies to it have become important diagnostic and predictive markers. IA-2 also is an intrinsic transmembrane component of dense core secretory vesicles and knock-out studies showed that IA-2 is a regulator of insulin secretion. Here we show that overexpression of IA-2 puts mouse insulinoma MIN-6 beta cells into a pre-apoptotic state and that exposure to high glucose results in G2/M arrest and apoptosis. Molecular study revealed a decrease in phosphoinositide-dependent kinase (PDK)-1 and Akt/protein kinase B (PKB) phosphorylation. Treatment of IA-2-transfected cells with IA-2 siRNA prevented both G2/M arrest and apoptosis and increased Akt/PKB phosphorylation. A search for IA-2 interacting proteins revealed that IA-2 interacts with sorting nexin (SNX)19 and that SNX19, but not IA-2, inhibits the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 and thereby suppresses the phosphorylation of proteins in the Akt signalling pathway resulting in apoptosis. We conclude that IA-2 acts through SNX19 to initiate the pre-apoptotic state. Our findings point to the possibility that in autoimmune diseases, tissue destruction may be autoantigen-induced, but not necessarily immunologically mediated.

Localization of Vacuolar Transport Receptors and Cargo Proteins in the Golgi Apparatus of Developing Arabidopsis Embryos

Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV.

Development and pharmacological modulation of embryonic stem cell-derived neuronal network activity

Embryonic stem cells can be differentiated into neurons of diverse neurotransmitter-specific phenotypes. While the time course of functional progression of ES cell-derived neural precursors towards mature neurons has been described in detail on single-cell level, the temporal development and pharmacological modulation of ES cell-derived neuronal network activity have not been explored yet. Neuronal network activity can be assessed by the microelectrode array (MEA) technology that allows simultaneous monitoring of the electrical activity exhibited by entire populations of neurons over several weeks or months in vitro. We demonstrate here that ES cell-derived neural precursors cultured on MEAs for 5 to 6 weeks develop neuronal networks with oscillating and synchronous spike patterns via distinct states of activity and change electrophysiological characteristics even after 5 to 6 weeks in culture pointing towards late maturational processes. These processes were accompanied by an increasing density of presynaptic vesicles. Furthermore, we demonstrated that ES cell-derived network activity was sensitive to synaptically acting drugs indicating that pharmacologically susceptible neuronal networks were generated. Thus, the MEA technology represents a powerful tool to describe the temporal progression of stem cell-derived neural populations towards mature, functioning neuronal networks that can be applied to investigate pharmacologically active compounds.

Proteomics of Neuroendocrine Secretory Vesicles Reveal Distinct Functional Systems for Biosynthesis and Exocytosis of Peptide Hormones and Neurotransmitters

Regulated secretory vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such secretory vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core secretory vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (microLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel secretory vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of secretory vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these secretory vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates secretory vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions.

Vesicle movements are governed by the size and dynamics of F-actin cytoskeletal structures in bovine chromaffin cells

Dense vesicles can be observed in live bovine chromaffin cells using fluorescent reflection confocal microscopy. These vesicles display a similar distribution, cytoplasmic density and average size as the chromaffin granules visualized by electron microscopy. In addition, the acidic vesicles labeled with Lysotracker Red comprised a subpopulation of the vesicles that are visualized by reflection fluorescence. A combination of fluorescence reflection and transmitted light images permitted the movements of vesicles in relation to the cortical cytoskeleton to be studied. The movement of vesicles located on the outside of this structure was restricted, with an apparent diffusion coefficient of 1.0±0.4×10−4 μm2/s. In contrast, vesicles located in the interior moved much more freely and escaped from the visual confocal plane. Lysotracker labeling was more appropriate to study the movement of the faster moving vesicles, whose diffusion coefficient was five times higher. Using this type of labeling we confirmed the restriction on cortical movement and showed a clear relationship between vesicle mobility and the kinetics of cytoskeletal movement on both sides of the cortical cytoskeleton. This relationship was further emphasized by studying cytoskeletal organization and kinetics. Indeed, an estimate of the size of the cytoskeletal polygonal cages present in the cortical region and in the cell interior agreed well with the calculation of the theoretical radius of the cages imprisoning vesicle movement. Therefore, these data suggest that the structure and kinetics of the cytoskeleton governs vesicle movements in different regions of chromaffin cells.

Multiple potential of single cell-derived colonies from human mesenchymal stem cells

Purpose: The importance of adult bone marrow (BM) resides in its capacity to generate cells that scientists put their great hope in for future therapies. Starting from the heterogeneous nature of BM cells, we checked the in vitro spontaneous differentiation of the clones generated by the main cell phenotypes. Methods: BM was aspirated from the posterior iliac crest from patients with anemia and mononuclear cells were isolated by using Ficoll gradient. The clones were generated from the main cell types of the mesenchymal stem cells (hMSC) starting from a density of 1 cell/well. Characterization and spontaneous differentiation of clones were performed by phase contrast, electron microscopy, flow cytometry, Western blot, and IHC. Results: After 3 weeks, most cells were positive for alkaline phosphatase and Stro-1 and were thus considered hMSC. The phase contrast microscopy revealed a heterogeneous culture at the time of passage, having mostly a fibroblast-like or a large flattened morphology. By culturing, each cell has generated not a uniform, but a heterogeneous population. Single cell-derived colonies proved to be positive for smooth muscle markers and connexin 43, but still expressed Stro-1. Electron microscopy examination confirmed the heterogeneous nature of the cultures, showing cells with high density of either secretory vesicles or enlarged mitochondria. Conclusions: The MSC from BM, at a single cell level, give rise to multiple cell types in vitro. In the absence of a specific inducer, the cells had a great proliferative capacity and gained a smooth muscle phenotype, while retaining their stem properties.

Synaptic vesicle pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon terminal, inactivity

Both spinal hemisection (SH) at C 2 and tetrodotoxin (TTX) phrenic nerve blockade result in diaphragm muscle paralysis and inactivity of the phrenic axon terminals. However, phrenic motoneuron somata are inactive with SH but remain active with TTX phrenic nerve blockade. Neuromuscular transmission failure with repeated activation decreases following SH and increases following TTX phrenic nerve blockade, suggesting that matching (or mismatching) of somal and synaptic inactivities of phrenic motoneurons differentially regulates synaptic vesicle pools at diaphragm neuromuscular junctions. At individual type-identified rat diaphragm presynaptic terminals, the size of the releasable pool of synaptic vesicles was analyzed by fluorescence confocal microscopy of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64) uptake and synaptic vesicle density at active zones was determined using transmission electron microscopy. After 14 days of SH and TTX-induced diaphragm muscle inactivity, neuromuscular junction size was not different at type I or IIa fibers, but increased at type IIx and/or IIb fibers (by 51% in SH and 35% in TTX) compared with control. With SH, synaptic vesicle pool size and density increased at presynaptic terminals innervating type I or IIa fibers (17 and 63%, respectively; P<0.001) and type IIx and/or IIb fibers (41 and 31%, respectively; P<0.001) when compared with controls. Following TTX, synaptic vesicle pool size and density decreased by 64 and 17%, respectively, at presynaptic terminals innervating type I or IIa fibers, and by 50 and 36%, respectively, at type IIx and/or IIb fibers ( P<0.001, for all comparisons). Thus, matching motoneuron soma and axon terminal inactivity (SH) increases the size and density of releasable synaptic vesicle pools at adult rat diaphragm neuromuscular junctions. Mismatching motoneuron soma and axon terminal inactivities (TTX) results in converse presynaptic adaptations. Inactivity-induced neuromuscular plasticity reflects specific adaptations in the size and density of synaptic vesicle pools that depend on motoneuron soma rather than axon terminal (or muscle fiber) inactivity.

Biphasic perimicrovillar membrane production following feeding by previously starved Dysdercus peruvianus (Hemiptera: Pyrrhocoridae)

The development of perimicrovillar membranes (PMM) from midgut cells of starved and fed Dysdercus peruvianus was studied by using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and assays for specific enzymatic markers of the perimicrovillar membranes (α-glucosidase), perimicrovillar space (aminopeptidase) and microvillar membranes (β-glucosidase). High activities of these enzymes were observed 6 h post-feeding and significant production of membranes was observed at 30 h post-feeding. In the gut cells of starved insects, the rough endoplasmic reticulum was organized in concentric bundles, with a greater number of mitochondria in the cellular apex. The presence of electron dense double-membrane vesicles and the production of PMM were not observed in this condition. Thirty hours post-feeding, a disorganization of the rough endoplasmic reticulum was observed, and it was possible to see double-membrane vesicles close to the cell apex. The membrane system formation was evident with a significant development of PMM in the midgut lumen. The luminal surface of the midgut during starvation and up to 48 h post-feeding was monitored using SEM. It was demonstrated that in the starved condition, the PMM was virtually absent from gut cells, except at the base of the microvilli. At 6 h post-feeding, the microvilli were already completely covered with PMM, but with a maximum of PMM formation seen at 30 h post-feeding. Signals of PMM degradation were observed 48 h after pulse feeding.

Loss of α-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.

Hypoglutamatergic activity in the STOP knockout mouse: A potential model for chronic untreated schizophrenia

In mice, the deletion of the STOP protein leads to hyperdopaminergia and major behavioral disorders that are alleviated by neuroleptics, representing a potential model of schizophrenia. The reduction of the glutamatergic synaptic vesicle pool in the hippocampus could reflect a disturbance in glutamatergic neurotransmission in this model. Here we examined potential disturbances in energy metabolism and interactions between neurons and glia in 15-week-old STOP KO, wild-type, and heterozygous mice. Animals received [1-(13)C]glucose and [1,2-(13)C]acetate, the preferential substrates of neurons and astrocytes, respectively. Extracts from the whole forebrain and midbrain were analyzed by HPLC, (13)C and (1)H NMR spectroscopy. Amounts and labeling of most metabolites were unchanged. However, glutamine concentration and amount of [4,5-(13)C]glutamine derived from [1,2-(13)C]acetate significantly decreased by 17% and 18%, respectively, in STOP KO compared with wild-type mice. The amount of [4-(13)C]glutamate was decreased in STOP KO and heterozygous compared with wild-type mice. gamma-Aminobutyric acid labeling was not influenced by the genotype. Because STOP-deficient mice have a lower synaptic vesicle density, less glutamate is released to the synaptic cleft, leading to decreased stimulation of the postsynaptic glutamate receptors, reflecting increased glutamine metabolism only in the vicinity of the postsynapse of STOP KO mice.

A qualitative and quantitative electron microscopic study of differences in adrenomedullary adrenaline cells between golden hamsters and rats, with special reference to the Golgi apparatus

Ultrastructural features of adrenomedullary adrenaline (A) cells in golden hamsters and rats were investigated qualitatively and quantitatively with special reference to the Golgi apparatus. The A cells displayed a characteristic follicular arrangement, with each cell showing structural polarity in hamsters, but not apparently in rats. In hamsters, the Golgi apparatus of A cells was larger (t-test: P<0.001) and more frequently showed large and complexly organized structures (chi(2)-test: P<0.005) compared with that of rats. Quantitative analysis of the Golgi apparatus revealed differences in the size and numerical density of Golgi vesicles in relation to the animal species and region. Two-way analysis of variance (ANOVA) confirmed species difference in the size of coated vesicles (P<0.005) and interaction between species and region concerning the size of smooth-clear vesicles (P<0.01) and numerical density of granular vesicles (P<0.05). One-way ANOVA revealed regional differences in the size and numerical density of smooth-clear vesicles in rats and hamsters (P<0.01 approximately 0.001), and in the numerical density of coated vesicles in hamsters (P<0.05). Data were further analyzed by Tukey-Kramer's method. These and other reported results suggest that, in hamster A cells, the Golgi apparatus has different structural, molecular, and functional mechanisms which are at least partly related to the distinct cellular polarity and higher concentration of peptide hormones in secretory vesicles, and that in rat A cells, in contrast, loading secretory vesicles with A during the post-Golgi stage is predominant. In conclusion, the Golgi apparatus in hamster A cells shows markedly different ultrastructural features compared with that in rat A cells.

Dissecting Multiple Steps of GLUT4 Trafficking and Identifying the Sites of Insulin Action

Insulin-stimulated GLUT4 translocation is central to glucose homeostasis. Functional assays to distinguish individual steps in the GLUT4 translocation process are lacking, thus limiting progress toward elucidation of the underlying molecular mechanism. Here we have developed a robust method, which relies on dynamic tracking of single GLUT4 storage vesicles (GSVs) in real time, for dissecting and systematically analyzing the docking, priming, and fusion steps of GSVs with the cell surface in vivo. Using this method, we have shown that the preparation of GSVs for fusion competence after docking at the surface is a key step regulated by insulin, whereas the docking step is regulated by PI3K and its downstream effector, the Rab GAP AS160. These data show that Akt-dependent phosphorylation of AS160 is not the major regulated step in GLUT4 trafficking, implicating alternative Akt substrates or alternative signaling pathways downstream of GSV docking at the cell surface as the major regulatory node.

Microinclusion-rich vesicular olivine in the Karoonda CK4 chondrite: transmission electron microscopy

The Karoonda CK4 chondrite exhibits pronounced silicate darkening of matrix and chondrule mesostases. Our scanning electron microscope study showed that the Karoonda matrix contains unusual olivine, with a high density of vesicles and inclusions of mainly magnetite and pentlandite, all between 0.1 and 3 μm in diameter. The vesicular and nonvesicular olivines occur as an intermixture, and vesicularity is particularly abundant in regions that exhibit a high degree of silicate darkening. Our transmission electron microscope study revealed that the vesicular olivine is entirely crystalline and contains a high density of much smaller (< 10 to 100 nm) vesicles and inclusions of mostly pentlandite. In contrast, nonvesicular olivine contains no such inclusions. The observations indicate that the silicate darkening is caused by the dispersion of not only opaque mineral inclusions but also vesicles, and the microinclusion-rich vesicular olivine is the principal cause of the silicate darkening. The texture and mineralogy of the vesicular olivine suggest that it was formed by crystallization of melt that was produced from nonvesicular olivine by shock at a high temperature.

An Ultrastructural Study of Tegumental Glands of the Fifth Pereiopods of Aegla Platensis (Anomura: Aeglidae)

Abstract Tegumental glands are a ubiquitous feature of the decapod cuticle. In the fifth pair of pereiopods (P5) of Aegla platensis Schmitt, 1942, these tegumental glands (PeTG's) are similar to those described in other decapod crustaceans. Type 1 PeTG's, with mucous and serous cells and type 2 PeTG's, with serous cells only are organized into proximal, medial and distal clusters along the appendage. Both types of PeTG's possess secretory cells arranged concentrically around a central duct. These secretory cells have well developed Rough endoplasmic reticulum (RER) and numerous Golgi Complexes, and possess electron-lucid and electron dense vesicles. The finely granular aspect of the electron-lucid vesicles suggest they contain a mucous-like substance, while electron-dense vesicles probably contain a substance of a proteinaceous nature. Despite various hypotheses, there are few indications that these substances act as cleaning agents. However, the cytoplasm of the secretory cells appear to be composed of c...

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging

As they are often designed for lysosomotropic, endosomotropic and/or transcellular delivery, an understanding of intracellular trafficking pathways is essential to enable optimised design of novel polymer therapeutics. Here, we describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics. Hepatoma (HepG2) cells were used as a model and cell breakage was optimised using a cell cracker to ensure assay of the whole cell population. After removal of unbroken cells and nuclei, the cell lysate as a post-nuclear supernatant (PNS) was layered onto an iodixanol (OptiPrep™) density gradient optimised to 5–20%. Early endosomes, late endosomes and lysosomes were identified from gradient fractions by immunoblotting for marker proteins early endosome antigen 1 (EEA 1) and lysosomal associated membrane protein 1 (LAMP 1) using horseradish peroxidase or fluorescently-labelled secondary antibodies. Lysosomes were also detected using N-acetyl-β-glucosamindase (Hex A) activity. In addition, cells were incubated with Texas-red labelled transferrin (TxR-Tf) for 5 min to specifically label early endosomes and this was directly detected from SDS-PAGE gels. Internalised macromolecules and colloidal particles can potentially alter vesicle buoyant density. To see if typical macromolecules of interest would alter vesicle density or perturb vesicle traffic, HepG2 cells were incubated with dextran or a polyethyleneglycol (PEG)-polyester dendron G4 (1 mg/ml for 24 h). The PEG-polyester dendron G4 caused a slight redistribution of endocytic structures to lower density fractions but immunofluorescence microscopy showed no obvious dendron effects. In conclusion, the combined subcellular fractionation with fluorescent imaging approach described here can be used as a tool for both fundamental cell biology research and/or the quantitative localisation of polymer therapeutics in the endocytic pathway.

Fine ultrastructure and biochemistry of PC12 cells: A comparative approach to understand neurotoxicity

The PC12 cell line is commonly used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons. Despite the broad use of this cell line, there are a number of points differing between PC12 cells and dopamine neurons in vivo which are missed out when translating in vitro data into in vivo systems. This led us to compare the PC12 cells with central dopamine neurons, aiming at those features which are predictors of in vivo physiology and degeneration of central dopamine neurons. We carried out this comparison, either in baseline conditions, following releasing or neurotoxic stimuli (i.e. acute or chronic methamphetamine), to end up with therapeutic agents which are suspected to produce neurotoxicity ( l-DOPA). Although the neurotransmitter pattern of PC12 cells is close to dopamine neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicles density, which parallels low catecholamine levels. Again, compartmentalization of secretory elements in PC12 cells is already pronounced in baseline conditions, while it is only slightly affected following catecholamine-releasing stimuli. This low flexibility is caused by the low ability of PC12 cells to compensate for sustained catecholamine release, due both to non-sufficient dopamine synthesis and poor dopamine storage mechanisms. This contrasts markedly with dopamine-containing neurons in vivo lending substance to opposite findings between these compartments concerning the sensitivity to a number of neurotoxins.

Rapid and reversible changes in intrahippocampal connectivity during the course of hibernation in European hamsters

The hippocampal formation is a highly plastic brain structure that undergoes structural remodeling in response to internal and external challenges such as metabolic imbalance and repeated stress. We investigated whether the extreme alterations in metabolic status that occur during the course of hibernation in European hamsters cause structural changes in the dendritic arborizations of the CA3 pyramidal neurons and their main excitatory afferents, the mossy fiber terminals (MFT), that originate in the dentate gyrus. We report that apical, but not basal, dendritic trees of Golgi-impregnated CA3 principal neurons are significantly shorter, less branched, and less spiny in hypothermic hamsters compared with active animals. After the induction of arousal from torpor, within 2 h, the apical dendritic lengths, branching patterns, and spine density estimations returned to levels found in active, euthermic hamsters. The ultrastructure of MFT in hibernating hamsters showed a significant reduction in synaptic vesicle density and in the percentage of MFT area covered by spine profiles. Awakened hamsters showed restoration of MFT morphology to that seen in active animals. MFT of torpid animals also showed a significant increase in the percentage area of mitochondrial profiles that remained higher 3 h after induced arousal from hibernation compared with euthermic controls. Thus, the torpid/awakening cycle of the hibernating European hamster causes a rapid and reversible morphological reorganization of intrahippocampal subregions involved in information processing. The reported reductions in morphological connectivity between the dentate gyrus and the CA3 subregions could underlie the cessation of exploratory activity and spatial navigation skills during hibernation.