Dystonin modifiers of junctional epidermolysis bullosa and models of epidermolysis bullosa simplex without dystonia musculorum.

The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice.

Diet-related changes of basal lamina fenestrations in the villous epithelium of the rat small intestine: Statistical analysis on scanning electron microscopy.

The epithelial basal lamina of the small intestine has numerous fenestrations for intraepithelial migration of leukocytes. We have reported dynamic changes of fenestrations in dietary conditions. To investigate this phenomenon, we performed statistical analyses using scanning electron microscopy images of the epithelial basal lamina of rat intestinal villi after removal of the villous epithelium by osmium maceration. We examined structural changes in the number and size of fenestrations in the rat jejunum and ileum under fasted and fed states for 24 h. Our findings revealed that, in the jejunum, the number of free cells migrating into the epithelium through fenestrations increased from 2 h after feeding, resulting in an increase in the fenestration size of intestinal villi; the number of free cells then tended to decrease at 6 h after feeding, and the fenestration size also gradually decreased. By contrast, the increase in the fenestration size by feeding was not statistically significant in the ileum. These findings indicate that the number of migrating cells increases in the upper part of the small intestine under dietary conditions, which may influence the absorption efficiency of nutrients including lipids, as well as the induction of nutrient-induced inflammation.

EGF Downregulates Presynaptic Maturation and Suppresses Synapse Formation In Vitro and In Vivo.

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation.

Scanning ion-conductance microscopy with a double-barreled nanopipette for topographic imaging of charged chromosomes.

Scanning ion conductance microscopy (SICM) is useful for imaging soft and fragile biological samples in liquids because it probes the samples' surface topography by detecting ion currents under non-contact and force-free conditions. SICM acquires the surface topographical height by detecting the ion current reduction that occurs when an electrolyte-filled glass nanopipette approaches the sample surface. However, most biological materials have electrically charged surfaces in liquid environments, which sometimes affect the behavior of the ion currents detected by SICM and, especially, make topography measurements difficult. For measuring such charged samples, we propose a novel imaging method that uses a double-barrel nanopipette as an SICM probe. The ion current between the two apertures of the nanopipette desensitizes the surface charge effect on imaging. In this study, metaphase chromosomes of Indian muntjac were imaged by this technique because, owing to their strongly negatively charged surfaces in phosphate-buffered saline, it is difficult to obtain the topography of the chromosomes by the conventional SICM with a single-aperture nanopipette. Using the proposed method with a double-barrel nanopipette, the surfaces of the chromosomes were successfully measured, without any surface charge confounder. Since the detailed imaging of sample topography can be performed in physiological liquid conditions regardless of the sample charge, it is expected to be used for analyzing the high-order structure of chromosomes in relation to their dynamic changes in the cell division.

Scanning ion conductance microscopy of isolated metaphase chromosomes in a liquid environment.

Scanning probe microscopy (SPM) uses a probing tip which scans over a sample surface for obtaining information on the sample surface characteristics. Among various types of SPM, atomic force microscopy (AFM) has been widely applied to imaging of biological samples including chromosomes. Scanning ion conductance microscopy (SICM) has been also introduced for visualizing the surface structure of biological samples because it can obtain "contact-free" topographic images in liquid conditions by detecting ion current flow through a pipette opening. However, we recently noticed that the consistent imaging of chromosomes is difficult by SICM. In this paper, the behaviors of the ion current on the sample surfaces were precisely investigated for obtaining SICM images of isolated muntjac metaphase chromosomes more consistently than at present. The present study revealed that application of positive potential to the pipette electrode was acceptable for obtaining the topographic image of chromosomes, while application of negative potential failed in imaging. The approach curves were then studied for analyzing the relationship between the ion current and the tip sample distance when the pipette is approaching chromosomes. The current-voltage (I-V) curve further provided us the accurate interpretation of the ion current behavior during chromosome imaging. These data were further compared with those for SICM imaging of HeLa cells. Our findings indicated that chromosomes are electrically charged and the net charge is strongly negative in normal Dulbecco's phosphate buffered saline. We finally showed that the ion concentration of the bath electrolyte is important for imaging chromosomes by SICM.

SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.

To elucidate the underlying mechanism of secretory leukocyte protease inhibitor (SLPI)-induced cell migration, we compared SLPI-deleted human gingival carcinoma Ca9-22 (ΔSLPI) cells and original (wild-type: wt) Ca9-22 cells using several microscopic imaging methods and gene expression analysis. Our results indicated reduced migration of ΔSLPI cells compared to wtCa9-22 cells. The lamellipodia/dorsal ruffles were smaller and moved slower in ΔSLPI cells compared to wtCa9-22 cells. Furthermore, well-developed intermediate filament bundles were observed at the desmosome junction of ΔSLPI cells. In addition, Galectin4 was strongly expressed in ΔSLPI cells, and its forced expression suppressed migration of wtCa9-22 cells. Taken together, SLPI facilitates cell migration by regulating lamellipodia/ruffles and desmosomes, in which Galectin4 plays an important role.

Novel concept for the epaxial/hypaxial boundary based on neuronal development.

Trunk muscles in vertebrates are classified as either dorsal epaxial or ventral hypaxial muscles. Epaxial and hypaxial muscles are defined as muscles innervated by the dorsal and ventral rami of spinal nerves, respectively. Each cluster of spinal motor neurons passing through dorsal rami innervates epaxial muscles, whereas clusters traveling on the ventral rami innervate hypaxial muscles. Herein, we show that some motor neurons exhibiting molecular profiles for epaxial muscles follow a path in the ventral rami. Dorsal deep-shoulder muscles and some body wall muscles are defined as hypaxial due to innervation via the ventral rami, but a part of these ventral rami has the molecular profile of motor neurons that innervate epaxial muscles. Thus, the epaxial and hypaxial boundary cannot be determined simply by the ramification pattern of spinal nerves. We propose that, although muscle innervation occurs via the ventral rami, dorsal deep-shoulder muscles and some body wall muscles represent an intermediate group that lies between epaxial and hypaxial muscles.

Diverse dystonin gene mutations cause distinct patterns of Dst isoform deficiency and phenotypic heterogeneity in Dystonia musculorum mice.

Loss-of-function mutations in dystonin (DST) can cause hereditary sensory and autonomic neuropathy type 6 (HSAN-VI) or epidermolysis bullosa simplex (EBS). Recently, DST-related diseases were recognized to be more complex than previously thought because a patient exhibited both neurological and skin manifestations, whereas others display only one or the other. A single DST locus produces at least three major DST isoforms: DST-a (neuronal isoform), DST-b (muscular isoform) and DST-e (epithelial isoform). Dystonia musculorum (dt) mice, which have mutations in Dst, were originally identified as spontaneous mutants displaying neurological phenotypes. To reveal the mechanisms underlying the phenotypic heterogeneity of DST-related diseases, we investigated two mutant strains with different mutations: a spontaneous Dst mutant (Dstdt-23Rbrc mice) and a gene-trap mutant (DstGt mice). The Dstdt-23Rbrc allele possesses a nonsense mutation in an exon shared by all Dst isoforms. The DstGt allele is predicted to inactivate Dst-a and Dst-b isoforms but not Dst-e There was a decrease in the levels of Dst-a mRNA in the neural tissue of both Dstdt-23Rbrc and DstGt homozygotes. Loss of sensory and autonomic nerve ends in the skin was observed in both Dstdt-23Rbrc and DstGt mice at postnatal stages. In contrast, Dst-e mRNA expression was reduced in the skin of Dstdt-23Rbrc mice but not in DstGt mice. Expression levels of Dst proteins in neural and cutaneous tissues correlated with Dst mRNAs. Because Dst-e encodes a structural protein in hemidesmosomes (HDs), we performed transmission electron microscopy. Lack of inner plaques and loss of keratin filament invasions underneath the HDs were observed in the basal keratinocytes of Dstdt-23Rbrc mice but not in those of DstGt mice; thus, the distinct phenotype of the skin of Dstdt-23Rbrc mice could be because of failure of Dst-e expression. These results indicate that distinct mutations within the Dst locus can cause different loss-of-function patterns among Dst isoforms, which accounts for the heterogeneous neural and skin phenotypes in dt mice and DST-related diseases.

Effect of methionine/choline-deficient diet and high-fat diet-induced steatohepatitis on mitochondrial homeostasis in mice.

Considering the increase in cases of non-alcoholic steatohepatitis (NASH), the use of appropriate animal model of NASH is essential to understand the underlying pathogenesis mechanism. To date, several mice models have been used; however, significant differences in the etiologies and food administered affected the results, with inconsistent conclusions. Therefore, it is necessary to understand these models and their differences to be able to choose appropriate models. Inspired by the fact that mitochondrial (mt)DNA content is changed in non-alcoholic fatty liver disease in humans, we investigated the mtDNA copy number in the NASH mice models induced by high-fat diet (HFD) and methionine/choline-deficient diet (MCD) to understand the differences between these models. Megamitochondria were observed in both MCD and HFD groups. However, the MCD group showed significant decrease in liver mtDNA content compared with that in the HFD group. These changes were associated with significant upregulation of mitochondrial biogenesis- and degradation-related genes in MCD model than in HFD model. Thus, stability of mtDNA is associated with the differences between MCD and HFD-induced NASH models often used in studies; these findings could help in choosing appropriate models for studies on NASH.

Three-dimensional configuration of apical epithelial compartments including stem cell niches in guinea pig cheek teeth.

OBJECTIVES: Continuously growing rodent incisors have an apically located epithelial stem cell compartment, known as an "apical bud" (AB). Few studies have described the morphological features of ABs and stem cell niches in continuously growing premolars/molars. We attempted to clarify the relationship between the three-dimensional configuration of ABs and the stem cell niches in guinea pig cheek teeth. METHODS: We perfusion-fixed four-week-old guinea pigs, then decalcified their premolars/molars to produce serial paraffin sections, which we immunostained for Sox2. We reconstructed the serial sections using image processing and analysis software. We processed undecalcified samples for scanning electron microscopy by KOH digestion. RESULTS: Two types of epithelia with M and Δ shapes surrounded the S-shaped dental papilla in the apical region of the premolars/molars, and there were three Sox2-positive epithelial bulges above the M- and Δ-shaped epithelia. Sox2-positive epithelial stem cell niches were restricted to the apical side, and cell proliferation and differentiation immediately proceeded in the crown-analogue dentin. The Sox2-positive epithelial stem cell niches were sparsely distributed and extended to the occlusal side. We also detected continuously proliferating cells in the cervical loop and Hertwig's epithelial root sheath of the root-analogue dentin. CONCLUSIONS: Our findings suggest that guinea pig cheek teeth have three ABs, and the complex configuration of these types of teeth may be attributed to the prompt formation of crown-analogue dentin followed by the long-term formation of root-analogue dentin.

Dynamic changes in basal lamina fenestrations in rat intestinal villous epithelium under high-fat diet condition.

The basal lamina of the villous epithelium in the small intestine has numerous fenestrations, which are produced by leukocytes for their intraepithelial migration. We previously showed that these fenestrations change due to the dynamics of migrating leukocytes in response to dietary conditions and suggested the possibility that this change is related to the regulation of the absorption of large-sized nutrients such as chylomicrons. The present study was, thus, designed to investigate structural changes in basal lamina fenestrations in response to a high-fat diet. The ultrastructure of the intestinal villi in the rat upper jejunum was investigated by electron microscopy of tissue sections in both the normal and the high-fat diet groups, and the fenestrations in the villous epithelium of rat upper jejunum were studied by scanning electron microscopy of osmium macerated/ ultrasonicated tissues. The present study showed that free cells adhering to the fenestrations increased in the upper jejunum two hours after feeding high-fat diet and the size of the fenestrations in this region also increased after feeding high-fat diet for 2 days. This enlargement of fenestrations may play an important role in increasing the efficiency of lipid absorption by facilitating the movement of chylomicrons from the intercellular space to the lamina propria.

The morphology, size and density of the touch dome in human hairy skin by scanning electron microscopy.

The touch domes of mammalian hairy skin are mechanoreceptors characterized by the accumulation of Merkel cell-neurite complexes at the epidermal base. In this study, we examined the shape, size, and density of the touch dome of human skin of the forearm and the abdomen through scanning electron microscopy (SEM). Human skin samples were obtained from donated bodies, as well as a patient who underwent biopsy. Skin pieces were treated with a KOH-collagenase method for the separation of the epidermis from the dermis. The basal surface of the separated epidermis was then observed using SEM. The touch dome was clearly determined as a concave area bordered by a thick epidermal ridge, where neural components accumulated. The touch dome was rather independent from hair follicles, although they were sometimes located beside the touch dome. The average size and density of the touch dome were 0.06 mm2 and 3.82 cm2 in the forearm, and 0.10 mm2 and 1.30 cm2 in the abdomen, respectively. Our results suggested that the regional difference in size and density of the touch dome might be related to the sensation's sensitivity as touch spots in human hairy skin.

Dual effect of polyphosphate on mineralization of rat osteoblast ROS17/2.8 cells in a dose-dependent manner.

Inorganic polyphosphate (polyP), a linear polymer of orthophosphate, is found at high concentrations in osteoblasts. We demonstrated the effects of various polyP concentrations on the mineralization of rat osteoblast ROS17/2.8 cells. Mineralization of ROS17/2.8 was induced by a high polyP concentration (1 mg/mL), which was accompanied by an upregulation of the bone sialoprotein and osteocalcin. In contrast, a low polyP concentration (1 × 10-2 mg/mL) reduced mineralization without affecting the osteogenic gene expression. Furthermore, gene expression profiling and forced expression analysis indicated that phosphodiesterase 11a could be a candidate involved in the dose-dependent effect of polyP on osteoblast mineralization.

A bacterial metabolite ameliorates periodontal pathogen-induced gingival epithelial barrier disruption via GPR40 signaling.

Several studies have demonstrated the remarkable properties of microbiota and their metabolites in the pathogenesis of several inflammatory diseases. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a bioactive metabolite generated by probiotic microorganisms during the process of fatty acid metabolism, has been studied for its protective effects against epithelial barrier impairment in the intestines. Herein, we examined the effect of HYA on gingival epithelial barrier function and its possible application for the prevention and treatment of periodontal disease. We found that GPR40, a fatty acid receptor, was expressed on gingival epithelial cells; activation of GPR40 by HYA significantly inhibited barrier impairment induced by Porphyromonas gingivalis, a representative periodontopathic bacterium. The degradation of E-cadherin and beta-catenin, basic components of the epithelial barrier, was prevented in a GPR40-dependent manner in vitro. Oral inoculation of HYA in a mouse experimental periodontitis model suppressed the bacteria-induced degradation of E-cadherin and subsequent inflammatory cytokine production in the gingival tissue. Collectively, these results suggest that HYA exerts a protective function, through GPR40 signaling, against periodontopathic bacteria-induced gingival epithelial barrier impairment and contributes to the suppression of inflammatory responses in periodontal diseases.

Dynamics of basal lamina fenestrations in the rat intestinal villous epithelium in response to dietary conditions.

The epithelial basal lamina of the small intestine forms a felt-like sheet at the base of the epithelium. Previous studies have shown that the basal lamina has numerous fenestrations, which are produced by leukocytes penetrating through the basal lamina. In this study, we aimed to directly visualize fenestrations of the rat basal lamina in intestinal villi by scanning electron microscopy (SEM) after removal of the villous epithelium by osmium maceration and ultrasonic treatment. Structural changes in fenestrations were then investigated in relation to dietary conditions. SEM of these tissues revealed the presence of fenestrations in the villous epithelial basal lamina in all segments of the small intestine, although the number was the highest in the jejunum. The present study also showed that the number and size of fenestrations increased after feeding in the jejunum, whereas changes were unclear in the ileum. These findings suggested that the basal lamina fenestrations were changed through the dynamics of migrating leukocytes in dietary conditions and may also be related to the regulation of nutrient absorption, particularly as lipids are transported from the intercellular space of the epithelium to the lamina propria.

Scanning ion conductance microscopy for visualizing the three-dimensional surface topography of cells and tissues.

Scanning ion conductance microscopy (SICM), which belongs to the family of scanning probe microscopy, regulates the tip-sample distance by monitoring the ion current through the use of an electrolyte-filled nanopipette as the probing tip. Thus, SICM enables "contact-free" imaging of cell surface topography in liquid conditions. In this paper, we applied hopping mode SICM for obtaining topographical images of convoluted tissue samples such as trachea and kidney in phosphate buffered saline. Some of the SICM images were compared with the images obtained by scanning electron microscopy (SEM) after drying the same samples. We showed that the imaging quality of hopping mode SICM was excellent enough for investigating the three-dimensional surface structure of the soft tissue samples. Thus, SICM is expected to be used for imaging a wide variety of cells and tissues - either fixed or alive- at high resolution under physiologically relevant liquid conditions.

Electron tomography of whole cultured cells using novel transmission electron imaging technique.

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research.

Integrative method for three-dimensional imaging of the entire Golgi apparatus by combining thiamine pyrophosphatase cytochemistry and array tomography using backscattered electron-mode scanning electron microscopy.

Thiamine pyrophosphatase (TPPase) cytochemistry is an established method for specific labeling of the trans-Golgi cisterns in tissue sections. Herein, we combined this enzyme cytochemical method with array tomography using scanning electron microscopy (SEM), a new imaging technique based on collection of backscattered electron (BSE) images of consecutive resin-embedded sections on glass slides, to detect the entire three-dimensional (3D) organization of the Golgi apparatus with sufficient spatial resolution. As the signal intensity of BSE depends on the atomic number of the materials, lead precipitates confined to the trans-Golgi cisterns after TPPase cytochemistry were clearly observed by BSE-mode SEM. The mild fixative used for TPPase cytochemistry also enabled accurate identification of target gonadotropes in the composite pituitary tissue by immunocytochemical staining. By 3D reconstruction of the entire trans-Golgi cisterns based on serial ultrathin section images of tissues after TPPase cytochemistry, we detected ultrastructural differences in the 3D configuration of the Golgi apparatus between cerebellar Purkinje cells and pituitary gonadotropes. The appropriate combination of enzyme cytochemistry and/or immunostaining with array tomography will further clarify the relationship between the organization and functional states of the Golgi apparatus.

Three-dimensional reconstruction of root cells and interdental cells in the rat inner ear by serial section scanning electron microscopy.

In the cochlea, a high K+ environment in the endolymph is essential for the maintenance of normal hearing function, and the transport of K+ ions through gap junctions of the cochlear epithelium is thought to play an important role in endolymphatic homeostasis. The aim of the present study was to demonstrate the three-dimensional (3D) ultrastructure of spiral ligament root cells and interdental cells, which are located at both ends of the gap junction system of the cochlea epithelium. Serial semi-thin sections of plastic-embedded rat cochlea were mounted on glass slides, stained with uranyl acetate and lead citrate, and observed by scanning electron microscopy (SEM) using the backscattered electron (BSE) mode. 3D reconstruction of BSE images of serial sections revealed that the root cells were linked together to form a branched structure like an elaborate "tree root" in the spiral ligament. The interdental cells were also connected to each other, forming a comb-shaped cellular network with a number of cellular strands in the spiral limbus. Furthermore, TEM studies of ultra-thin sections revealed the rich presence of gap junctions in both root cells and interdental cells. These findings suggest the possibility that both root cells and interdental cells contribute to K+ circulation as the end portion of the epithelial cell gap junction system of the cochlea.

Changes in the three-dimensional ultrastructure of membranous organelles in male rat pituitary gonadotropes after castration.

The increased discharge of gonadotropin releasing hormone (GnRH) from hypothalamic neurons after castration specifically stimulates pituitary gonadotropes. To elucidate the putative effects of GnRH on the three-dimensional ultrastructure of gonadotropes, we examined osmium-macerated pituitary tissues of male rats at various time points after castration by high resolution scanning electron microscopy (SEM) combined with immunocytochemistry. Two days after castration, the Golgi apparatus was disassembled into small stacks; patch-like, tubuloreticular clusters of endoplasmic reticulum (ER) membranes were present; and spherically enlarged mitochondria were accumulated in the central area of the stimulated gonadotropes. These acute changes were indiscernible by 1 week after castration, and then the pituitary gonadotropes of castrated animals gradually became hypertrophic, finally exhibiting the characteristic "signet-ring" appearance, with markedly dilated cisterns of the rough ER. Upon SEM observation, the inner surface of the cavity was mostly flat, and openings connecting adjacent lumens of the ER were sparse. Proliferation of the osmiophilic tubular network of the ER-Golgi intermediate compartment was observed in the persistently stimulated gonadotropes, indicating a marked increase in trafficking of secretory proteins between the Golgi and ER. The acute and chronic changes in the gonadotropes after castration revealed in the present study by SEM provide evidence for a putative link between the intracellular signaling events evoked by GnRH and the ultrastructural dynamics of the organelles of the secretory pathway.