Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution of water in native starch granules—a multinuclear NMR study

The microscopic distribution and dynamic state of water in native potato, maize and pea starch granules are investigated with NMR relaxometry and diffusometry. Besides extra-granular water, three water populations can be identified inside native potato starch granules. These are assigned to water in the amorphous growth rings; water in the semi-crystalline lamellae and “channel water”, which is located in the hexagonal channels within the B-type amylopectin crystals. The first two water populations are orientationally disordered and exchange with each other on a millisecond timescale at 290 K. NMR diffusometry shows that the water in packed granule beds is undergoing translational diffusion in a 2-dimensional space, either in thin layers between granules and/or in amorphous growth rings within the granules. The “channel water” is uniquely characterised by a 1 kHz deuterium doublet splitting and is in slow exchange with water in the other compartments on the NMR timescale. In the smaller maize granules all intra-granular water populations are in fast exchange and there is no evidence for “channel water” in the A-type crystal lattice. The NMR water proton and deuterium data for pea starch are consistent with a composite A and B-type crystal structure.     

Trans-epidermal Transport and Storage of Calcium in Holthuisana transversa (Brachyura; Sundathelphusidae) During Premoult

Holthuisana transversa reabsorbs much of its exoskeletal calcium in the last 3 days before ecdysis and stores it in circulating granules in the haemocoel and in non-circulating granules in the subepidermal connective tissue. Calcium enters the epidermal cells from the moulting fluid, probably through their apical microvilli and is either incorporated into intracellular calcium granules or exits the cell via the basolateral membranes to be used in formation of two other granule types. Intracellular granules (0.4–2 μm long) form in large masses in the apical cytoplasm of the epidermal cells. They are formed as membrane-bound vesicles by the Golgi, and calcium and organic matrix material are added from the surrounding cytoplasm. As development proceeds, lamellae appear and calcium carbonate is deposited in the matrix. Granule masses move basally and are stored in the connective tissue. Calcium is also incorporated into extracellular large granules (0.8–3.8 μm long) which are formed in narrow intercellular channels between epidermal cells. A third granule type (small granules, 0.26 μm diameter) is formed in subepidermal connective tissue cells and released into the haemolymph in very large numbers. Calcium was identified in the two larger granule types using X-ray microanalysis and significant amounts of phosphorus and potassium were also present in the large granules. A model for ion cycling between the exoskeleton and granules is presented.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study

Summary The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 m in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled. From these findings, it is suggested that the granules of rat Clara cells consist of two types of granules of distinct origin; one appears to derive from condensing vacuoles of Golgi origin, whereas the other may be formed by membranefusions with small cytoplasmic vesicles of unknown source.     

The fine structure of granules in eosinophil leucocytes from aquatic and terrestrial birds

The ultrastructure of eosinophil granules from various aquatic and terrestrial birds has been described. Granules of three basic types were found. The first had a crystalline internum and was found only in the order Anseriformes, which included the black-necked screamer, ducks, geese and swans. The crystals occurred in three morphological forms. The second and least common granule examined contained a non-crystalline internum which was either homogeneous or composed of microfilaments or microtubules. The largest and most common group of birds had a homogenous granule with no internum shown. Homogeneous granules occurred less frequently than did those with interna.     

Electron microscopic study on the thyroid gland of the salamander Hynobius nebulosus in the breeding season

Summary The thyroid gland of adult salamanders, Hynobius nebulosus, in the breeding season was studied by electron microscopy. The follicular cells are different in cell height and fine structures; the taller cells with many cell organelles and granules and the lower cells with a few cell organelles and granules are both present in the same follicle. In the cytoplasm, three types of membrane-bounded granules, namely, cytosomes, colloid droplets, and vacuolar bodies and circular membrane complexes occur. The vacuolar bodies are subdivided into two types; the ordinary type having loosely distributed particles and the specific type containing tubules and/or closely packed filaments, crystalloid structures, except for the particles. The chromophobe colloids within the Bensley-cells correspond to extremely large, ordinary type vacuolar bodies, while the Langendorff-colloid cells possess increased numbers of granular cisternae of endoplasmic reticulum and a ribosome-rich, dense cytoplasmic matrix but not extremely large colloid. The intracytoplasmic circular membrane complexes appear in the Golgi area of cytosome-rich cells. It is suggested that they originate from the Golgi apparatus which was activated to produce many cytosomes. Intranuclear inclusions consisting of microtubules and filaments and tight junctions between two adjacent lateral plasma membranes are occasionally encountered.     

Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16

A two‐hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule‐associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium‐sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA‐binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp–PhaP5 fusion localized at the cell poles in a PHB‐negative background and overexpression of eYfp–PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule‐associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.     

Morphological Characterization of Three Phenotypes of the Isopod Armadillidium vulgare

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black-grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron-dense central region surrounded by an electron-lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large-sized vesicles containing a small amount of electron-dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.     

Binding of microtubules to pituitary secretory granules and secretory granule membranes

Microtubules assembled in vitro were bound to purified porcine pituitary secretory granules and to isolated granule membranes. The interaction between microtubules and whole secretory granules was demonstrated by alteration in the sedimentation properties of the microtubules. Incubation of secretory granules with microtubules resulted in pelleting of microtubules which increased as a function of the number of granules added. Binding was quantitated by measurement of the tubulin remaining in the supernate after centrifugation. The interaction of secretory granules and microtubules was inhibited by nucleoside triphosphates and augmented by adenosine 5'-monophosphate and adenosine. When depolymerized protein from microtubules was incubated with secretory granules, the granules did not appear to bind the soluble tubulin dimer present in these preparations. However, the high molecular weight protein associated with microtubules was adsorbed by secretory granules during the binding process. Incubation of isolated secretory granule membranes with microtubules followed by centrifugation to density equilibrium in a discontinuous sucrose density gradient caused pelleting of the membranes, which otherwise banded higher in the gradient. The visible alteration in membrane sedimentation was confirmed by measurements of the membrane-associated magnesium-ATPase activity and by a shift in radioactivity in iodinated membrane preparations. Our data suggest a role for microtubules in the intracellular movement of secretory granules; this movement is perhaps brought about by dynein-like cross bridges which link the tubulin backbone and granule surface.     

An ultrastructural study of the prostate gland of megascolicid earthworm Lampito mauritii (Kinberg)

The ultrastructure of prostate gland of Lampito mauritii revealed two types of secretory cells. Type 1 cells with a broad basal region and a long apical region contain electron dense oval secretory granules with an increased density at the core region. Numerous electron lucent granules with fine filamentous and electron dense amorphous materials also occur at the basal region of these cells. Type 2 cells contain electron lucent mucous-like secretory granules. This cell type contains exceptionally large Golgi complexes having 20-23 stacked cisternae. Both cell types open into a common lumen and numerous microtubules are visible at the apical end. Junctional complexes, such as desmosomes and septate junctions, are observed in this glandular tissue.     

Fine structure of the kidney and characterization of secretory products inDentalium rectius (Mollusca,Scaphopoda)

Summary The ultrastructure of the scaphopod kidney and secretory product composition is described, for the first time, inDentalium rectius. The kidney epithelium consists of two primarily secretory cell types. The first exhibits extensive vacuolation, and scattered granules are formed within the vacuolar space by a process of surface accretion; the incorporation of glycogen particles in this process is associated with very fine, electronopaque threads which radiate from the granule. The second cell type possesses granules enclosed individually within secretory vesicles, and intermediate stages in their growth are characterized by needle-like crystals on the granule surface. The secretory vesicles in some cases coalesce to form a large central vacuole filled with granules. This cell type possesses an apical membrane with sparse microvilli, which may indicate a secondary reabsorptive capacity. Granules in both cell types show a concentric ring ultrastructure, and are composed primarily of calcium phosphate with a small amount of zinc; there is also an organic component of protein, mucopolysaccharide and a large amount of glycogen. Ultrastructural and histochemical observations indicate a lysosomal origin for the granules, although granules of the second cell type develop intracellularly to a greater extent than those of the first. All granules are extruded into the kidney lumen by a process of merocrine secretion prior to release into the mantle cavity via an externally ciliated, muscular excretory pore.     

Atomic force microscopy of pea starch granules: granule architecture of wild-type parent, r and rb single mutants, and the rrb double mutant

AFM studies have been made of the internal structure of pea starch granules. The data obtained provides support for the blocklet model of starch granule structure (Carbohydr. Polym. 32 (1997) 177-191). The granules consist of hard blocklets dispersed in a softer matrix material. High-resolution images have yielded new insights into the detailed structure of growth rings within the granules. The blocklet structure is continuous throughout the granule and the growth rings originate from localised defects in blocklet production distributed around the surface of spheroidal shells within the granules. A mutation at the rb locus did not lead to significant changes in granule architecture. However, a mutation at the r locus led to loss of growth rings and changed blocklet structure. For this mutant the blocklets were distributed within a harder matrix material. This novel composite arrangement was used to explain why the granules had internal fissures and also changes in gelatinisation behaviour. It is suggested that the matrix material is the amylose component of the granule and that both amylose and amylopectin are present within the r mutant starch granules in a partially-crystalline form. Intermediate changes in granule architecture have been observed for the double mutant rrb.     

Mechanism of Intranuclear Crystal Formation of Herpes Simplex Virus as Revealed by the Negative Staining of Thin Sections

Structural alterations induced in HeLa cells by herpes simplex virus and the mechanism whereby the virus is formed in the nucleus in crystal arrays were studied by electron microscopy with both the usual and negatively stained sections. Aggregates of granular and filamentous material were observed in the cytoplasm of infected cells with both sections. On the other hand, no remarkable alterations in appearance of the cytoplasmic ground substance were observed with the usual sections of infected cells. However, the cytoplasmic ground substance of infected cells when negatively stained consisted of granular material which was different in appearance from the spongy material constituting the cytoplasmic matrix of uninfected cells. In the nucleus of infected cells, complexes consisting of round bodies, amorphous material, aggregates of uniform granules in rows, and viral crystals were often observed near the nuclear membrane in both types of sections. Examinations of the granular aggregates with negatively stained sections suggested that each granule represents a subunit and that the several adjoining subunits (approximately eight) constitute the requirement for formation of a single viral capsid with a core. Thus, rapid and simultaneous formation of the core and capsid within the aggregate would replace the rows of the granules with the viral crystal. The advantages of negative staining of thin sections for visualization of fine structural alterations are discussed.     

Ferritin forms dynamic oligomers to associate with microtubules in vivo: implication for the role of microtubules in iron metabolism

Ferritin, a ubiquitously distributed iron storage protein, has been reported to interact with microtubules in vitro (Hasan et al., 2005, FEBS journal 272:822-831). Here, we demonstrate that ferritin binds with the microtubules in an oligomeric form and that the microtubule-bound ferritin contains more than two-fold amount of iron compared to the unbound ferritin fraction in vitro. Indirect immunofluorescence microscopy showed that a significant fraction of the ferritin molecules colocalized with the microtubules as oligomers in a wide variety of cell lines. These findings are consistent with the immediate oligomerization of rhodamine-labeled ferritin, microinjected in living human hepatoma cells. Ferritin oligomers were dynamic in the cytoplasm, and an anti-microtubule drug significantly inhibited their intracellular movement. Treatment of cells with an iron donor, ferric ammonium citrate, remarkably increased the number of cells containing ferritin oligomers. On the other hand, when the cells, such as mouse neuroblastoma cells, were deprived of iron, ferritin oligomers were localized in the microtubule dense, neurite shafts, but were disappeared from the microtubule deficient neurite tips. These data indicate that the microtubules provide a scaffold for the cytoplasmic distribution and transport of the iron-rich ferritin and implicate the role of microtubules in iron metabolism.     

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.     

Characteristics of mast cells in Chediak-Higashi mice: light and electron microscopic studies of connective tissue and mucosal mast cells

The beige mouse, a homologue of the Chediak-Higashi syndrome in man, possesses abnormally large granules in many tissue cells. The granules in the mucosal mast cells (MMC) of the small intestine of beige and littermate C57BL/6J mice were examined after infecting the mice with the intestinal parasite, Nippostrongylus brasiliensis. MMC in both beige and littermate mice had irregular granules which contained paracrystalline substructures embedded in an amorphous matrix. Granules were not observed in fusion with the cell membrane. Instead, in late-stage mast cells, the granule membrane broke down, the granule contents were spread throughout the cytoplasm, and the cell organelles disintegrated. Unlike connective tissue mast cells, MMC were poorly demonstrated with formalin fixation and toluidine blue staining.     

Cortical granule exocytosis is coupled with membrane retrieval in the egg of Brachydanio

Activation of the teleost (Brachydanio) fish egg includes the exocytosis of cortical granules, the construction of a mosaic surface consisting of the unfertilized egg plasma membrane and the limiting membranes of the cortical granules, and the appearance of coated and smooth vesicles in the cytoplasm (Donovan and Hart, '82). Unfertilized and activated eggs were incubated in selected extracellular tracers to (1) determine experimentally if cortical granule exocytosis was coupled with the endocytosis of membrane during the cortical reaction, and (2) establish the intracellular pathway(s) by which internalized vesicles were processed. Unfertilized eggs incubated in dechlorinated tap water or Fish Ringer's solution containing either horseradish peroxidase (HRP; 10 mg/ml), native ferritin (12.5 mg/ml), or cationized ferritin (12.5 mg/ml) were activated as judged by cortical granule breakdown and elevation of the chorion. Cells treated with HRP and native ferritin exhibited a delay in cortical granule exocytosis when compared with water-activated eggs lacking the tracer. Each tracer was internalized through the formation of a coated vesicle from a coated pit. Since coated pits appeared to be topographically restricted to the perigranular membrane domain of the mosaic egg surface, their labeling, particularly with cationized ferritin, strongly suggested that the retrieved membrane was of cortical granule origin. Cationized ferritin and concanavalin A (Con A) coupled with either hemocyanin or ferritin labeled the surface of the unactivated egg and both domains of the mosaic egg surface. Transformation of the deep evacuated cortical granule crypt into later profiles of exocytosis was accompanied by increased Con A binding. Within activated egg cortices, HRP reaction product, native ferritin, and cationized ferritin were routinely localized in smooth vesicles, multivesicular bodies, and autophagic vacuoles. Occasionally, each tracer was found in small coated vesicles adjacent to the Golgi and within Golgi cisternae. The intracellular distribution of HRP, native ferritin, and cationized ferritin suggests that internalized membrane is primarily processed by organelles of the lysosomal compartment. A second and less significant pathway is the Golgi complex.     

L-bodies are RNA–protein condensates driving RNA localization in Xenopus oocytes

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.     

APPEARANCE AND DISTRIBUTION OF FERRITIN IN MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF HETEROLOGOUS ERYTHROCYTES

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.     

The effects of colchicine on the ultrastructure of the dental epithelium and odontoblasts of teleost tooth buds

Secretory granule ultrastructure of teleost inner dental epithelial (IDE) cells has been reported to be similar to procollagen granules of other cells synthesizing collagen. This study describes the ultrastructure of secretory products in odontogenic cells during enameloid matrix formation in cichlids after inhibition of granule secretion with colchicine. Thirty-six fish were injected with 0.1 mg colchicine, then three were killed first at 2-hr intervals for 12 hr, then daily for 5 days. Tooth buds were processed for transmission electron microscopy, and ultrastructural alterations were assessed for each post-injection interval. Four hours post-injection, IDE cells contained increased numbers of secretory granules, lightly stained granules, dilated cisternae of the granular endoplasmic reticulum, and intercellular amorphous material. After 6 hr, the IDE intercellular amorphous material additionally contained electron dense deposits, and after 8 hr, the intercellular material had fibers similar in appearance to enameloid collagen. No ultrastructural changes were detected in odontoblasts that were in close proximity to the enameloid matrix. Only odontoblasts synthesizing predentin were affected by colchicine, and the observed alterations were similar to those seen in IDE cells. It is concluded that IDE cells synthesize and secrete ectodermal enameloid matrix collagen.