Large Number Of Small Vesicles Research Articles

Development of highspeed and super-resolution microscopy

Live cell imaging is a powerful tool to understand the molecular mechanisms of a wide range of cellular processes. However, it is difficult to detect very small objects such as transport vesicles (< 100 nm in diameter) by light microscopy because of the diffraction limit of light. To overcome this problem, several superresolution technologies such as STED, SIM, and STORM/PALM has been developed so far, but the time resolution of these methods is not sufficient to track the small vesicles undergoing fast movement inside the cell. In the present study, we developed a highspeed, superresolution, and multicolor optical microscope, which we call SCLIM2 (the second generation superresolution confocal live cell imaging microscopy). SCLIM2 consists of an inverted microscope equipped with piezo actuator for objective lens, a spinningdisk confocal scanner, an emission splitter unit, cooled image intensifiers, and ultrafast CMOS cameras (1000 frames/s). Using this system, we acquired fluorescence images of specimens at a single photon precision. We also developed a mathematical method for 4D (xyz and time) image reconstruction and novel deconvolution algorism based on the probability calculation. The resulting lateral resolution was less than 71 nm. We then applied SCLIM2 to observe a variety of membrane traffic events in live cells. We succeeded in visualizing a large number of small vesicles (< 100 nm in diameter) were moving around the Golgi apparatus.

Generation of a Transgenic Rabbit Model of Retinal Degeneration

To generate a transgenic (Tg) rabbit model of retinal degeneration and to characterize the pattern of degeneration by using histology and electrophysiology. Rhodopsin Pro347Leu Tg rabbits were generated by BAC transgenesis. Tg rabbits were identified by Southern blot analysis, and the expression levels were measured by quantitative RT-PCR. Retinal histology was examined by light and electron microscopy and immunohistochemistry. Retinal function was assessed by full-field electroretinograms (ERGs). Six lines of Tg rabbits were generated, and two lines with higher levels of expression showed rod-dominant progressive retinal degeneration. Retinal histology indicated a marked regional variation in the loss of photoreceptors with the central retina more severely affected than the peripheral retina. The characteristics of the ERGs of transgenic rabbits indicated that the rod components of the ERGs were reduced to only 5% by 48 weeks, whereas the cone components remained at 35% in the wild-type at the same time point. The retinal ultrastructure of Tg rabbits showed a large number of small vesicles that accumulated in the extracellular space of the photoreceptors. To the best of the authors' knowledge, this is the first rabbit model of progressive retinal degeneration. Because rabbits have large eyes and are easy to handle and breed, they will provide a useful animal model for the study of the pathophysiology of and new treatments for retinal degeneration.

Fusion Gains Independence

Fusion Gains Independence

Ultrastructure of Oogenesis in Dryopteris crassirhizoma Nakai

Abstract: The ultrastructure of oogenesis in Dryopteris crassirhizoma Nakai has been investigated using transmission electron microscopy. The nucleus in the young egg is rounded with an uneven outline. As it develops, it becomes amoeboid and extends nuclear protrusions that are not only sac-like nuclear evaginations like those often seen in the oogenesis of other ferns, but also mushroom-like and finger-like, with an opening at their end allowing the nucleolus material to flow out from the openings. This has not been observed previously. The nuclear protrusions differ from Dryopteris filix-mas (L.) Schott. in the absence of sheets of nuclear membrane in the form of a closed ring. As the egg matures, the nucleus transforms into a tuber-like structure with a smooth surface, lying transversely in the egg cell. In the immature egg, vesicles almost encircle the nucleus twice and are most remarkable. In the maturing egg, the vesicles are distributed at the periphery, except for at the top of the egg, and affect the formation of the separation cavity and extra egg membrane. Simultaneously, vesicles from the venter canal cell move to the egg and take part in the formation of separation cavity and extra egg membrane. In the mature egg, a large number of small vesicles containing fragments of lamellae or osmiophilic material emerge from the cytoplasm. The origin of these vesicles is obscure. Irregular plastids containing a cylindrical starch grain dedifferentiated progressively. Mitochondria seem to have been undeveloped during the process, but return to normal at later stages of oogenesis. There is a high frequency of ribosomes in the mature egg. Microtubules, rarely seen in the eggs of D. filix-mas (L.) Schott. and Pteridium aquilinum (L.) Kuhn, have been observed inside the plasmalemma of the maturing egg in D. crassirhizoma. ( Managing editor: Wei WANG)

Fine Structure of the Mature Plasmodium of Intoshia variabili (Phylum Orthonectida), a Parasite of the Platyhelminth Macrorhynchus crocea

AbstractThe fine structure of the mature plasmodia of Intoshia variabili, found in the kalyptorhynch flatworm Macrorhynchus crocea, was studied with transmission electron microscopy. As the Plasmodium matures it forms long finger‐like extensions inside the host. These extensions eventually take the place of the genital system of the host turbellarian. The mature Plasmodium is separated from the host tissues by a continuous membrane. The entire Plasmodium is surrounded by modified host cells, which are thin and overlapping. These cells also have a rather homogenous cytoplasm with limited endoplasmic reticulum. The surface of the Plasmodium is mostly smooth and has numerous small extensions. Within the cytoplasm of the Plasmodium, different stages of development of both males and females could be observed. The cytoplasm also contains numerous small nuclei with distinct nucleoli. Scattered throughout the cytoplasm, in addition to nuclei, are mitochondria, various granules, multivesiculate bodies with lamellar structures, and a large number of small vesicles. The nature of the formation of the Plasmodium is discussed. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved

Appearance of mouse-lethal toxin in liquid cultures of Bordetella pertussis.

The mouse-lethal toxin present in liquid cultures of most smooth strains of Bordetella pertussis is known to originate in the cytoplasm of the organism but to be most lethal for mice when released into the supernatant fluid. It is also recognized that cell degeneration and lysis occur in liquid cultures during the stationary and decline phases of growth. For these reasons, it is generally believed that most of the toxicity demonstrable in liquid cultures at the time of harvest is released during the later stages of cultivation, when high alkalinity and aging of cells favor lysis. However, the results reported here have indicated that high levels of mouse-lethal toxicity arise during very early log phase and that the peak of toxicity is reached before the end of the log phase. No further increase in toxicity was observed during stationary and decline phases. The very early appearance of toxicity could not be explained by the presence in the inoculum of a proportion of dead and degenerating cells, and it is concluded that the toxin is produced mainly by actively growing cells. This was confirmed by tests on organisms growing in continuous culture. Electron-microscopic examination of cells from a very early log-phase culture revealed the presence of large numbers of small vesicles on the cell walls of about 5% of the population. It is suggested that these vesicles may be associated with the releases of toxin from living cells. It is concluded that no useful reduction in the toxicity of cultures would result from harvesting before the end of the log phase of growth.

Appearance of Mouse-Lethal Toxin in Liquid Cultures of Bordetella pertussis

The mouse-lethal toxin present in liquid cultures of most smooth strains of Bordetella pertussis is known to originate in the cytoplasm of the organism but to be most lethal for mice when released into the supernatant fluid. It is also recognized that cell degeneration and lysis occur in liquid cultures during the stationary and decline phases of growth. For these reasons, it is generally believed that most of the toxicity demonstrable in liquid cultures at the time of harvest is released during the later stages of cultivation, when high alkalinity and aging of cells favor lysis. However, the results reported here have indicated that high levels of mouse-lethal toxicity arise during very early log phase and that the peak of toxicity is reached before the end of the log phase. No further increase in toxicity was observed during stationary and decline phases. The very early appearance of toxicity could not be explained by the presence in the inoculum of a proportion of dead and degenerating cells, and it is concluded that the toxin is produced mainly by actively growing cells. This was confirmed by tests on organisms growing in continuous culture. Electron-microscopic examination of cells from a very early log-phase culture revealed the presence of large numbers of small vesicles on the cell walls of about 5% of the population. It is suggested that these vesicles may be associated with the releases of toxin from living cells. It is concluded that no useful reduction in the toxicity of cultures would result from harvesting before the end of the log phase of growth.

Photoperiodic and osmotic influences on the ultrastructure of the hypothalamic neurosecretory system of the white-crowned sparrow, Zonotrichia leucophyrs gambelii.

An attempt was made to correlate functional changes in the neurohypophysis of the White-crowned Sparrow, Zonotrichia leucophrys gambelii, with morphologic features on the light- and electron-microscope levels. The aldehyde-fuchsin-staining anterior median eminence possesses essentially the same ultrastructural features as the non-staining posterior median eminence. The axon terminals are characterized by the presence of a large number of small vesicles (approximately 400 A in diameter) and occasional electron-dense granules. The more-or-less “depleted” anterior median eminence occasionally evident in the photosensitive bird showing testicular development is indistinguishable ultrastructurally from the more intensely staining median eminence generally characteristic of the photorefractory bird. In the median eminence, stainability and functional state do not seem to be correlated with changes in the type, size or number of vesicles. A slight increase in the number of granules was noted in the photorefractory bird but this was considered insufficient basis to account for differences in stainability.

Electron microscopic observation on the hypothalamo-hypophyseal system in the rat. I. The ultrafine structure of the contact region between the external layer of the infundibulum and pars tuberalis of the anterior pitutary.

The ultrafine structure of the contact region between the infundibulum and Pars tuberalis of the anterior pituitary was investigated in rats, and following results were obtained.1) Pars tuberalis of the anterior pituitary consists of several layers of relatively small and variously shaped cells with large nuclei and conspicuous cell membrane. They contain neither secretory granules nor vacuoles except sparse mitochondria.2) Around both the capillary loops and the capillaries of the primary plexus of the portal vessels, a wide pericapillary space is always present. Its maximal width measures several micra. Collagen fibrills and numerous extremely fine granules are distributed in this space, and fibroblasts as well as the endfeets of glial processes are also occasionally observed.3) The external layer of the infundibular wall consists of numerous fibres with various calibre range. Most of these fibres are nothing but the processes of the astro lia which contain very few cytoplasmic element. The presence of nerve fibres in this layer was asccrtained. They are arranged verticaliy to the outer limiting surface of the external layer and thus parallel to each other. In addition to the sparse mitochondria, they contain a large number of small vesicles. The vesicles measure about 300-500A in diameter and even the maximal diameter does not exceed 1000A. They resemble “synaptic vesicles”. The fibre which contains neurosecretory granules of high electron density could not be found in the external layer.These characteristic structures of the contact region strongly support the concept that some chemical substance (s) is released from the nerve endings in the external layer of the infundibulum into the capillaries in the median eminence. Although the small vesicles described above, which might be regarded as the morphological representation of this chemical substance, are similar in many respects to the so-called “synaptic vesicles”, the final conclusion on the nature of the small vesicles cannot be derived at present. From the present observation, however, it is very likely that, even if the small vesicles are not identical with the synaptic vesicles, but are of neurosecretory nature, they are somewhat different in character from the ordinary neurosecretory granules contained in the nerve axons of the hypochalamo-neurohypophyseal tractus.

The fine structure of Meissner's touch corpuscles of human fingers.

Thin slices of the finger pads of six individuals were fixed in buffered 1 per cent osmic acid, embedded in deaerated, nitrogenated methacrylate, and cut into thin sections for electron microscopic study. Before embedding, the slices were trimmed so as to include several digital tactile corpuscles. Some thin sections were stained in 10 per cent aqueous phosphotungstic acid solution. The principal part of Meissner's corpuscle is made up of flattened laminar cells stretching across the corpuscle in irregular layers. The perinuclear cytoplasm of these cells contains numerous small mitochondria, a sparse granular endoplasmic reticulum, and a large number of small vesicles. Nerve fibers enter the side or base of the corpuscle, lose their myelin sheaths, and follow a meandering course between the laminar cell plates. The nerve endings enter into a close appositional relationship with the flattened portions of the laminar cells. In some areas the apposed axolemma and cell membranes are slightly thickened with small vesicles located along the cell membrane or on both surfaces. These regions are interpreted as synapses. The most prominent feature of the nerve endings is an extraordinary accumulation of small mitochondria which vary in size and internal density. The nerve endings also contain vacuoles, groups of dense concentric membranes, and small dense vesicles of irregular distribution. The laminar cells are separated from one another by a dense intercellular substance of uniform thickness which also envelops the entire corpuscle. This material contains randomly oriented collagen fibers and fine fibrils bound together by a dense material at nodal points recurring at regular intervals of approximately 120 mmicro. These findings are discussed in relation to the problems of the function of Meissner's corpuscle, neural material loss and replacement, and the presence of synapses.

An electron microscope study of the rat ovum.

This paper reports on the fine structure of rat oocytes at stages before ovulation, during maturation, fertilization, and early cleavage. The study includes parallel observations on light and electron microscope preparations with attempted correlations. The follicular cells of the ovarian egg are described as sending long processes through the zona pellucida to the egg surface where they mingle with thin projections from the egg itself. No open communication between follicle cell cytoplasm and egg cytoplasm was observed. During maturation and fertilization both types of processes are withdrawn from the zona. The germinal vesicle and later the pronuclei of the fertilized egg are characterized by numerous large nucleoli. These have the form of thick walled vesicles with diameters as great as 8 to 10 micro. The wall is dense in the EM image and appears to consist in part of small granules. The cytoplasm shows several inclusions including mitochondria of usual form and a Golgi component which has the typical fine structure and the distribution described by earlier light studies. Small dense particles, presumably RNP particles, are distributed throughout the cytoplasmic matrix and show no preference for membranes. The endoplasmic reticulum of the oocyte is represented by a scattering only of vesicles, but begins a more extensive and elaborate development with the onset of segmentation. One inclusion of the ooplasm, similar in size to mitochondria, receives special attention. It is a vesicular structure, containing a large number of small vesicles (10 to 50 mmicro in diameter) and frequently a central density or nucleoid. They are referred to as multivesicular bodies. Such bodies are found in small number in the ovarian egg, but increase greatly in number during maturation and fertilization. It appears from the micrographs of eggs in these latter stages that these vesicular bodies break down and liberate their content of small vesicles to the surrounding ooplasm. Comments are provided on the apparent significance of the various observations.