LYSOSOME FUNCTION IN THE REGULATION OF THE SECRETORY PROCESS IN CELLS OF THE ANTERIOR PITUITARY GLAND

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH¹ was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products.     

FUNCTIONS OF COATED VESICLES DURING PROTEIN ABSORPTION IN THE RAT VAS DEFERENS

The role of coated vesicles during the absorption of horseradish peroxidase was investigated in the epithelium of the rat vas deferens by electron microscopy and cytochemistry. Peroxidase was introduced into the vas lumen in vivo. Tissue was excised at selected intervals, fixed in formaldehyde-glutaraldehyde, sectioned without freezing, incubated in Karnovsky's medium, postfixed in OsO₄, and processed for electron microscopy. Some controls and peroxidase-perfused specimens were incubated with TPP,¹ GP, and CMP. Attention was focused on the Golgi complex, apical multivesicular bodies, and two populations of coated vesicles; large (> 1000 A) ones concentrated in the apical cytoplasm and small (<750 A) ones found primarily in the Golgi region. 10 min after peroxidase injection, the tracer is found adhering to the surface plasmalemma, concentrated in bristle-coated invaginations, and within large coated vesicles. After 20–45 min, it is present in large smooth vesicles, apical multivesicular bodies, and dense bodies. Peroxidase is not seen in small coated vesicles at any interval. Counts of small coated vesicles reveal that during peroxidase absorption they first increase in number in the Golgi region and later, in the apical cytoplasm. In both control and peroxidase-perfused specimens incubated with TPP, reaction product is seen in several Golgi cisternae and in small coated vesicles in the Golgi region. With GP, reaction product is seen in one to two Golgi cisternae, multivesicular bodies, dense bodies, and small coated vesicles present in the Golgi region or near multivesicular bodies. The results demonstrate that (a) this epithelium functions in the absorption of protein from the duct lumen, (b) large coated vesicles serve as heterophagosomes to transport absorbed protein to lysosomes, and (c) some small coated vesicles serve as primary lysosomes to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies.     

EXTRUSION OF NUCLEOLI FROM PRONUCLEI OF THE RAT

Electron microscope observations of osmium tetroxide-fixed rat eggs indicate that small nucleoli are extruded from pronuclei in a sharply demarcated time period after sperm penetration. Approximately 4½ hours after sperm penetration, fine fibrous material aggregated in distinct loci along the inner surface of the nuclear envelope and condensed into small, dense bodies. The term tertiary nucleolus or extrusion body is used to designate the forming bodies. The small tertiary nucleoli form distinct protrusions from the pronuclei during the following developmental period and finally bud off into the cytoplasm, carrying with them a small portion of the double nuclear envelope. The extrusion bodies can be observed only in the vicinity of the pronuclei and have not been seen near the cell membrane. The fate of the tertiary nucleoli is not known; apparently they transform or disappear after they have passed into the cytoplasm. Eleven hours after sperm penetration, tertiary nucleoli are not present near the nuclear membrane and the extrusion activity has apparently ceased. Large and small nucleoli react similarly to cytochemical reagents: they are Feulgen negative; they are positive to the Millon, Sakaguchi, brom-phenol blue, and PAS reactions. Azure B stain combined with nuclease extraction indicates the presence of small amounts of RNA in the nucleoli.     

Fine structural localization of peroxidase activity in the epithelium and the gland of the rat larynx

Summary Endogenous peroxidase activity was demonstrated in ciliated cells and secretory cells of the laryngeal epithelium and gland of rats, using the diaminobenzidine method for cytochemical demonstration of peroxidase activity. The intensity of peroxidase activity was greatly varied from cell to cell, but the fine structural localization of the activity was similar in various cell types. The activity was localized in cisternae of rough-surfaced endoplasmic reticulum including nuclear envelope, some vesicles and saccules of the Golgi complex, large membrane-limited granules, multivesicular bodies and probable lysosomes. In secretory cells, the activity was also found in secretory granules.The significance of peroxidase activity is not unclear, while the activity, at least a part of it, seems to be secreted into the cavity of the larynx. The possibility that peroxidase participates bactericidal mechanism, deserves further investigation.     

The hyphae ofUromyces appendiculatus within the leaf tissue after high pressure freezing and freeze substitution

Summary The fine structure of the intercellular dikaryotic hyphae of the biotrophic fungusUromyces appendiculatus was studied. High pressure freezing and freeze substitution were used to achieve a closer approximation of the native state than with conventional fixation and dehydration techniques. In addition to organelles previously described in rust fungi, heavily decorated multivesicular bodies (star bodies) were found close to the nuclei. Two types of tubular-vesicular complexes were distributed randomly within the cytoplasm of the hyphae. Furthermore, a more or less pronounced brush-like fibrillar layer on the hyphal walls was detected. The possibility that the latter two structures are correlated with the biotrophic phase of this fungus is discussed.Abbreviations TVC tubular-vesicular complex - MVB multivesicular body - M mitochondrion - N nucleus - NP nuclear pore - S septum - MT microtubule     

Death of mitochondria during programmed cell death of leaf mesophyll cells

The role of plant mitochondria in the programmed cell death (PCD) is widely discussed. However, spectrum and sequence of mitochondrial structural changes during different types of PCD in leaves are poorly described. Pea, cucumber and rye plants were grown under controlled growing conditions. A part of them were sprinkled with ethylene releaser to accelerate cell death. During yellowing the palisade parenchyma mitochondria were attracted to nuclear envelope. Mitochondrial matrix became electron translucent. Mitochondria entered vacuole by invagination of tonoplast and formed multivesicular bodies. Ethephon treatment increased the frequency of sticking of mitochondria to the nuclear envelope or chloroplasts and peroxisomes. Mitochondria divided by different mechanisms and became enclosed in Golgi and ER derived authopagic vacuoles or in the central vacuole. Several fold increase of the diameter of cristae became typical. In all cases mitochondria were attached to nuclear envelope. It can be considered as structural mechanism of promoting of PCD.     

THE INTRACELLULAR MEMBRANES OF BLASTOMYCES DERMATITIDIS

Edwards , George A., and Mercedes R. Edwards . (Div. Labs, and Research, N.Y.S. Dept. of Health, Albany.) The intracellular membranes of Blastomyces dermatitidis. Amer. Jour. Bot. 47 (8): 622–632. Illus. 1960.—The yeast cells of Blastomyces dermatitidis have been studied in thin sections with the electron microscope. The cell is multinucleate, and the nuclei are frequently interconnected by their outer limiting membranes. The cell is bordered by a cell wall and the plasma membrane, which may be seen in direct continuity with the nuclear envelope. The cytoplasm contains numerous mitochondria, many profiles of the endoplasmic reticulum, and few multivesicular bodies. The membranes of all the constant cellular components are interconnected. Mitochondria appear to be formed from any of several membrane systems. The micromorphology of the cell suggests efficiency of communication and cytoplasmic mobility.     

多泡体形成过程的细胞化学研究

本实验用酶细胞化学和示踪细胞化学方法观察了睾丸间质细胞中多泡体的形成过程及其与溶酶体的关系。实验结果表明,睾丸间质细胞中多泡体的形成可分三个阶段:首先,一些含内吞物质的泡状结构进入高尔基体区域,与那里的小泡融合,形成内含少量小泡的前多泡体;然后,前多泡体互相融合,形成体积较大、基质电子密度低、内含小泡排列稀疏的低电子密度多泡体;最后,低电子密度多泡体通过表面长出微绒毛样结构并不断断裂的方式去除多余的界膜,形成体积较小、基质电子密度高、内含小泡排列紧密的高电子密度多泡体。因此,多泡体的形成既与内吞活动有关,又与高尔基体区域小泡有关。前多泡体和低电子密度多泡体不含溶酶体酶。在多泡体形成过程中,只有到高电子密度多泡体阶段,才与溶酶体发生关系,从溶酶体中获取溶酶体酶。多泡体形成后,常与自体吞噬泡靠近,可能参与睾丸间质细胞的自体吞噬活动。     

Role of the transmembrane domain of marburg virus surface protein GP in assembly of the viral envelope

The major protein constituents of the filoviral envelope are the matrix protein VP40 and the surface transmembrane protein GP. While VP40 is recruited to the sites of budding via the late retrograde endosomal transport route, GP is suggested to be transported via the classical secretory pathway involving the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network until it reaches the plasma membrane where most filoviral budding takes place. Since both transport routes target the plasma membrane, it was thought that GP and VP40 join there to form the viral envelope. However, it was recently shown that, upon coexpression of both proteins, GP is partially recruited into peripheral VP40-enriched multivesicular bodies, which contained markers of the late endosome. Accumulation of GP and VP40 in this compartment was presumed to play an important role in the formation of the filoviral envelope. Using a domain-swapping approach, we were able to show that the transmembrane domain of GP was essential and sufficient for (i) partial recruitment of chimeric glycoproteins into VP40-enriched multivesicular bodies and (ii) incorporation into virus-like particles (VLPs) that were released upon expression of VP40. Only those chimeric glycoproteins which were targeted to VP40-enriched endosomal multivesicular bodies were subsequently recruited into VLPs. These data show that the transmembrane domain of GP is critical for the mixing of VP40 and GP in multivesicular bodies and incorporation of GP into the viral envelope. Results further suggest that trapping of GP in the VP40-enriched late endosomal compartment is important for the formation of the viral envelope.     

THE ULTRASTRUCTURE OF A MAMMALIAN CELL DURING THE MITOTIC CYCLE

With a technique of preselecting the mitotic cell in the living state for subsequent electron microscopy, it has been possible to examine the ultrastructure of the various stages of mitosis with greater precision than has been reported previously. The early dissolution of the nuclear envelope has been found to be preceded by a marked undulation of this structure within the nuclear "hof." This undulation appears to be intimately related to the spindle-forming activity of the centriole at this time. Marked pericentriolar osmiophilia and extensive arrays of vesicles are also prominent at this stage, the former continuing into anaphase. Progression of the cell through prophase is accompanied by a disappearance of these vesicles. A complex that first makes its appearance in prophase but becomes most prominent in metaphase is a partially membrane-bounded cluster of dense osmiophilic bodies. These clusters which have a circumferential distribution in the mitotic cell are shown to be derived from multivesicular bodies and are acid phosphatase-positive. The precise selection of cells during the various stages of anaphase has made it possible to follow chronologically the morphological features of the initiation of nuclear membrane reformation. The nuclear membrane appears to be derived from polar aggregates of endoplasmic reticulum, and the process begins less than 2 minutes after the onset of karyokinesis. While formation of the nuclear envelope is initiated on the polar aspects of the chromatin mass, envelope elements appear on the equatorial aspect long before the polar elements fuse. Apparently interfering with this fusion are continuous spindle tubules which traverse the chromatin mass in striking density at characteristic points. Several cortical changes, also most pronounced in anaphase, have been described, as has the kinetochore which is seen to good advantage only in this stage. The Golgi complex has been found to disappear both morphologically and histochemically during mitosis and to reappear rapidly in telophase. Evidence is presented which implicates the continuous spindle tubules in certain phases of chromosome movement.     

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

THE FINE STRUCTURE OF THE NUCLEUS AND NUCLEAR ASSOCIATIONS OF DEVELOPING CARPOSPORANGIA IN POLYSIPHONIA NOVAE-ANGLIAE (RHODOPHYTA)1

A high degree of activity of the nuclei in the developing carposporangia of the red alga Polysiphonia novae-angliae Taylor is described. Profiles of the nucleus are greatly convoluted, resulting in a much increased surface area. Regions where endoplasmic reticulum substitutes for the nuclear envelope are frequently observed. Various cytoplasmic reserves are associated with the nucleus during carposporangium maturation. Lipid bodies, fibrillar bodies, and striated vesicles (or cylindrical bodies) may occur within the nucleoplasm beneath areas of the nuclear envelope substituted by ER. Granules of Floridean starch are observed in proximity to the outer surface of these same areas as well as the nuclear membrane. The homologous nature of the nuclear envelope with the ER is stressed. The role of the nucleus as being actively involved in the synthesis of materials is suggested.     

Viral release and membrane acquisition by budding through the nuclear envelope of hemocytes of the gypsy moth, Porthetria dispar

Viral particles of the nuclear polyhedrosis virus (Baculovirus) of the gypsy moth, Porthetria dispar, appear to be released from hemocyte nuclei by budding through both inner and outer lamellae of the nuclear envelope. As a result of budding, the virus particle acquires its envelope from the inner lamella of the nuclear envelope. The outer lamella, which forms a membrane-limited vesicle around the enveloped particles, may fuse with the plasma membrane during viral release from host cells by exocytosis. These observations differ from two other reported cases of nuclear budding in NPV-infected cells in that the process occurred in the absence of nuclear inclusion bodies.     

Zinc iodide-osmium tetroxide (ZIO) reactive sites in the extrafloral nectary of Citharexylum mirianthum Cham. (Verbenaceae)

This paper reports on a study of the zinc iodide-osmium tetroxide method (ZIO) applicability to formaldehyde-glutaraldehyde prefixed extrafloral nectary tissues of Citharexylum mirianthum Cham. (Verbenaceae). The ZIO solution impregnates the dictyosome stacks and adjacent vesicles, smooth endoplasmic reticulum, nuclear envelope, multivesicular bodies, and peroxisomes. The use of this method greatly facilitates the observation and recognition of organelles in each nectary region. It also allows the correlation between structure and function in nectariferous cells.     

钙离子浓度对暗适应时的中华绒螯蟹光感受器超微结构的影响

用透射电镜研究了暗适应时中华绒螯蟹的光感受器超微结构与外界钙离子浓度的关系,结果显示出与培育在生理溶液中的光感受器相比,细胞外钙离子浓度升高,使得感杆束的直径急剧缩小,感杆束周围胞质增厚,胞饮泡增加,膜下猪泡囊极度减小。胞质中多囊体的数量和直径减小,而板模体和溶酶体的数量增加,同时细胞内的色素颗粒增多。分布在小网膜细胞的远端。细胞的结构表现为类似光适应状态,与之相反,细胞外钙离子浓度降低时小眼的感     

ESCRTs are everywhere

The ESCRT proteins are an ancient system that buds membranes and severs membrane necks from their inner face. Three “classical” functions of the ESCRTs have dominated research into these proteins since their discovery in 2001: the biogenesis of multivesicular bodies in endolysosomal sorting; the budding of HIV-1 and other viruses from the plasma membrane of infected cells; and the membrane abscission step in cytokinesis. The past few years have seen an explosion of novel functions: the biogenesis of microvesicles and exosomes; plasma membrane wound repair; neuron pruning; extraction of defective nuclear pore complexes; nuclear envelope reformation; plus-stranded RNA virus replication compartment formation; and micro- and macroautophagy. Most, and perhaps all, of the functions involve the conserved membrane-neck-directed activities of the ESCRTs, revealing a remarkably widespread role for this machinery through a broad swath of cell biology.     

THE FINE STRUCTURE OF MITOSIS AND CELL DIVISION IN THE CHRYSOPHYCEAN ALGA OCHROMONAS DANICA1

At the ultrastructural level, cell division in Ochromonas danica exhibits several unusual features. During interphase, the basal bodies of the 2 flagella replicate and the chloroplast divides by constriction between its 2 lobes. The rhizoplast, which is a fibrous striated root attached to the basal body of the long flagellum, extends under the Golgi body to the surface of the nucleus in interphase cells. During proprophase, the Golgi body replicates, apparently by division, and a daughter rhizoplast, appears. During prophase, the 2 pairs of flagellar basal bodies, each with their accompanying rhizoplast and Golgi body, begin to separate. Three or 4 flagella are already present at this stage. At the same time, there is a proliferation of microtubules outside the nuclear envelope. Gaps then appear in the nuclear envelope, admitting the microtubules into the nucleus, where they form a spindle. A unique feature of mitosis in O. danica is that the 2 rhizoplasts form the poles of the spindle, spindle microtubules inserting directly onto the rhizoplasts. Some of the spindle microtubules extend from pole to pole; others appear to attach to the chromosomes. Kinetochores, however, are not present. The nuclear envelope breaks down, except, in the regions adjacent, to the chloroplasts; chloroplast ER remains intact throughout mitosis. At late anaphase the chromosomes come to lie against part of the chloroplast ER. This segment of the chloroplast ER appears to be incorporated as part of the reforming nuclear envelope, thus reestablishing the characteristic nuclear envelope—chloroplast ER association of the interphase cell.     

THE FINE STRUCTURE OF THE PURKINJE CELL

This paper describes the fine structure of the Purkinje cell of the rat cerebellum after fixation by perfusion with 1 per cent buffered osmium tetroxide. Structures described include a large Golgi apparatus, abundant Nissl substance, mitochondria, multivesicular bodies, osmiophilic granules, axodendritic and axosomatic synapses, the nucleus, the nucleolus, and the nucleolar body. A new and possibly unique relationship between mitochondria and subsurface cisterns is described. Possible functional correlations are discussed.     

STRUCTURE AND DEVELOPMENT OF SIEVE ELEMENTS IN THE LEAF OF ISOETES MURICATA

Leaf tissue of Isoetes muricata Dur. was fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. The very young sieve elements can be distinguished from contiguous parenchyma cells by their distinctive plastids and the presence of crystalline and fibrillar proteinaceous material in dilated cisternae of the rough ER. During differentiation, the portions of ER enclosing this proteinaceous substance become smooth surfaced and migrate to the cell wall. Along the way they apparently form multivesicular bodies which then fuse with the plasmalemma, discharging their contents to the outside. At maturity, the sieve element contains an elongate nucleus, which consists of dense chromatin material, and remnants of the nuclear envelope. In addition, the mature sieve element is lined by a plasmalemma and a parietal, anastomosing network of smooth ER. Both plastids and mitochondria are present. P-protein is lacking at all stages of development. Tonoplasts are. not discernible in mature sieve elements. The end walls of mature sieve elements contain either plasmodesmata or sieve pores or both, but only plasmodesmata occur in the lateral walls.