多泡体形成过程的细胞化学研究

Multivesicular bodies were observed frequently in electron microscope photographs of Leydig cells from normal adult rat testes. Their formation, evolution and fate were analyzed morphologically in preparations treated to show cytidine monophosphatase (CMPase) activity and in animals sacrificed at various time intervals ranging from 5 min to 2 hrs after a single intratesticular injection of cationic ferritin (CF). Analysis of morphological and cytochemical data led to the following interpretation for the origin and fate of the multivesicular bodies in Leydig cells. The formation of multivesicular bodies in Leydig cells can be divided into three steps. Step 1, some endocytic vacuoles in Golgi region fuse with small vesicles to form pre-multivesicular bodies. Step 2, the pre-multivesicular bodies fuse together to form pale multivesicular bodies which are characterized by their large size, pale matrix and paucity of internal vesicles. Step 3, the pale multivesicular bodies remove their surplus enveloping membrane to become dense multivesicular bodies which are characterized by their smaller size, dense matrix and filling with internal vesicles. The pre-multivesicular bodies and pale multivesicular bodies do not contain hydrolytic enzymes, the dense multivesicular bodies acquire their hydrolytic enzymes by fusion with lysosomes and show CMPase activity. The dense multivesicular bodies often show a very close association with autophagosomes, and they might be involved in the autophagic activity of Leydig cells.     

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。     

Acyltransferase and acid hydrolase activities of the abalone photoreceptor cell

Summary In order to study the synthesis and degradation processes of the photoreceptor membranes in the abalone, Nordotis discus, the localization of acyltransferase and acid hydrolase activities, respectively, were determined at the electron-microscopic level. Acyltransferase activity was localized on the cytoplasmic sides of thick (>10 nm) membranes of the following organelles: a few cisternae at the trans (or concave) side of Golgi apparatus, Golgi and probably related vesicles, short tubules, curved pentalaminar disks and limiting membranes of the phagosomal multivesicular bodies; all organelles were scattered in the peri- to supranuclear cytoplasm. The phospholipids, which are major components of the photoreceptor membrane, are considered to be synthesized by these membranes. Acid phosphatase activity was localized in the lumina of Golgi cisternae and vesicles, lysosomes, and smaller multivesicular and related bodies, but not in multilamellar bodies. The matrices of the larger multivesicular bodies and of the pigment granule complexes showed arylsulfatase activity. Vesiculated and autophagocytosed photoreceptor microvilli seemed to be degraded by acid hydrolases, forming multivesicular and related bodies. Supporting cells also showed acyltransferase and acid hydrolase activities.Abbreviations used in this Paper AcP acid phosphatase - ArS arylsulfatase - AT acyltransferase - ER endoplasmic reticulum - GERL Golgi-endoplasmic reticulum-lysosomal complex - MEB meshwork body - MLB multilamellar body - MVB multivesicular body - VLB vesiculolamellar body     

A protein's final ESCRT

In eukaryotic cells, delivery of transmembrane proteins into the lumen of the lysosome for degradation is mediated by the multivesicular body pathway. The function of the ESCRT protein complexes is required for both the formation of multivesicular body lumenal vesicles and the sorting of endosomal cargo proteins into these vesicles. Recent studies have identified additional factors that seem to function as an upstream cargo retention system feeding into the ESCRT machinery, given new insights into the dynamic structure of multivesicular bodies, and identified a potential mechanism for multivesicular body vesicle formation.     

Membrane production and yolk degradation in the early fly embryo (Calliphora erythrocephala meig.): An ultrastructural analysis

Formation of nuclear envelopes during the last cleavage mitosis and the formation of the cell membranes during the cellularization of the blastoderm have been studied ultrastructurally in the blowfly egg. Dense bodies arising from yolk granules by budding could contain membrane material destined to be incorporated into the new membranes of the blastoderm. The presence of transitional structures indicates that these bodies can be converted into dark multivesicular bodies. Large amounts of endoplasmic reticulum are found around the mitotic nuclei. Clusters or branched chains of vesicles associated with this are interpreted as evidence for the formation of endoplasmic reticulum by the breakdown of dark multivesicular bodies. Nuclear envelopes of mitotic daughter nuclei probably originate from endoplasmic reticulum. The egg contains both intranuclear and extranuclear annulate lamellae. The main events of cytokinesis are furrow initiation and cell membrane growth during the slow first phase, but probably only cytokinetic movement during the rapid second phase. On the assumption that cell membrane growth occurs by incorporation of complete membrane pieces, the addition of coated vesicles and/or light multivesicular bodies is definitely most probable. Some intermediate profiles indicate that light and dark multivesicular bodies are related. The membrane needed for second phase cytokinesis could well be provided by the unfolding of surface microvilli and protuberances of the furrow canal.     

THE FORMATION OF MULTIVESICULAR BODIES FROM THE NUCLEAR ENVELOPE

Cells of the gas gland of the perch Perca fluviatilis L., stimulated to increased generation of gas by the repeated emptying of the swim-bladder, were examined in the electron microscope. Intense activity of the nuclear envelope was demonstrated. Simple vesicles originating from the external nuclear membrane and the so-called multivesicular bodies derived from the outpocketings of both membranes of the nuclear envelope were observed. The multivesicular bodies were filled with numerous fine vesiculae arising from the active proliferation of their internal membrane. The authors offer two alternative mechanisms of formation of fine vesiculae inside the multivesicular bodies and the mechanism of the tearing away of these bodies from the nuclear envelope.     

Multitubular bodies in intestinal cells of Amphiuma means/tridactylum (Urodela): ultrastructural characterization

Summary The jejunal absorptive cells of the salamander Amphiuma, when examined using transmission electron microscopy, were found to possess a unique type of intracellular vacuole containing membranous tubules. These vanoles, tentatively named multitubular bodies, were located in the cytoplasm between the nucleus and the brush-border membrane, and were seen with greatest frequency in the summer and fall. The vacuoles containing multitubular bodies had an average diameter of 0.6 m, and the membranous tubules within had an average diameter of 30 nm. The tubules differed morphologically from the vesicles in the multivesicular bodies, and from the primary lysosomes in the polylysosomal vacuoles. The tubules did not exhibit acid phosphatase activity, and were of similar diameter and membrane thickness as the Golgi saccules. In contrast to the multivesicular bodies, the multitubular bodies did not take up exogenous horseradish peroxidase. Early forms of autophagosomes resembling these vacuoles were often seen in the para-Golgi region of the cell. The multitubular bodies may represent a distinct type of autophagosome. Although the exact origin of the tubules as well as their role in cellular activity is unclear, their seasonal appearance within the multitubular bodies of the absorptive cells suggests a unique means of selective down-regulation of Golgi-like organelles.     

Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.     

Regional differences in the pattern of vitellogenesis in the painted frog Discoglossus pictus

During oocyte growth in the frog Discoglossus pictus two patterns of vitellogenesis are described. The first one consists of the transformation of multivesicular bodies into yolk platelets; the second is the result of a typical endocytotic process, as described in other species. The peculiarity in Discoglossus vitellogenesis consists of a regional difference of these features of vitellogenesis in vitellogenic oocytes: the multivesicular bodies transforming into yolk platelets are found only in the germinative area—the central portion of the animal half—whereas deep crypts with numerous endocytotic pits are found only in the vegetal half. The probable meaning of this regional difference in vitellogenic oocytes is discussed.     

Receptor-mediated and adsorptive endocytosis by male germ cells of different mammalian species

Summary The routes for adsorptive and receptor-mediated endocytosis were studied in vivo after microinjection of tracers into the lumen of the seminiferous tubules, and in vitro in isolated germ cells of different mammals. Cationic ferritin was located on the plasma membrane, in vesicles, in tubules, in multivesicular bodies and in lysosome-like granules of mouse spermatocytes. In these cells the number of multivesicular bodies varied during spermatogenesis. Spermatids and to a lesser extent residual bodies also performed adsorptive endocytosis. In the rat and monkey (Macaca fascicularis) diferric transferrin was specifically taken up by germ cells via receptor-mediated endocytosis. The labelling was observed subsequently in membrane pits, vesicles, endosome-like bodies and pale multivesicular bodies. A progressive decrease in the frequency of the labelling of the germ cells by transferrin-gold particles was observed from spermatogonia to spermatocytes and to early spermatids, which could indicate that iron is particularly required by germ cells during the mitotic and meiotic processes. Adsorptive and receptor-mediated endocytosis therefore occurs in all classes of germ cells. These endocytic processes are most probably required for germ cell division, differentiation and metabolism.     

Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: A quantitative and spatial analysis

Cytokinesis in plants involves both the formation of a new wall and the partitioning of organelles between the daughter cells. To characterize the cellular changes that accompany the latter process, we have quantitatively analyzed the cell cycle-dependent changes in cell architecture of shoot apical meristem cells of Arabidopsis thaliana. For this analysis, the cells were preserved by high-pressure freezing and freeze-substitution techniques, and their Golgi stacks, multivesicular bodies, vacuoles and clathrin-coated vesicles (CCVs) characterized by means of serial thin section reconstructions, stereology and electron tomography techniques. Interphase cells possess ∼35 Golgi stacks, and this number doubles during G2 immediately prior to mitosis. At the onset of cytokinesis, the stacks concentrate around the periphery of the growing cell plate, but do not orient towards the cell plate. Interphase cells contain ∼18 multivesicular bodies, most of which are located close to a Golgi stack. During late cytokinesis, the appearance of a second group of cell plate-associated multivesicular bodies coincides with the onset of CCV formation at the cell plate. During this period a 4× increase in CCVs is paralleled by a doubling in number and a 4× increase in multivesicular bodies volume. The vacuole system also undergoes major changes in organization, size, and volume, with the most notable change seen during early telophase cytokinesis. In particular, the vacuoles form sausage-like tubular compartments with a 50% reduced surface area and an 80% reduced volume compared to prometaphase cells. We postulate that this transient reduction in vacuole volume during early telophase provides a means for increasing the volume of the cytosol to accommodate the forming phragmoplast microtubule array and associated cell plate-forming structures.     

Ultrastrukturen der zellulären Autophagie

Zusammenfassung Im Laufe der Cytoplasmareduktion während der Spermiogenese von Eisenia foetida sind zwei Vorgänge zu unterscheiden: 1. Die Bildung autophagischer Vakuolen. Sie entstehen, indem Teile des Grundcytoplasmas in das Kompartiment des ER verlagert werden. Da sie keine Reaktion auf saure Phosphatase geben, sind sie als nicht lysosomale Anfangstadien der zellulären Autophagie zu betrachten. 2. Die Bildung primärer Lysosomen. Sie entstehen in Form von lytische Enzyme enthaltenden Golgivesikeln, die von einer neu im Cytoplasma entstehenden Membran zu größeren Einheiten zusammengefaßt werden: den multivesicular bodies. Autophagische Vakuolen und multivesicular bodies gelangen ins Cytophor das am Ende der Spermiogenese den Charakter eines ausgedehnten Autophagosoms annimmt. Als Struktureigentümlichkeit entstehen in ihm undulierende Tubulikörper. Der coat an den Hüllmembranen junger multivesicular bodies und am Plasmalemm der Spermatidenverbindung zum Cytophor wird in Zusammenhang mit der Membrandifferenzierung diskutiert.

---

Ultrastructural equivalents of cellular autophagyElectronmicroscopical observations on spermatids of Eisenia foetida during the cytoplasmic reduction

Summary During the cytoplasmic reduction phase in the spermiogenesis of Eisenia foetida two different processes may be defined: 1. The formation of autophagic vacuoles, which arise by the displacement of cytoplasmic portions into the cisternae of the endoplasmic reticulum. Since they exhibit no acid phosphatase activity they are considered to be early stages in cellular autophagy. 2. The formation of primary lysosomes. They originate in Golgi vesicles and are then enveloped by a membrane, formed in the cytoplasm de novo, which transforms them into multivesicular bodies. Autophagic vacuoles and multivesicular bodies subsequently transfer to the cytophor, which contains at the end of the spermiogenesis the characteristics of a large autophagosom, showing aggregates of undulating tubules. The outer coat of the limiting membranes in the early multivesicular bodies and of the cell membrane of the connecting piece between spermatid and cytophor appear to be associated with the membrane development.

     

Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes

Hrs and the endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are involved in the endosomal sorting of membrane proteins into multivesicular bodies and lysosomes or vacuoles. The ESCRT complexes are also required for formation of intraluminal endosomal vesicles and for budding of certain enveloped RNA viruses such as HIV. Here, we show that Hrs binds to the ESCRT-I subunit Tsg101 via a PSAP motif that is conserved in Tsg101-binding viral proteins. Depletion of Hrs causes a reduction in membrane-associated ESCRT-I subunits, a decreased number of multivesicular bodies and an increased size of late endosomes. Even though Hrs mainly localizes to early endosomes and Tsg101 to late endosomes, the two proteins colocalize on a subpopulation of endosomes that contain lyso-bisphosphatidic acid. Overexpression of Hrs causes accumulation of Tsg101 on early endosomes and prevents its localization to late endosomes. We conclude that Hrs mediates the initial recruitment of ESCRT-I to endosomes and, thereby, indirectly regulates multivesicular body formation.     

Ultrastructural effects of silicic acid on primary lung fibroblasts in tissue culture

Transmission and scanning electron microscopic examination of primary lung fibroblasts exposed in tissue culture to polymeric silicic acid (PSA) revealed profound cellular changes in the cell surface membranes, resulting in rapid endocytosis of affected membranes and formation of multivesicular bodies. Exposure to monomeric silicic acid did not appear to exhibit any immediate adverse effects. Appearance of numerous cytoplasmic vacuoles within 1 h of PSA exposure was easily visible by light microscopy. Electron microscopy revealed that PSA exposure caused formation of an 'osmiophilic' cell surface membrane. Numerous osmiophilic cytoplasmic blebs on the surface and subsequent endocytotic vesicles appeared to collapse and aggregate into multivesicular bodies. This study provides ultrastructural evidence of the direct interaction between lung fibroblasts and polymeric silicic acid, which has a dramatic effect the surface membrane, its subsequent internalization and cytoplasmic processing. This interaction could be one of the key steps in the damaging effects of silica containing dust.     

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.     

Lamellar bodies are the intracellular site of membrane turnover in insect fat body

Analysis of the time course of highly cationic ferritin uptake by fat body cells has shown that the tracer bound to the plasma membrane and was pinocytosed by coated vesicles. The first sites of intracellular accumulation were multivesicular bodies which became filled with ferritin between 30-60 min after cells were exposed to the tracer. At no time during the experiments were any parts of the Golgi complex labeled by the tracer. By 60 min, the ferritin was increasingly found in lamellar bodies. The different types of 'light' and 'dark' multivesicular bodies suggest that lamellar bodies form from multivesicular bodies as they fill with tracer. The occurrence of lamellar bodies in many different cell types suggests an important role in membrane dynamics.     

Photoreceptor membrane breakdown in the spider Dinopis: The fate of rhabdomere products

Summary Photoreceptor membrane breakdown at dawn in the posterior median eyes of the spider Dinopis is described. Coated and smooth vesicles are shed into the receptor cytoplasm and are assembled into multivesicular bodies of two kinds: (i) Coated vesicles form loosely-assembled multivesicular bodies (mvbs) whose bounding membranes are derived from endoplasmic reticulum. (ii) Smooth vesicles generated by the mass disintegration of membrane aggregate to yield tightly-assembled multivesicular bodies which are not membrane-bound. Both types are either lysed in the inter-rhabdomeral cytoplasm, or degrade via multi-lamellar bodies to residual bodies (rbs) while they are being transported to the intermediate segments. Two systems are associated with lysis. Nebenkerne produced by the rapid differentiation of GERL in the intermediate segments fuse with membrane-bound mvbs or rbs and may inject them with hydrolases. Partially-differentiated rigid tubules (Blest et al., 1978) travel to the receptive segments together with RER from the intermediate segments and also fuse with or engulf mvbs. Both systems may contain pro-enzymes which are activated at their target sites. No evidence of a close or necessary geometrical relationship between GERL and Golgi bodies has been seen, and there is no clear demarcation between RER, smooth ER and GERL which is entering into continuity with or engulfing mvbs. The implications of these findings for hypotheses about the origins of isolation membranes and autolytic systems in invertebrate systems are briefly discussed.The authors thank Professor D.T. Anderson, F.R.S., for our use of field facilities at the Crommelin Biological Field Station of Sydney University at Warrah, Pearl Beach, N.S.W., Andrew & Sally Austin and Sally Stowe for help in the field, and Joanne Maples for technical assistance. Professor T.H. Waterman and Dr. V.B. Meyer-Rochow kindly gave us access to certain of their results prior to publication. We are indebted to Rod Whitty and the Electron Microscopy Unit for advice and support throughout these studies     

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     

DISTRIBUTION OF PHOSPHATASES IN THE GOLGI REGION AND ASSOCIATED STRUCTURES OF THE THORACIC GANGLIONIC NEURONS IN THE GRASSHOPPER, MELANOPLUS DIFFERENTIALIS

The neuronal perikarya of the grasshopper contain sudanophilic lipochondria which exhibit an affinity for vital dyes. These lipochondria are membrane-delimited and display acid phosphatase activity; hence they correspond to lysosomes. Unlike those of most vertebrates, these lysosomes also hydrolyze thiamine pyrophosphate and adenosine triphosphate. Like vertebrate lysosomal "dense bodies," they are electron-opaque and contain granular, vesicular, or lamellar material. Along with several types of smaller dense bodies, they are found in close spatial association with the Golgi apparatus. The Golgi complexes are frequently arranged in concentric configurations within which these dense bodies lie. Some of the smaller dense bodies often lie close to or in association with the periphery of dense multivesicular bodies. Further, bodies occur that display gradations in structure between these multivesicular bodies and the dense lysosomes. Acid phosphatase activity is present in the small as well as the larger dense bodies, in the multivesicular bodies, and in some of the Golgi saccules, associated vesicles, and fenestrated membranes; thiamine pyrophosphatase is found in both the dense bodies and parts of the Golgi complex. The close spatial association of these organelles, together with their enzymatic similarities, suggests the existence of a functional or developmental relationship between them.     

Formation of primordial yolk granules in the avian oocyte

Summary Electron and light microscopical investigations of early oocytes (between 1.0 mm and 5.0 mm in diameter) from the ovary of 28–30 week-old chickens, suggested the formation of primordial yolk granules from cytoplasmic vesicles. These vesicles formed an aggregation which was observed to be surrounded by membranes, giving the aggregate a multivesicular body-like appearance. At a later stage the vesicles inside the membrane disintegrated and the multivesicular bodies acquired the appearance of primordial yolk granules. The contribution of other structures to the formation of yolk granules is discussed.For constructive criticism I am very grateful to Dr. Hadar Emanuelsson, Institute of Zoophysiology, Lund. The excellent technical assistance of Miss Inger Antonsson and Mrs. Annagreta Petersen is gratefully acknowledgedThis work was supported by Kungliga Fysiografiska Sällskapet, Lund