Endoplasmic Reticulum Sheets Research Articles

Further assembly required: construction and dynamics of the endoplasmic reticulum network

The endoplasmic reticulum (ER) is a continuous membrane system comprising the nuclear envelope, ribosome-studded peripheral sheets and an interconnected network of smooth tubules extending throughout the cell. Although protein biosynthesis, transport and quality control in the ER have been studied extensively, mechanisms underlying the notably diverse architecture of the ER have only emerged recently; this review highlights these new findings and how they relate to ER functional specializations. Several protein families, including reticulons and DP1/REEPs/Yop1, harbour hydrophobic hairpin domains that shape high-curvature ER tubules and mediate intramembrane protein interactions. Members of the atlastin/RHD3/Sey1 family of dynamin-related GTPases mediate the formation of three-way junctions that characterize the tubular ER network, and additional classes of hydrophobic hairpin-containing ER proteins interact with and remodel the microtubule cytoskeleton. Flat ER sheets have a different complement of proteins implicated in shaping, cisternal stacking and microtubule interactions. Finally, several shaping proteins are mutated in hereditary spastic paraplegias, emphasizing the particular importance of proper ER morphology and distribution for highly polarized cells.

Formation of the Fertilization Pore during Oogenesis of the Fern Ceratopteris thalictroides

The development of the fertilization pore during oogenesis of the fern Ceratopteris thalictroides was followed using transmission electron microscopy. The newly formed egg is appressed closely to the adjacent cells. There are well-developed plasmodesmata between the egg and the ventral canal cell, but none between the egg and the jacket cells of the archegonium. During maturation, a separation cavity is formed around the egg. However, a pore region persistently connects the egg and the ventral canal cell. The extra egg membrane is formed by deposition of sheets of endoplasmic reticulum (ER), but no ER is deposited on the inner surface of the pore region. Thus, a fertilization pore, covered by a layer of plasmalemma, is formed. The ventral canal cell undoubtedly participates the formation of the fertilization pore, probably by absorbing the sheets of ER beneath the pore region. The functional significance of the ventral canal cell in formation of the fertilization pore is discussed. The features of the mature egg include that abundant concentric membranes and osmiophilic vesicles occur in the cytoplasm of the mature egg. The initial, round nucleus of the egg eventually becomes cup-shaped. This investigation gives some new insights about the cells participating oogenesis in ferns.

Mechanisms Shaping the Membranes of Cellular Organelles

Cellular organelles have characteristic morphologies that arise as a result of different local membrane curvatures. A striking example is the endoplasmic reticulum (ER), which consists of ER tubules with high curvature in cross-section, peripheral ER sheets with little curvature except at their edges and the nuclear envelope with low curvature except where the nuclear pores are inserted. The ER may be shaped by several mechanisms. ER tubules are often generated through their association with the cytoskeleton and stabilized by two families of integral membrane proteins, the reticulons and DP1/Yop1p. Similar to how curvature is generated in budding vesicles, these proteins may use scaffolding and hydrophobic insertion mechanisms to shape the lipid bilayer into tubules. In addition, proteins of the dynamin family may deform the ER membrane to generate a tubular network. Mechanisms affecting local membrane curvature may also shape peripheral ER sheets and the nuclear envelope as well as mitochondria and caveolae.

Direct observation of molecular arrays in the organized smooth endoplasmic reticulum

BackgroundTubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization.ResultsHere, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation - do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets.ConclusionOur results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.

Cellular differentiation in moss protonemata: a morphological and experimental study.

Previous studies of protonemal morphogenesis in mosses have focused on the cytoskeletal basis of tip growth and the production of asexual propagules. This study provides the first comprehensive description of the differentiation of caulonemata and rhizoids, which share the same cytology, and the roles of the cytoskeleton in organelle shaping and spatial arrangement. Light and electron microscope observations were carried out on in vitro cultured and wild protonemata from over 200 moss species. Oryzalin and cytochalasin D were used to investigate the role of the cytoskeleton in the cytological organization of fully differentiated protonemal cells; time-lapse photography was employed to monitor organelle positions. The onset of differentiation in initially highly vacuolate subapical cells is marked by the appearance of tubular endoplasmic reticulum (ER) profiles with crystalline inclusions, closely followed by an increase in rough endoplasmic reticulum (RER). The tonoplast disintegrates and the original vacuole is replaced by a population of vesicles and small vacuoles originating de novo from RER. The cytoplasm then becomes distributed throughout the cell lumen, an event closely followed by the appearance of endoplasmic microtubules (MTs) in association with sheets of ER, stacks of vesicles that subsequently disperse, elongate mitochondria and chloroplasts and long tubular extensions at both poles of the nucleus. The production of large vesicles by previously inactive dictysomes coincides with the deposition of additional cell wall layers. At maturity, the numbers of endoplasmic microtubules decline, dictyosomes become inactive and the ER is predominantly smooth. Fully developed cells remain largely unaffected by cytochalasin; oryzalin elicits profound cytological changes. Both inhibitors elicit the formation of giant plastids. The plastids and other organelles in fully developed cells are largely stationary. Differentiation of caulonemata and rhizoids involves a remarkable series of cytological changes, some of which closely recall major events in sieve element ontogeny in tracheophytes. The cytology of fully differentiated cells is remarkably similar to that of moss food-conducting cells and, in both, is dependent on an intact microtubule cytoskeleton. The disappearance of the major vacuolar apparatus is probably related to the function of caulonema and rhizoids in solute transport. Failure of fully differentiated caulonema and rhizoid cells to regenerate is attributed to a combination of endo-reduplication and irreversible tonoplast fragmentation. The formation of giant plastids, most likely by fusion, following both oryzalin and cytochalasin treatments, suggests key roles for both microtubules and microfilaments in the spatial arrangement and replication of plastids.

Transmembrane domain–dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein–protein and protein–lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail–anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER–Golgi boundary by simple receptor-independent mechanisms based on partitioning.

Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells

Plasma membrane blebbing is a defining characteristic of apoptosis, but its significance is not understood. Using live-cell imaging we have identified two phases of apoptotic blebbing. The early phase is restricted to adherent cells, and is prevented by the Rho-activated kinase inhibitor Y27632. The late phase is partially resistant to Y27632, and generates morphologically distinct membrane protrusions that are likely precursors to apoptotic bodies. Late blebbing is observed in all apoptotic cells tested. It occurs at a fixed period before phosphatidyl serine exposure, indicating that it is a universal and important feature of apoptosis. Late blebs contain a cortical layer of endoplasmic reticulum that often surrounds condensed chromatin, while other organelles are excluded. The appearance in some apoptotic cells of partially formed sheets of endoplasmic reticulum suggest that these cortical layers are newly formed by the remodelling of the endoplasmic reticulum of interphase cells. Formation of endoplasmic reticulum and chromatin-containing blebs requires both actin and microtubules, and is prevented by the caspase-6 inhibitor zVEID.fmk.

Ultrastructure of tetrasporogenesis in the red alga Osmundea spectabilis var. spectabilis (Rhodomelaceae: Ceramiales: Rhodophyta)

Tetrasporogenesis in Osmundeaspectabilis (Postels and Ruprecht) K.W. Nam var. spectabilis proceeds through three developmental stages. A uninucleate tetraspore mother cell with synaptonemal complexes depicted during early prophase of meiosis I comprises the youngest stage. Subsequently, the nuclear envelope obtains a highly irregular profile and is surrounded by perinuclear endoplasmic reticulum (ER) and numerous mitochondria. Proplastids with peripheral thylakoid and numerous straight-profiled dictyosomes are present. Dictyosome vesicles discharge their contents, initiating tetraspore wall formation. A four-nucleate tetraspore mother cell is formed prior to tetrahedral cleavage. During the second stage of tetraspore formation the nuclei are very irregular in outline and move toward the central region where they fuse. The formation of fibrous vacuoles originates from the fibrous vacuole associated organelles (FVAOs). Thylakoid formation from concentric lamellar bodies (CLBs) seems to occur in plastids. Extremely hypertrophied dictyosomes with two tightly compressed and densely laminated mid-regions produce vesicles which contribute to enlargement of the fibrous vacuoles. During the second stage of tetraspore development striated vesicles are formed and starch granule formation increases. Mature tetraspores are characterized by numerous cored vesicles and abundant starch granules. Plastids have a fully developed system of internal thylakoids, and cleavage is usually complete at this stage. Mature tetraspores are surrounded by a bi-layered wall and the tetrasporangial wall. Unbranched, tubular structures connecting the plasmalemma with the peripheral sheets of ER are possibly involved in wall deformation. A distinct blebbing nuclear envelope appears to sequester vesicles into the cytoplasm.

Three dimensional configuration of the secretory pathway and segregation of secretion granules in the yeast Saccharomyces cerevisiae.

The structural elements of the secretory pathway in the budding yeast Saccharomyces cerevisiae were analyzed by 3D stereo-electron microscopy using relatively thick sections in which membranes were selectively impregnated. In a wild-type strain, tubular networks of various sizes and staining properties were distributed throughout the cytoplasm. As a rule, wide-meshed, lightly stained polygonal networks were connected to more or less fenestrated sheets of endoplasmic reticulum (ER). Some of these networks were continuous with more intensely stained networks and narrower meshes that displayed at their intersections nodular dilations that progressively increased in size and staining properties to reach those of secretion granules. Such networks presumably corresponded to Golgi elements. Indeed, stacked cisternae typical of the mammalian Golgi apparatus are rarely found in wild-type cells. However, if it is assumed that the Golgi apparatus plays a key role in the segregation and maturation of secretion granules, then tubular networks with nodular dilations should be equivalent to parts of this organelle. In correlation with the increase in size and density of the nodules there was a decrease in diameter and staining intensity of the interconnecting tubules. These results parallel observations on the formation of secretory granules in mammalian cells and suggest that the segregation of secretory material is concomitant with the progressive perforation and tubulization of previously unperforated sheets. When the sec21-3 thermosensitive mutant was examined at the nonpermissive temperature (37 degrees C), the secretory pathway was blocked at exit from the ER, which started to accumulate as clusters of narrow, anastomosed, unperforated ribbon-like elements. When the block was released by shifting down to permissive temperature (24 degrees C), tubular networks of various sizes and caliber, presumably Golgi in nature, formed as soon as 5 minutes after release of the block. At later time intervals, granules of various sizes and densities appeared to be released by rupture of these tubular networks or even to form at the edges of ER fenestrae. These observations support a dynamic maturation process in which the formation of secretion granules occurs by means of an oriented series of membrane transformations starting at the ER and culminating with the liberation of secretion granules from Golgi networks.

Nodal endoplasmic reticulum, a specialized form of endoplasmic reticulum found in gravity-sensing root tip columella cells.

The endoplasmic reticulum (ER) of columella root cap cells has been postulated to play a role in gravity sensing. We have re-examined the ultrastructure of columella cells in tobacco (Nicotiana tabacum) root tips preserved by high-pressure freezing/freeze-substitution techniques to gain more precise information about the organization of the ER in such cells. The most notable findings are: the identification of a specialized form of ER, termed "nodal ER," which is found exclusively in columella cells; the demonstration that the bulk of the ER is organized in the form of a tubular network that is confined to a peripheral layer under the plasma membrane; and the discovery that this ER-rich peripheral region excludes Golgi stacks, vacuoles, and amyloplasts but not mitochondria. Nodal ER domains consist of an approximately 100-nm-diameter central rod composed of oblong subunits to which usually seven sheets of rough ER are attached along their margins. These domains form patches at the interface between the peripheral ER network and the ER-free central region of the cells, and they occupy defined positions within central and flanking columella cells. Over one-half of the nodal ER domains are located along the outer tangential walls of the flanking cells. Cytochalasin D and latrunculin A cause an increase in size and a decrease in numbers of nodal ER domains. We postulate that the nodal ER membranes locally modulate the gravisensing signals produced by the sedimenting amyloplasts, and that the confinement of all ER membranes to the cell periphery serves to enhance the sedimentability of the amyloplasts in the central region of columella cells.

Developmental Transitions and Dynamics of the Cortical ER of Arabidopsis Cells Seen with Green Fluorescent Protein

Arabidopsis thaliana plants were stably transformed with DNA encoding green fluorescent protein and with sequences ensuring retention in the endoplasmic reticulum (ER). Confocal laser scanning microscopy shows fluorescent ER in many cells of seedlings so allowing developmental changes to be documented. The arrangement of the cortical ER changes as cells mature in the hypocotyl and root epidermis. In the root, cells that have completed expansion have reticulate cortical ER resembling the ER described in many previous studies. Expanding cells, however, show extensive perforated sheets of cortical ER which transform quite abruptly into a loose reticulum at the basipetal end of the elongation zone. The reticulum compacts in trichoblasts beginning at sites where root hairs are about to emerge. The compacted form is maintained throughout the hair until growth ceases and the open reticulate form returns. All forms of cortical ER are dynamic and we use a color overlay method to distinguish stable and moving structures in a single composite image. Reticulate ER continuously rearranges its polygonal layout and perforations move and change their shape in the ER sheets of younger cells. ER deeper in the cell (i.e. not close to the plasma membrane) moves more actively so that almost no tubules remain stable even over short periods of less than one minute. The function of the perforated sheets of cortical ER present in growing cells is unknown.

Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization

The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization.

Studies on the Sporogenous Lineage in the Moss Timmiella barbuloides IX. Development of the Tapetum

The ultrastructure of tapetal cells in Timmiela barbuloides was investigated in relation to events of sporogenesis. After their establishment both internally and externally to the sporogonial initials, tapetal cells enlarge and assume a permanently polarized organization after completion of meiosis. A large vacuole is formed in the cell region distal to the spore sac, the nucleus becomes centrally located, and amyloplasts lie in the cytoplasm adjacent to the spore sac. An extensive endomembrane system develops in tapetal cells during the stage of exine deposition in spore tetrads. Sheets of rough endoplasmic reticulum develop first around the nucleus then also in close proximity to the plasma membrane abutting the spore sac. Concomitantly, intervening dictyosomes produce a variety of vesicles. Unusual structures with vesicle-like profiles also occur in the inner tapetum cell walls abutting the spore sac. At the same time most of the starch is lost from the plastids in which grana-fretwork systems develop. A massive secretion of extremely electron-opaque material is associated with perine deposition onto the free spore surfaces. Degeneration of the tapetal cells during the terminal stages of spore maturation is marked by distortion of the organelles, increase in vacuolation and the appearance of electron-opaque material between the sheets of endoplasmic reticulum.

Polarity of the ascidian egg cortex before fertilization.

The unfertilized ascidian egg displays a visible polar organization along its animal-vegetal axis. In particular, the myoplasm, a mitochondria-rich subcortical domain inherited by the blastomeres that differentiate into muscle cells, is mainly situated in the vegetal hemisphere. We show that, in the unfertilized egg, this vegetal domain is enriched in actin and microfilaments and excludes microtubules. This polar distribution of microfilaments and microtubules persists in isolated cortices prepared by shearing eggs attached to a polylysine-coated surface. The isolated cortex is further characterized by an elaborate network of tubules and sheets of endoplasmic reticulum (ER). This cortical ER network is tethered to the plasma membrane at discrete sites, is covered with ribosomes and contains a calsequestrin-like protein. Interestingly, this ER network is distributed in a polar fashion along the animal-vegetal axis of the egg: regions with a dense network consisting mainly of sheets or tightly knit tubes are present in the vegetal hemisphere only, whereas areas characterized by a sparse tubular ER network are uniquely found in the animal hemisphere region. The stability of the polar organization of the cortex was studied by perturbing the distribution of organelles in the egg and depolymerizing microfilaments and microtubules. The polar organization of the cortical ER network persists after treatment of eggs with nocodazole, but is disrupted by treatment with cytochalasin B. In addition, we show that centrifugal forces that displace the cytoplasmic organelles do not alter the appearance and polar organization of the isolated egg cortex. These findings taken together with our previous work suggest that the intrinsic polar distribution of cortical membranous and cytoskeletal components along the animal-vegetal axis of the egg are important for the spatial organization of calcium-dependent events and their developmental consequences.

The disruption of dictyosomal integrity by caffeine in the green algal flagellate, Gloeomonas Kupfferi

ABSTRACT The endomembrane system of the green algal flagellate, Gloeomonas kupfferi, is a highly ordered complex of endoplasmic reticulum sheets, 14–18 dictyosomes, vesicles and 8–12 peripheral vacuoles centered around a large nucleus. Each dictyosome is a highly polar cisternal stack with distinct cis and trans faces and an assortment of different-sized vesicles. The methylxanthine, caffeine, in concentrations of 1–2 mM, has been shown to be an effective disrupting agent of dictyosome integrity. For short treatments (1 h), caffeine causes an inhibition of typical trans-face vesiculation. Dictyosomes expand laterally and trans-face cisternal edges fuse. The resultant morphological products includeexaggerated circular profiles and multidictyosomal masses. During longer treatment times (3–4 h), cisface cistemae swell and ultimately disintegrate into large vesicles. Trans-face cistemae also vesiculate but do so into masses of tiny vesicles. The caffeine effects are reversible for up to 12 h, after which time cytological changes result in cell death. Phosphatase cytochemistry was also employed in this study to help reveal localized effects of caffeine. A putative explanation of the action of caffeine is presented.

Structure and Operation of the Feeding Apparatus in a Colorless Euglenoid, Entosiphon sulcatum1

ABSTRACTEntosiphon sulcatum is a phagotrophic euglenoid. The tubular ingestion apparatus, called a siphon, is composed of three microtubular rods extending the length of the cell. Within the tube are four large striated vanes arranged much like the blades in a pinwheel. The vanes arise from the microtubular rods and curve towards the center of the feeding apparatus. Sheets of endoplasmic reticulum are positioned adjacent to each of the vanes and surround the perimeter of the apparatus. A cap, supported by a scaffold and anchored into the cytoplasm, covers the opening of the siphon. An elongate invagination of the plasma membrane is positioned adjacent to the edge of the cap and extends downward into the siphon forming the opening. The vanes converge at the anterior end of the siphon and surround the invagination. During feeding, the siphon protrudes from the cell. As the apparatus protrudes the cap is withdrawn to the side, opening the siphon. The vanes spread apart expanding the invagination of the plasma membrane into a large cavity into which ingested food particles are taken.

Cytoplasmic deletion processes during spermatogenesis in mosses

AbstractComparative ultrastructural observations reveal that cytoplasmic deletion during spermatogenesis in Sphagnum and other mosses (Bryopsida) has two distinct phases. In young spermatids, Golgi‐derived vesicles produce the mucopolysaccharide sheaths in which the gametes are liberated. Golgi bodies, however, play no part in removal of cytoplasm during gamete maturation. Rounding off of the cells during this process results in a 50% reduction in volume.Mid‐spermatid stages in Sphagnum are characterised by the sequential loss of Golgi bodies and endoplasmic reticulum (ER) but no further diminution of the cytoplasm. The final stages of nuclear metamorphosis and chromatin condensation, in late spermatids, are marked by the sudden appearance, in the otherwise featureless central cytoplasm, of a membrane vesicle complex (MVC) comprising cisternae, tubules, and smooth and coated vesicles. Following repositioning of the MVC beneath the plasma membrane, rapid shrinkage of the cytoplasm is associated with the presence of vesicle fusion profiles at the cell surface. The MVC is considered to be intimately involved in cytoplasmic breakdown and loss. Acid phosphatase activity can be detected throughout spermatogenesis. Spermatogenous cells and young spermatids possess relatively low levels of the enzyme, restricted to the ER and perinuclear space, but particularly high levels occur in the MVC region of late spermatids of Sphagnum.The deletion process in Bryopsida is much more gradual than that of Sphagnum. Mid‐spermatids contain sheets of ER, Golgi with small vesicles, and irregular cisternae associated with coated vesicles. Vacuoles derived either from dilation of the ER or the coated vesicle complexes gradually increase in size and number at the expense of the cytoplasm. During the early stages of chromatin condensation, a large central vacuole opens onto the anterior face of the gametes. Further discharge of vesicles continues throughout gamete maturation.A comparative survey of spermatogenesis in land plants indicates that cytoplasmic deletion is achieved in different ways in different groups. We speculate that the spermatozoids of the common ancestor of archegoniate plants probably possessed large amounts of cytoplasm. The deletion mechanisms may have originated from a contractile vacuole apparatus.

Ultracytochemical localization of X-prolyl-dipeptidyl (amino)peptidase in microglobules and endoplasmic membranes accumulated in pep4-3 mutant of Saccharomyces cerevisiae

The ultracytochemical localization of X-prolyl-dipeptidyl (amino)peptidase (DPP) activity was studied in a late exponential culture of a haploid (alpha) wild-type strain of Saccharomyces cerevisiae and its pep4-3 mutant. Yeast cells were fixed for 20 min in cold 1% glutaraldehyde buffered with 50 mM TES buffer to pH 7.0 and then incubated for 80 min with 1.2 mM L-alanyl-L-proline-4-methoxy-2-naphthylamide (Ala-Pro-MNA) or Lys-Pro-MNA as cytochemical substrates plus 0.06% hexazonium p-rosaniline (HPR) buffered with 160 mM cacodylate to pH 7.0. The osmiophilic azoindoxyl complex was formed by coupling HPR with MNA liberated by DPP activity and was then osmicated during an overnight post-fixation of cells in cold 1% OsO4. In the wild-type strain, conspicuous deposits of DPP reaction product were observed in vacuolar membranes. When compared with the parent strain, the pep4-3 mutant cells were enriched in endoplasmic reticulum (ER), cytoplasmic lipoprotein, and microcompartments: membranous vesicles and microglobules. In the mutant, DPP reaction product was found in about 50% of non-vacuolated cells at the following sites: the nuclear envelope, polar layers of ER sheets and of membranous vesicles (diameter, 40-90 nm), the surface or the lumen of these vesicles, the cytoplasmic membrane (under some bud scars) and the periplasmic space. The largest amount of reaction product was found in microglobules (diameter, 20-50 nm) that were mainly observed in the cytoplasmic matrix but were also present in nuclei (nucleoli) and mitochondria. These microglobules had a single-line boundary and appeared to be composed of lipoprotein. The surface ultrastructure of sectioned microglobules in the cytoplasmic matrix was similar to that of the coated vesicles found in mammalian cells. Only sparse amounts of DPP reaction product were seen in budding yeast. In all pep4-3 cells with electron-lucent vacuoles, the reaction product was confined to the vacuolar membranes (i.e. homologous to the ER), microglobules and the periplasmic space. Polysaccharides with free vic-groups were shown by the cytochemical reaction to be present on the surface of ER membranes, in microglobules, in the periplasmic space and in the cell wall. Our cytochemical results indicate that microglobules participate in the exocytosis of both DPP and glycoproteins, and reveal new features of vacuolar morphogenesis in yeast.

The ultrastructure of the zoospore of Hyphochytrium catenoides

The ultrastructure of the zoospore of Hyphochytrium catenoides Karling is described. The zoospore has a single, anterior, tinsel flagellum. The nucleus is elongate and convoluted with an indentation at the anterior end in which the Golgi cisternae are located. There are large lipid globules in the posterior end of the cell. The ribosomes are loosely enclosed by endoplasmic reticulum, the nuclear envelope, and mitochondria. The mitochondria have tubular cristae in a dense matrix. Microbodies are found appressed to the nuclear envelope and also free in the ribosomal region. Endoplasmic reticulum sheets traverse the ribosome region. The vesiculate cytoplasm has several distinct types of membrane-bound inclusions: (i) multivesicular bodies, (ii) vesicles containing presumptive mastigonemes, (iii) vesicles having an electron-dense cortex with an electron-transparent center, and (iv) electron-opaque vesicles whose contents seem condensed and only partially fill the vesicles. The transition zone from the flagellum to the kinetosome has three segments: a distal set of struts extending from the axonemal doublets into the axoneme core, a midsection of electron-opaque rings, and a distinctive "disclike" terminal plate with a thickened portion between the doublets and the flagellar membrane. The three-part rootlet system has (i) a "ribbed" pair of microtubules on one side of the kinetosome, (ii) a curved "ribbed," single microtubule with electron-opaque backing which originates near the nonfunctional centriole, and (iii) a straight doublet of microtubules without ribs extending from the nonfunctional centriole posteriorly to the midregion of the zoospore.

か牛脂質代謝に関する研究 Ｉ オートラジオグラフィー的研究

The lipid metabolism in the guinea pig cochlea was studied by means of autoradiography with 3H-glycerol. Following results were obtained.1) In this autoradiographic study, the most heavily labelled cells were the outer hair cells, especially intensive along the cell membrane, where there were the sheets of endoplasmic reticulum (subsurface cisterna).2) In the hair cells the radioactivity increased gradually as the apex was approached, but in the other cells this longitudinal gradient was not seen.3) Kanamycin depressed the incorporation of 3H-glycerol into the hair cells, especially outer hair cells. These results, together with the results obtained by biochemical and histochemical studies (ref. 2nd report), will be discussed in 2nd report.