Autophagy Is Involved in the Reduction of Myelinating Schwann Cell Cytoplasm during Myelin Maturation of the Peripheral Nerve

Peripheral nerve myelination involves dynamic changes in Schwann cell morphology and membrane structure. Recent studies have demonstrated that autophagy regulates organelle biogenesis and plasma membrane dynamics. In the present study, we investigated the role of autophagy in the development and differentiation of myelinating Schwann cells during sciatic nerve myelination. Electron microscopy and biochemical assays have shown that Schwann cells remove excess cytoplasmic organelles during myelination through macroautophagy. Inhibition of autophagy via Schwann cell-specific removal of ATG7, an essential molecule for macroautophagy, using a conditional knockout strategy, resulted in abnormally enlarged abaxonal cytoplasm in myelinating Schwann cells that contained a large number of ribosomes and an atypically expanded endoplasmic reticulum. Small fiber hypermyelination and minor anomalous peripheral nerve functions are observed in this mutant. Rapamycin-induced suppression of mTOR activity during the early postnatal period enhanced not only autophagy but also developmental reduction of myelinating Schwann cells cytoplasm in vivo. Together, our findings suggest that autophagy is a regulatory mechanism of Schwann cells structural plasticity during myelination.     

Programmed-cell-death events during tapetum development of angiosperms

Summary Programmed-cell-death events in the tapetum of two angiosperms (Lobivia rauschii Zecher andTillandsia albida Mez et Purpus) are described by ultrastructural methods. Tapetum degradation appears to be a type of programmed cell death, with the cellular remnants necessary for pollen development, acting as products of holocrine secretion. Diagnostic features of apoptosis during tapetum development are: general shrinkage of the whole cell and the nuclei; condensation of the chromatin at the periphery of the internal nuclear membrane; the enlargement of the endoplasmicreticulum cisternae to circumscribe portions of the cytoplasm; the persistence of mitochondria together with microfilament bundles until the last stages of tapetal degeneration.     

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis)

Summary. In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine. Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, People’s Republic of China.     

Francisella tularensis Harvests Nutrients Derived via ATG5-Independent Autophagy to Support Intracellular Growth

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.     

Identification, subcellular localization, and developmental studies of oleosins in the anther of Brassica napus

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15-residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of ～ 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N-terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of low-density organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule-filled plastids and lipid-containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low-density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil-body oleosins share common epitopes at the generally non-conserved C-terminal domain with the sporophytic tapetum oleosins.     

The Formation of Pollenkitt in Apium nodiflorum (Apiaceae)

Apium nodiflorum produces a considerable amount of pollenkittwithin a secretory tapetum. ER and plastids (elaioplasts) participatein pollenkitt formation. Both organelles deposit precursorswithin vesicles, which finally fuse to form pollenkitt. Apium nodiflorum L., Apiaceaesecretory tapetum, pollenkitt, ER, plastids, elaioplasts     

Self-eating to grow and kill: autophagy in filamentous ascomycetes

Autophagy is a tightly controlled degradation process in which eukaryotic cells digest their own cytoplasm containing protein complexes and organelles in the vacuole or lysosome. Two types of autophagy have been described: macroautophagy and microautophagy. Both types can be further divided into nonselective and selective processes. Molecular analysis of autophagy over the last two decades has mostly used the unicellular ascomycetes Saccharomyces cerevisiae and Pichia pastoris. Genetic analysis in these yeasts has identified 36 autophagy-related (atg) genes; many are conserved in all eukaryotes, including filamentous ascomycetes. However, the autophagic machinery also evolved significant differences in fungi, as a consequence of adaptation to diverse fungal lifestyles. Intensive studies on autophagy in the last few years have shown that autophagy in filamentous fungi is not only involved in nutrient homeostasis but in other cellular processes such as cell differentiation, pathogenicity and secondary metabolite production. This mini-review focuses on the specific roles of autophagy in filamentous fungi.     

Cellular organisation and differentiation of organelles in pre-meiotic rice anthers

Pre-meiotic cellular organisation of rice anthers has a great significance in pollen formation. We have used a combination of confocal laser and transmission electron microscopy (TEM) to characterise and differentiate organelles in pre-meiotic rice anthers. Along with the characteristic organelles in the cytoplasm the epidermal cells of the pre-meiotic rice anther are coated on their outer surface by a conspicuous bi-lamellate cuticle. Chloroplasts of the endothecium contain immature grana, thylakoids and also starch granules. These plastids clearly contain photosynthetic pigments as shown by autofluorescence in confocal microscope studies. Both confocal and TEM studies reveal clusters of mitochondria in the middle layer. The tapetum contains electron opaque ribosomes, bundles of mitochondria and plastids. The nuclei of the tapetum occupy a large volume of the cytoplasm indicating the onset of mitotic prophase. Intense Rhodamine 123 staining reveals that a major portion of the structurally indistinguishable organelles that were seen throughout the densely ribosomic cytoplasm of sporogenous cells are mitochondria.     

Ultrastructural Studies of Microsporogenesis in Phaseolus vulgaris L.

Microsporogenesis in dwarf Phaseolus vulgaris was studied under the electron microscope. Before meiosis the microspore mother cell had a lot of organelles especially plastids and ER in its cytoplasm. There were many osmiophilic granules adhering to the membranes of the plastids and vesicular ER until meiosis began. Some cytoplasmic channels were present between adjacent microsporocytes from pachytene to telophase Ⅱ. The organelles were at early stage in the early rnlcrospore, the plastids and mitochondria of which showed regional distribution. Original vacou[es were produced by smooth ER. The organelles in the tapetum cells were mainly mitochondria, plastids and ER. The ER was concentric circles in shape in transverse section.     

Differentiation of the Tapetum During Microsporogenesis in Tomato (Lycopersicon esculentum Mill.), with Special Reference to the Tapetal Cell Wall

During microsporogenesis and pollen maturation, the tapetumin anthers of tomato (Lycopersicon esculentum) underwent severalultrastructural changes and ultimately degenerated. The changesobserved related to the secretory function of the tapetum andto the transfer of materials from the cytoplasm to the surfaceof tapetal cells. Electron dense deposits, initially in thevacuoles, disappeared coincident with the appearance of orbiculeson the cell wall. The fibrillar wall of the tapetal cells loosened,presumably to facilitate transfer of materials through the wall.In Addition, membranous fragments were a consistent featurein the tapetum wall and may play a role in transport of materials.The cells of the inner tapetum (towards the connective) andouter tapetum (towards the epidermis) had different ultrastructuralfeatures. The cytoplasm of the outer tapetum was more electrondense and had a higher proportion of dictyosomes and mitochondriathan the inner tapetum, indicating the greater secretory natureof the outer tapetum. The plastids and mitochondria also differedin morphology between the two regions. Degenerations of thetapetal cytoplasm began by the vacuolate microspore stage. Atanthesis, cytoplasm was absent but the orbicular wall of thetapetum remained appressed to the wall of the middle layer ofthe anther.Copyright 1993, 1999 Academic Press Lycopersicon esculentum, microsporogenesis, pollen development, tapetum development, tomato, ultrastructure     

Amorphous areas in the cytoplasm of Dendrobium tepal cells

In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy.     

Fate of green plastids in Dinophysis caudata following ingestion of the benthic ciliate Mesodinium coatsi: Ultrastructure and psbA gene

Phototrophic Dinophysis species are known to acquire plastids of the cryptophyte Teleaulax amphioxeia through feeding on the ciliate Mesodinium rubrum or M. cf. rubrum. In addition, several molecular studies have detected plastid encoding genes of various algal taxa within field populations of Dinophysis species. The trophic pathway by which Dinophysis species acquire plastids from algae other than the cryptophyte genus Teleaulax, however, is unknown. In this study, we examined the fate of prey organelles and plastid genes obtained by Dinophysis caudata through ingestion of Mesodinium coatsi, a benthic ciliate that retains green plastids of Chroomonas sp. Transmission electron microscopy and molecular analysis revealed relatively rapid digestion of prey-derived plastids. Following digestion of M. coatsi, however, photodamaged D. caudata cells having olive-green rather than reddish-brown plastids were able to recover some of their original reddish-brown pigmentation. Results further suggest that plastid genes of various algal taxa detected in field populations of Dinophysis species may reflect prey diversity rather than sequestration of multiple plastid types. Ingestion and digestion of prey other than M. rubrum or M. cf. rubrum may also provide nutritional requirements needed to repair and perhaps maintain sequestered T. amphioxeia plastids.     

The role of autophagy in the midgut epithelium of Eubranchipus grubii (Crustacea,Branchiopoda, Anostraca)

Eubranchipus grubii (Crustacea, Branchiopoda, Anostraca) is an omnivorous filter feeder whose life span lasts no more than 12 weeks. Adult males and females of E. grubii were used for ultrastructural studies of the midgut epithelium and an analysis of autophagy. The midgut epithelium is formed by columnar digestive cells and no regenerative cells were observed. A distinct regionalization in the distribution of organelles appears – basal, perinuclear and apical regions were distinguished. No differences in the ultrastructure of digestive cells were observed between males and females. Autophagic disintegration of organelles occurs throughout the midgut epithelium. Degenerated organelles accumulate in the neighborhood of Golgi complexes, and these complexes presumably take part in phagophore and autophagosome formation. In some cases, the phagophore also surrounds small autophagosomes, which had appeared earlier. Fusion of autophagosomes and lysosomes was not observed, but lysosomes are enclosed during autophagosome formation. Autophagosomes and autolysosomes are discharged into the midgut lumen due to apocrine secretion. Autophagy plays a role in cell survival by protecting the cell from cell death.     

Pollen and Tapetum Development in Male Fertile Rosmarinus Officinalis L. (Lamiaceae)

The development of microspores/pollen grains and tapetum was studied in fertile Rosmarinus officinalis L. (Lamiaceae). Most parts of the cell walls of the secretory anther tapetum undergo modifications before and during meiosis: the inner tangential and radial cell walls, and often also the outer tangential and radial wall, acquire a fibrous appearance; these walls become later transformed into a thin poly-saccharidic film, which is finally dissolved after microspore mitosis. Electron opaque granules found within the fibrous/lamellated tapetal walls consist of sporopollenin-like material, but cannot be interpreted as Ubisch bodies. The middle lamella and the primary wall of the outer tangential and radial tapetal walls remain unmodified, but get covered by an electron opaque, sporopollenin-like layer. Pollenkitt is formed only by lipid droplets from the ground plasma and/or ER profiles, the plastids do not form pollenkitt precursor lipids. Tapetum maturation (“degeneration”) does not take place before late vacuolate stage.

The apertures are determined during meiosis by vesicles or membrane stacks on the surface of the plasma membrane. The procolumellae are conical, but at maturity the columellae are more cylindrical in shape. The columellar bases often fuse, but a genuine foot layer is lacking. The formation of the endexine starts with sporopollenin-accumulating white lines adjacent to the columellar bases. Later, the endexine grows more irregularly by the accumulation of sporopollenin globules. In mature pollen the intine is clearly bilayered.

Generative cells (GCs) and sperm cells contain a comparatively large amount of cytoplasm, and organelles like mitochondria, dictyosomes, ER, and multi-vesicular bodies, but no plastids; GCs and sperms are separated from the vegetative cell only by two plasma membranes.     

Ultrastructural aspects of cytoplasmic male sterility inPetunia hybrida

Summary Anther development of isogenic male fertile and cytoplasmic male sterile types ofPetunia hybrida cv. Blue Bedder is studied by electron microscopy. First deviation in sporogenesis of the sterile type, is observed during leptotene stage of the meiocytes. Initial aberration is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells. These vacuoles reveal the first aspects of degeneration; no other ultrastructural differences are observed. Vacuolation is accompanied by the condensation of cytoplasmic organelles. The tapetal cells become distorted and ultrastructural aberrations in mitochondria do occur. The mitochondria elongate and contain several tubular cristae.Substantial evidence suggests, that cytoplasmic male sterility in petunia is encoded by the mitochondrial genome (Boeshore el al. 1983). However, before degeneration becomes manifest, no consistent ultrastructural differences in mitochondrial organization are observed.Abortion of the tapetum and the sporogenous tissue in cytoplasmic male sterile plants, generally follows a corresponding pattern. Ultimately, the cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present.     

Cytological evidence for preservation of mitochondrial and plastid DNA in the mature generative cells of Chlorophytum spp. (Liliaceae)

Summary.  Following 4′,6-diamidino-2-phenylindole staining of mature pollen grains of Chlorophytum comosum, fluorescence microscopy confirmed that cytoplasmic nucleoids (DNA aggregates) were present in the generative cells, which indicated the possibility of biparental cytoplasmic inheritance. Electron and immuno-electron microscopy showed that both plastids and mitochondria were present in the generative cells, and both organelles contained DNA. These results indicate that mitochondria and plastids of C. comosum have the potential for biparental inheritance. Similar results were obtained with mature pollen grains of C. chinense. Therefore, we conclude the coincident biparental inheritance for mitochondria and plastids in the members of the genus Chlorophytum. Received June 28, 2002; accepted September 26, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: College of Life Science, Peking University, Bejing 100871, People's Republic of China.     

蕨类植物紫萁绒毡层细胞凋亡的细胞学特征

绒毡层凋亡过程是小孢子发生中的重要事件,以往的研究主要集中在被子植物,蕨类植物尚未见此方面的报道。该研究首次采用透射电镜和免疫荧光技术对蕨类植物紫萁(Osmunda japonica Thunb.)绒毡层细胞凋亡的细胞学过程进行了观察,以明确紫萁绒毡层细胞的发育类型和凋亡特征,为蕨类植物绒毡层细胞凋亡的深入研究以及孢子发育研究提供依据。结果显示：(1)紫萁的绒毡层属于复合型,即外层绒毡层为分泌型,该层细胞发育过程中液泡化,营养物质被吸收;内层绒毡层为原生质团型,经历了细胞凋亡的过程。(2)绒毡层内层细胞在凋亡过程中细胞壁和细胞膜降解,细胞质浓缩且空泡化;细胞核内陷、变形,染色质浓缩凝聚,形成多数小核仁,DAPI荧光由强变弱;线粒体、质体、内质网、高尔基体等细胞器逐渐退化,液泡中多包含纤维状物、絮状物、黑色嗜锇颗粒和小囊泡等;出现多泡体、多膜体和细胞质凋亡小体,上述特征与种子植物绒毡层凋亡特征基本一致。(3)与种子植物相比,紫萁绒毡层的细胞凋亡开始得早,在整个凋亡过程中没有核凋亡小体的产生;除了产生孢粉素外,绒毡层细胞内产生了大量的丝状物质、絮状物质和电子染色暗的颗粒物,这些物质可能用于...     

红盖鳞毛蕨孢子囊早期发育与质体分化的研究

利用光学显微镜和透射电子显微镜观察了红盖鳞毛蕨(Dryopteris erythrosora(Eaton)O.Ktze.)孢子囊的发育及在此期间质体的分化过程。研究表明:(1)红盖鳞毛蕨孢子囊的发育类型属于薄囊蕨型;(2)绒毡层为混合型,即内层绒毡层为原生质团型,外层绒毡层为腺质型;(3)孢子囊原始细胞中的质体通过3条路径分化,其一,原始细胞中含淀粉粒的质体通过分裂分配到下方细胞,继而进入孢子囊柄;其二,原始细胞分裂产生的新生质体被分配到上方细胞,进而被分配到除顶细胞外的原基细胞中,顶细胞将含淀粉粒的质体通过分裂分配到外套层原始细胞中;其三,顶细胞也将具淀粉粒的质体通过分裂分配到内部细胞,使分裂产生的孢原细胞和绒毡层原始细胞具新生质体;造孢细胞和孢子母细胞的质体具淀粉粒,孢子母细胞还具油体,新生孢子中具造粉体和油体;两层绒毡层具新生质体,随着退化外层绒毡层出现造粉体,内层绒毡层出现油体;(4)红盖鳞毛蕨与少数被子植物小孢子发育阶段质体分化模式类似,由前质体分化为造粉体再到油体。研究结果为蕨类植物质体在孢子囊发育过程不同组织细胞中的差异分化提供了新观察资料,为蕨类植物发育生物学和系统演化研究提供科学依据。     

Apoptosis in the Initial Leaf of Etiolated Wheat Seedlings: Influence of the Antioxidant Ionol (BHT) and Peroxides

Apoptosis was observed in the initial leaf of 5-8-day-old etiolated wheat seedlings. A condensation of cytoplasm in apoptotic cells, formation of myelin-like structures, specific fragmentation of cytoplasm, appearance in vacuoles of specific vesicles containing subcellular organelles, condensation and margination of chromatin in the nucleus, and internucleosomal fragmentation of nuclear DNA are ultrastructural features of apoptosis in the initial wheat leaf. Single-membrane vesicles detected in vacuoles of the leaf cells resemble in appearance the vacuolar vesicles in the coleoptile apoptotic cells described earlier (Bakeeva, L. E., et al. (1999) FEBS Lett., 457, 122-125); they contain preferentially plastids but not mitochondria as was observed in coleoptile. The vacuolar vesicles are specific for the apoptotic plant cells. Thus, apoptosis in various tissues is an obligatory element of plant (wheat) growth and development even in the early stages of ontogenesis. Contrary to strong geroprotecting action in coleoptile, the known antioxidant BHT (ionol, 2.27·10^–4 M) does not prevent in the leaf cells the apoptotic internucleosomal DNA fragmentation and appearance of specific vacuolar vesicles containing subcellular organelles. Therefore, the antioxidant action on apoptosis in plants is tissue specific. Peroxides (H₂O₂, cumene hydroperoxide) stimulated apoptosis (internucleosomal DNA fragmentation) in coleoptile and induced it in an initial leaf when apoptosis in a control seedling leaf was not yet detected. Thus, apoptosis that is programmed in plant ontogenesis and controlled by reactive oxygen species (ROS) can be modulated by anti- and prooxidants.