Membrane contacts of the endoplasmic reticulum and their possible functions in the plant cell

Close contacts of the endoplasmic reticulum membrane and plasmalemma have been visualized inside plant cells by means of electron microscopy. The qualitative similarity of these contacts to high-permeable intercellular contacts in animals has been shown. New data confirming the hypothesis of the identity of stromules, i.e., dynamic tubular protuberances of the plastid membrane of the plant cell, and tubular elements of the endoplasmic reticulum have been presented. New possible functions of the contacts of the endoplasmic reticulum membrane with other membranes inside the cell have been discussed on the basis of this hypothesis.     

Structures of the plant trophic tract: Plastid stromules and cell-wall plasmodesmata

Plastid stromules and cell-wall plasmodesmata are special plant-cell stsructures. They were discovered a century and a half apart: stromules at the beginning of the 21st century and plasmodesmata the end of the 19th. The former and latter are intra- and intercellular fragments, respectively, of endoplasmic reticulum, which is a network for photosynthesis distribution in the plant body. Methods and history of discovery, structural similarities and differences, and series of functional interpretations are discussed. The origins of both structures are connected with photosynthesis and photosynthate export. Their tubular structure and transport function are similar. The mobility of both is under control of the actomyosin cytoskeleton. The temperature regimes of formation and functioning also are the same. Photosynthesis is possible at 0°C and even lower. The structures of the exporting network—stromules and plasmodesmata—do not form below 10°C, and after 20°C the numbers of the former and latter significantly increase in relation to growth of cytoskeleton plasticity. The structural and functional continuity of stromules and plasmodesmata are postulated as the mobile trophic tract of vascular plants.     

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.     

Organelle Identification and Characterization in Plant Cells: Using a Combinational Approach of Confocal Immunofluorescence and Electron Microscope

The plant secretory and endocytic pathways consist of several functionally distinct membrane-bounded compartments. The ultra structures of the endoplasmic reticulum, the Golgi apparatus, and central vacuoles have been well characterized via traditional structural electron microscope (EM). However, the identification of plant prevacuolar compartments (PVCs) and early endosomes (EEs) had not been achieved until more recently because of the lack of specific markers for these organelles. Recent development of fluorescent reporters for PVCs and EEs expressing in transgenic tobacco BY-2 cells and Arabidopsis plants has allowed their dynamic characterization in living cells via confocal microscopy and drug treatment, which led to their subsequent morphological identification via structural and immunogold EM. Thus, in this review, we will use our studies on PVCs and EEs as examples to present an efficient approach for organelle identification in plant cells via primary characterization of fluorescent-marked organelles in living cells and their dynamic response to drug treatments, which then serves as the basis for subsequent immunogold and structural EM studies for organelle identification. Such strategy thus represents a powerful approach in future research for the identification of novel organelles and transport vesicles in plant cells.     

Plastids and stromules interact with the nucleus and cell membrane in vascular plants

The various metabolic activities of plastids require continuous exchange of reactants and products with other organelles of the plant cell. Physical interactions between plastids and other organelles might therefore enhance the efficiency of plant metabolism. We have observed a close apposition of plastids and nuclei in various organs of Nicotiana tabacum and Arabidopsis thaliana. In hypocotyl epidermal cells, plastids and stromules, stroma-filled tubular extensions of the plastid envelope membrane, were observed to reside in grooves and infoldings of the nuclear envelope, indicating a high level of contact between the two organelle membranes. In a number of non-green tissues, including suspension-cultured cells, perinuclear plastids were frequently associated with long stromules that extended from the cell center to the cell membrane. In cotyledon petioles, cells lying adjacent to one another frequently contained stromules that met on either side of the shared cell wall, suggesting a means of intercellular communication. Our results therefore suggest that stromules have diverse roles within plant cells, perhaps serving as pathways between nuclei and more distant regions of the cell and possibly even other cells.     

Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics

Stromules are stroma-filled tubules extending from plastids whose rapid extension toward or retraction from other plastids has suggested a role in interplastidic communication and exchange of metabolites. Several studies point to sporadic dilations, kinks, and branches occurring along stromule length but have not elucidated the underlying basis for these occurrences. Similarly, although specific details on interacting partners have been missing, a consensus viewpoint suggests that stromules increase the interactive surface of a plastid with its cytoplasmic surroundings. Here, using live imaging, we show that the behavior of dynamic, pleomorphic stromules strongly coincides with that of cortical endoplasmic reticulum (ER) tubules. Covisualization of fluorescent protein-highlighted stromules and the ER in diverse cell types clearly suggests correlative dynamics of the two membrane-bound compartments. The extension and retraction, as well as directional changes in stromule branches occur in tandem with the behavior of neighboring ER tubules. Three-dimensional and four-dimensional volume rendering reveals that stromules that extend into cortical regions occupy channels between ER tubules possibly through multiple membrane contact sites. Our observations clearly depict coincidental stromule-ER behavior and suggest that either the neighboring ER tubules shape stromules directly or the behavior of both ER and stromules is simultaneously dictated by a shared cytoskeleton-based mechanism. These new observations strongly implicate the ER membrane in interactions with stromules and suggest that their interacting surfaces might serve as major conduits for bidirectional exchange of ions, lipids, and metabolites between the two organelles.     

WatershedCounting3D: a new method for segmenting and counting punctate structures from confocal image data

Current research in cell biology frequently uses light microscopy to study intracellular organelles. To segment and count organelles, most investigators have used a global thresholding method, which relies on homogeneous background intensity values within a cell. Because this is not always the case, we developed WatershedCounting3D, a program that uses a modified watershed algorithm to more accurately identify intracellular structures from confocal image data, even in the presence of an inhomogeneous background. We give examples of segmenting and counting endoplasmic reticulum exit sites and the Golgi apparatus.     

Organelle extensions in plant cells

The life strategy of plants includes their ability to respond quickly at the cellular level to changes in their environment. The use of targeted fluorescent protein probes and imaging of living cells has revealed several rapidly induced organelle responses that create the efficient sub-cellular machinery for maintaining homeostasis in the plant cell. Several organelles, including plastids, mitochondria, and peroxisomes, extend and retract thin tubules that have been named stromules, matrixules, and peroxules, respectively. Here, I combine all these thin tubular forms under the common head of organelle extensions. All extensions change shape continuously and in their elongated form considerably increase organelle outreach into the surrounding cytoplasm. Their pleomorphy reflects their interactions with the dynamic endoplasmic reticulum and cytoskeletal elements. Here, using foundational images and time-lapse movies, and providing salient information on some molecular and biochemically characterized mutants with increased organelle extensions, I draw attention to their common role in maintaining homeostasis in plant cells.

Dynamic tubules extended from different organelles are integral components of the rapid subcellular response machinery involved in maintaining optimal working conditions within plant cells.     

The role of plastids and assimilate transport system in the control of plant development

Phylogenetic and ontogenetic relationships between the plastids, cell endoplasmic reticulum, and plant transport communication have been reviewed. The initiating role of plastids (endosymbionts) in the origin of endoplasmic reticulum (buffer zone of endosymbiogenesis) has been shown, as well as a similar role of endoplasmic reticulum in the development of transport communication of xylem and phloem. Plastids, sugars and transport system for their distribution can be interpreted as leading sections in the mechanism of developmental control: gene expression of nuclear genome and genome of organelles, cell and tissue differentiation, and plant morphogenesis. The conflict between the bulk of plant genome and low percentage of its realization is explained as a result of limitation of the nuclear genome realization by plastid genome. The concept of development as applied to plant ontogenesis has been critically analyzed. The possibilities of the concept correction by with the help of symbiogenetic hypothesis are discussed.     

朱顶红花粉萌发过程中营养细胞质的超微结构变化

本文应用透射电镜对朱顶红成熟花粉水合、活化和萌发的动态过程中营养细胞质的结构和组成变化进行了观察。成熟花粉具质体、线粒体、内质网、高尔基体。微丝束以聚集体的形式存在。花粉活化后,细胞器的数目和结构发生显著变化：质体和线粒体的片层明显增加,内质网片层狭窄,高尔基体活跃产生小泡,脂体降解及微丝聚集体散开。花粉萌发后,细胞质中出现周质微管和被刺小泡,此期细胞器的变化不明显。微丝以纤丝状遍布整个花粉管中。     

Aspects of the three-dimensional intracellular organization of mesocarp cells as revealed by scanning electron microscopy

Summary The osmium-ligand binding technique and scanning electron microscopy have been applied to the study of the three-dimensional organization of mesocarp cells of a mature avocado fruit. Using this approach the mitochondria of the cells appear as elongated, branching structures and the endoplasmic reticulum consists of a complex of tubular strands, vesiculated strands and lamellar sheets. Associations of the endoplasmic reticulum with other organelles are also apparent. It is suggested that this approach provides a valuable means to assess the structural transitions in cell organization that occur during development or with functional changes.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

取食转Bt基因水稻对稻纵卷叶螟幼虫中肠的组织病理学效应

利用透射电镜观察了稻纵卷叶螟 Cnaphalocrocis medinalis（Guenée）幼虫取食转Bt基因水稻后中肠的组织病理变化。结果表明：稻纵卷叶螟幼虫取食转cry1Ab基因水稻后,中肠上皮细胞的线粒体先发生形态变化,随连续取食时间的延长线粒体出现凝聚、内嵴稀疏、空泡化等,在后期还呈凝聚态随突起脱落或沿杯腔边沿单一排列。内质网的变化也很明显,病变过程中伴随着粗糙内质网的肿胀、核糖体脱落,粗糙内质网增多等现象。细胞核的变化较小,在处理后期出现细胞核拉长、核仁聚集等变化。组织病变程度不一,有的细胞在病变早期就出现了空泡化。     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC₆ (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10–70 nm for MDCK cells and 20–90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.     

The Role of Plastids and Assimilate Transport System in the Control of Plant Development

Phylogenetic and ontogenetic relationships between the plastids, cell endoplasmic reticulum, and plant transport communication have been reviewed. The initiating role of plastids (endosymbionts) in the origin of endoplasmic reticulum (buffer zone of endosymbiogenesis) has been shown, as well as a similar role of endoplasmic reticulum in the development of transport communication of xylem and phloem. Plastids, sugars and transport system for their distribution can be interpreted as leading sections in the mechanism of developmental control: gene expression of nuclear genome and genome of organelles, cell and tissue differentiation, and plant morphogenesis. The conflict between the bulk of plant genome and low percentage of its realization is explained as a result of limitation of the nuclear genome realization by plastid genome. The concept of development as applied to plant ontogenesis has been critically analyzed. The possibilities of the concept correction by with the help of symbiogenetic hypothesis are discussed.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 165–181.Original Russian Text Copyright © 2005 by Gamalei.     

Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus

Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na2CO3 or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are "embedded" within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell.     

Intercellular bridges in ovaries of the newborn gerbil

This study describes intercellular bridges in the ovaries of neonatal gerbils. Electron microscopy has revealed the presence of true intercellular bridges, connecting oogonia or oocytes, in ovaries of newborn gerbils. The cytoplasm of the intercellular channels is similar to that of the connected cells, with mitochondria, smooth and rough endoplasmic reticulum, and free ribosomes present. Lysosomes are also occasionally present in the intercellular bridges and they may be involved in early waves of oocyte atresia. An electron-dense substance, 350-500 A thick, is located immediately beneath the unit membrane of the intercellular bridges. Accumulation of electron-dense material increases the thickness of the walls of the intercellular bridges, supporting and maintaining the patency of the channels. It is suggested that the intercellular channels probably allow the interchange of nutrients, organelles, and possibly regulatory materials as well.     

Stromuli. Plastiden‐Brücken im Netzwerk der Zelle

Plastidic bridges in the plant cell network: Stromules Stromules are mobile protrusions emanating from plastids. They might form bridges between plastids and connect them also with other compartments of the plant cell. They could be involved in coordination of plastid activities and in signalling. Stromules have been first observed in the water fern Selaginella more than 100 years ago. Later improved light microscopy enabled the visualization of stromules in higher plant plastids. 15 years ago, since plants accumulating the green fluorescing proteins (GPF) in the stroma became available they have been newly detected and are now studied intensively. Formation of stromules differs among plant tissues, developmental stages and environmental situations. Actin and myosin are required for the formation of stromules.     

Myosin in onion (Allium cepa) bulb scale epidermal cells: involvement in dynamics of organelles and endoplasmic reticulum

Studied with the fluorochrome 3,3-dihexyloxacarbocyanine iodide [(DIOC₆(3)], the dynamic system of the endoplasmic reticulum (ER) in epidermal cells of onion bulb scales consists of long, tubular strands moving together with organelles in the deeper cytoplasm, and of a less mobile network composed of tubular and lamellar elements at the cell periphery. Treatment with the sulfhydryl-reagent N-ethylmaleimide (NEM) inhibited organelle and ER movement, and caused the fusion of ER-tubules into flat sheets. Fixed, long, tubular ER strands were formed by lowering the cytosolic pH of NEM-treated cells. Both these observations indicate the involvement of myosin in the dynamics of organelles and ER. Using a monoclonal antibody against murine skeletal muscle myosin (known to cross-react with plant myosin; Tang et al. 1989, J. Cell Sci. 92: 569–574), myosin was identified by immunofluorescence microscopy. Mapping the distribution of myosin, actin filaments, ER, and organelles in different phases of recovery after centrifugation of epidermal cells, co-localization of myosin with ER and organelles but not with actin filaments was observed, supporting the hypothesis that a membrane bound motor protein exists in onion epidermal cells, which translocates organelles and the endoplasmic reticulum along actin filaments.