Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga Cyanidioschyzon merolae

In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions. Received: 15 April 1999 / Accepted: 12 May 1999     

Organization of interphase chromatin

The organization of interphase chromatin spans many topics, ranging in scale from the molecular level to the whole nucleus, and its study requires a concomitant range of experimental approaches. In this review, we examine these approaches, the results they have generated, and the interfaces between them. The greatest challenge appears to be the integration of information on whole nuclei obtained by light microscopy with data on nucleosome–nucleosome interactions and chromatin higher-order structures, obtained in vitro using biophysical characterization, atomic force microscopy, and electron microscopy. We consider strategies that may assist in the integration process, and we review emerging technologies that promise to reduce the “resolution gap.” This article is dedicated to the memory of Hans Ris.     

Environmental toxins and α-synuclein in Parkinson’s disease

In recent years, environmental influences have been thought to play an important role in Parkinson’s disease (PD). Evidence from epidemiological investigations suggests that environmental factors might take part in the disease process. Intriguingly, most of environmental toxins share the common mechanism of causing mitochondria dysfunction by inhibiting complex I and promoting α-synuclein aggregation, a key factor in PD. Therefore, understanding the mechanism of interactions between α-synuclein and environmental factors could lead to new therapeutic approaches to PD.     

Morphology, Anatomy and Ontogeny of Female Cones in Acmopyle pancheri(Brongn. & Gris) Pilg. (Podocarpaceae)

The developmental morphology and anatomy of the female conesof Acmopyle pancheri(Brongn. & Gris) Pilg. (Podocarpaceae)are described and illustrated, based on observations, histology,scanning electron microscopy (SEM) and nuclear magnetic resonance(NMR) imaging. Ovulate development is typically podocarpaceous.Ovules are unitegmic, and horizontal or inclined upwards throughoutontogeny; the pollination drop is inverted because of the declinatemicropyle. Ontogeny of the epimatium-ovule complex is acropetal,the epimatium developing first. A terminal, distal sterile bractcreates a pollen-scavenging area. During development, the wholecone re-orientates through some 270°, and the seed realignsapprox. 60° with respect to the receptacle axis. The ‘receptacle’or podocarpium supporting the seed is formed by gradual fusionof initially free bracts. The structures adnate to these bractsrepresent homologues of ovuliferous scales; they bear vestigialepimatia which may develop into supernumerary ovules or non-functionalepimatia. Thus, female cones ofA. pancheri are vestigially multi-ovulate.NMR imaging effectively and non-invasively revealed the three-dimensionalarrangement of vascular bundles and resin canals in the cones.Copyright 2001 Annals of Botany Company Acmopyle pancheri(Brongn. & Gris) Pilg., anatomy, developmental morphology, gymnosperms, histology, nuclear magnetic resonance (NMR) imaging, ontogeny, ovules, Podocarpaceae, scanning electron microscopy (SEM), seed cones     

Structure, morphology, and composition of silicon biocomposites in the palm tree Syagrus coronata (Mart.) Becc.

Summary.  Syagrus coronata is an economically important palm tree grown as an ornament, for the oil extracted from its seeds, and the wax from its leaves which has several applications in industry. Silicon biocomposites were analyzed in leaves of S. coronata. Silica bodies were found as extracellular silica masses between the hypodermal-layer cell walls and in granules present in the vacuoles of palisade cells. Scanning electron microscopy of the hypodermal layer of cells showed a collection of spherical bodies embedded in enveloping cavities that outlined the general structure of the bodies. Globular subunits with sharp edges formed the spherical bodies that ranged from 6 to 10 μm in diameter (average, 7.8 μm). X-ray microanalysis detected only silicon and oxygen homogeneously distributed throughout the bodies. Vacuoles of palisade cells contained a large number of granules ranging from 20 nm to 1.2 μm in size (average, 300 nm). Transmission electron microscopy associated with electron spectroscopic imaging and electron energy loss spectroscopy were used to determine the elemental composition of the granules. Vacuolar granules were amorphous and composed of silicon and oxygen, suggesting they consist of amorphous silica biominerals. No nitrogen, indicative of organic matter, was detected in the granules. Received November 26, 2001; accepted July 1, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saude, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.     

The regulatory role of α-synuclein and parkin in neuronal cell apoptosis; possible implications for the pathogenesis of Parkinson’s disease

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder beyond Alzheimer’s disease, affecting approximately 1% of people over the age of 65. The major pathological hallmarks of PD are significant loss of nigrostriatal dopaminergic (DA) neurons and the presence of intraneuronal protein inclusions termed Lewy bodies. Sporadic cases represent more than 90% of total patients with PD, while there exist several inherited forms caused by mutations in single genes. Identification and characterization of these causative genes and their products can help us understand the molecular mechanisms of DA neuronal cell death and design new approaches to treat both the inherited and sporadic forms of PD. Based on the finding that a point mutation in the gene encoding α-synuclein (αSyn) protein causes a rare familial form of PD, PARK1, it is now confirmed that αSyn is a major component of Lewy bodies in patients with sporadic PD. Abnormal accumulation of αSyn protein is considered a neurotoxic event in the development of PD. PARK4, another dominantly inherited form of familial PD, is caused by duplication or triplication of the αSyn gene locus. This genetic mutation results in the production of large amounts of wild-type αSyn protein, supporting the αSyn-induced neurodegeneration hypothesis. On the other hand, the recessively inherited early-onset Parkinsonism is caused in about half of the cases with loss-of-function mutations in PARK2, which encodes E3 ubiquitin ligase parkin in the ubiquitin–proteasome system. These findings have shed light on DA neurodegeneration caused by accumulation of toxic protein species that can be degraded and/or detoxicated through parkin activity. In this review, we will focus on the regulatory roles of αSyn and parkin proteins in DA neuronal cell apoptosis and provide evidence for the possible therapeutic action of parkin in sporadic patients with PD.     

The timing and manner of disassembly of the apparatuses for chloroplast and mitochondrial division in the red alga Cyanidioschyzon merolae

The timing and manner of disassembly of the apparatuses for chloroplast division (the plastid-dividing ring; PD ring) and mitochondrial division (the mitochondrion-dividing ring; MD ring) were investigated in the red alga Cyanidioschyzon merolae De Luca, Taddei and Varano. To do this, we synchronized cells both at the final stage of and just after chloroplast and mitochondrial division, and observed the rings in three dimensions by transmission electron microscopy. The inner (beneath the stromal face of the inner envelope) and middle (in the inter-membrane space) PD rings disassembled completely, and disappeared just before completion of chloroplast division. In contrast, the outer PD and MD rings (on the cytoplasmic face of the outer envelope) remained in the cytosol between daughter organelles after chloroplast and mitochondrial division. The outer rings started to disassemble and disappear from their surface just after organelle division, initially clinging to the outer envelopes at both edges before detaching. The results suggest that the two rings inside the chloroplast disappear just before division, and that this does not interfere with completion of division, while the outer PD and MD rings function throughout and complete chloroplast and mitochondrial division. These results, together with previous studies of C. merolae, disclose the entire cycle of change of the PD and MD rings. Received: 19 May 2000 / Accepted: 3 August 2000     

Extending the knowledge in histochemistry and cell biology

Central to modern Histochemistry and Cell Biology stands the need for visualization of cellular and molecular processes. In the past several years, a variety of techniques has been achieved bridging traditional light microscopy, fluorescence microscopy and electron microscopy with powerful software-based post-processing and computer modeling. Researchers now have various tools available to investigate problems of interest from bird’s- up to worm’s-eye of view, focusing on tissues, cells, proteins or finally single molecules. Applications of new approaches in combination with well-established traditional techniques of mRNA, DNA or protein analysis have led to enlightening and prudent studies which have paved the way toward a better understanding of not only physiological but also pathological processes in the field of cell biology. This review is intended to summarize articles standing for the progress made in “histo-biochemical” techniques and their manifold applications.     

Orderly formation of the double ring structures for plastid and mitochondrial division in the unicellular red alga Cyanidioschyzon merolae

The formation of the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) was studied in a highly synchronous culture of the unicellular red alga Cyanidioschyzon merolae. The timing and the order of formation of the MD and PD rings were determined by observing organelles around the onset of their division, using transmission electron microscopy. In  C. merolae, there is one chloroplast and one mitochondrion per cell, and the shape of the chloroplast changes sequentially from acorn-like, to round, to trapezoidal, to peanut-shaped, in that order, during the early stage of chloroplast division. None of the cells with acorn-shaped or round chloroplasts contained organelles with PD rings or MD rings, while all of the cells with peanut-shaped chloroplasts contained organelles with both PD rings and MD rings. In cells with peanut-shaped chloroplasts, the PD and MD rings were double ring structures, with an outer ring located on the cytoplasmic face of the outer membrane of the organelle, and an inner ring located in the matrix beneath the inner membrane. These results suggested that the double ring structures of the PD ring and the MD ring form when chloroplasts are trapezoidal in shape. Detailed three-dimensional observation of cells with trapezoidal chloroplasts revealed the following steps in the formation of the double ring structures of the PD and MD rings: (i) the inner ring of the PD ring forms first, followed by the outer ring; (ii) then the MD ring forms and becomes visible; (iii) when the double ring structures of the two rings have formed, the microbody then moves from its remote location to the plane of division of the mitochondrion and contraction of the PD and MD rings commences. These steps were also confirmed by computer-aided three-dimensional reconstruction of the images from serial thin sections. This study reveals the order of formation of the double ring structures of the PD and MD rings, and the behavior of the microbody around the onset of division of plastids and mitochondria. The results also provide the first evidence that the inner PD ring is not a tension element formed by the contractile pressure but a definite structure, independent of the outer ring. Received: 31 March 1998 / Accepted: 14 May 1998     

Structure and microtubule-nucleation activity of isolated Drosophila embryo centrosomes characterized by whole mount scanning and transmission electron microscopy

Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles are on average 0.75 μm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification of the number of microtubules nucleated showed that 50–300 active nucleation sites are present. We examined via electron microscopy immunogold labeling the distribution of γ-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density. In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly and protein composition.     

Advantages of intermediate X-ray energies in Zernike phase contrast X-ray microscopy

Understanding the hierarchical organizations of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. Light microscopy is a powerful tool for observations of the dynamics of live cells, its resolution attainable is limited and insufficient. While electron microscopy can produce images with astonishing resolution and clarity of ultra-thin (< 1 μm thick) sections of biological specimens, many questions involve the three-dimensional organization of a cell or the interconnectivity of cells. X-ray microscopy offers superior imaging resolution compared to light microscopy, and unique capability of nondestructive three-dimensional imaging of hydrated unstained biological cells, complementary to existing light and electron microscopy.     

Fluorescence imaging and targeted distribution of bacterial magnetic particles in nude mice

Bacterial magnetic particles (BMPs) are of interest as potential carriers of bioactive macromolecules, drugs, or liposomes. In this study, a high-pressure homogenizer was used to disrupt Magnetospirillum gryphiswaldense strain MSR-1 cells, and BMPs were purified. BMPs were labeled with fluorescence reagent 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocianin perchlorate (DiI) and injected into the tail vein of BALB/c nude mice. Distribution of fluorescence signals of DiI–BMPs in vivo was examined using a whole-body fluorescence imaging system. The result showed that fluorescence signals were detected in liver, stomach, intestine, lungs, and spleen. However, transmission electron microscopy of ultrathin sections indicated that BMPs were mainly present in liver and lungs, but not in the other organs. BMPs could be useful as carriers for targeted drug therapy of diseases of the liver or lung.     

Proliferation of the hyperthermophilic archaeon Pyrobaculum islandicum by cell fission

Pyrobaculum islandicum is a hyperthermophilic archaeon. P. islandicum cells have been suggested to multiply by constriction, budding and branching, as no septa were observed in cells by phase-contrast light microscopy. In this study, we observed the cells using transmission electron microscopy, scanning electron microscopy, and light microscopy with dark-field image analyses, and we report binary fission via septum formation to be the main mode of P. islandicum’s proliferation. “Long cells” reported previously were found to comprise several cylindrical cells that align in tandem.     

Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation

The accumulation of protein aggregates containing amyloid fibrils, with α-synuclein being the main component, is a pathological hallmark of Parkinson's disease (PD). Molecules which prevent the formation of amyloid fibrils or disassociate the toxic aggregates are touted as promising strategies to prevent or treat PD. In the present study, in vitro Thioflavin T fluorescence assays and transmission electron microscopy imaging results showed that gallic acid (GA) potently inhibits the formation of amyloid fibrils by α-synuclein. Ion mobility-mass spectrometry demonstrated that GA stabilises the extended, native structure of α-synuclein, whilst NMR spectroscopy revealed that GA interacts with α-synuclein transiently.     

Real-time analyses of chloroplast and mitochondrial division and differences in the behavior of their dividing rings during contraction

The time courses of chloroplast and mitochondrial division and the morphological changes in the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) during chloroplast and mitochondrial division were studied in Cyanidioschyzon merolae De Luca, Taddei and Varano. To accomplish this, chloroplast and cell division of living cells were continuously video-recorded under light microscopy, and the morphological changes in the PD and MD rings were analyzed quantitatively and three-dimensionally by transmission electron microscopy (TEM). Under the light microscope, the diameters of the chloroplast and the cell decreased at uniform velocities, the speed depending on the temperature. To study in detail the sequential morphological change of the mitochondrion in M phase and the contractile mechanism in the divisional planes of the chloroplast and the mitochondrion, we observed the PD and MD rings, which are believed to promote contraction, under TEM, using the diameter of the chloroplast as an index of the time. Three PD rings (an outer PD ring on the cytoplasmic face of the outer envelope, a middle PD ring in the intermembrane space, and an inner PD ring on the stromal face of the inner envelope) were clearly observed, but only the outer MD ring could be observed. The PD ring started to contract soon after it formed, while the contraction of the MD ring did not occur immediately after formation, but was delayed until the contraction of the PD ring was almost complete. Once the MD ring began to contract, the rate of decrease of its circumference was 4 times as high as that of the PD ring. As the outer PD and MD rings contracted, they grew thicker and maintained a constant volume, while the thickness of the inner PD ring did not change and its volume decreased at a constant rate with contraction. In the early stage of contraction, the widths of the three PD rings increased in order, from the outer to the inner ring. With contraction, their widths changed at different rates until they came to have much the same width. In cross-section, the MD ring was wider where it was next to the chloroplast than at the opposite side, adjacent to the nucleus in the early stage of contraction. By the late stage, the widths of the two sides became equal. In our observations, the microbody elongated along the outer MD ring and touched the outer PD ring during contraction of the PD and MD rings. These results clearly revealed differences between the mode of contraction of the outer, middle, and inner PD rings, and between the PD and the MD rings. They also revealed the coordinated widening of the three PD rings, and suggested that the microbody plays a role in the contraction of the PD and MD rings. Received: 1 July 1998 / Accepted: 1 September 1998     

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4 weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12 h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.     

Do symplasmic networks in cambial zones correspond with secondary growth patterns?

The plasmodesmal (PD) network in the cambial zone of Arabidopsis thaliana hypocotyls was analysed using electron microscopy and dye-coupling studies and compared to those of internodes of Populus nigra and Solanum lycopersicum. In all species, PD densities and frequencies undergo alterations in topologically successive cambial walls reflecting species-specific patterns of PD degradation and PD insertion during cell development. Longitudinal PD fission is responsible for an abrupt increment of PD numbers in specific walls of the youngest derivatives at the xylem and/or phloem side. Here, PDs seem to mediate positional signalling to control tissue fate and early cell determination. PD numbers at all cambial interfaces of A. thaliana correspond to those of the herbaceous tomato, but are higher with the woody poplar. This suggests a positive correlation between PD frequencies and the rapidity of cell division activity. Photoactivated green fluorescent protein (26 kDa) did not diffuse through cambial PDs of A. thaliana. This is in keeping with the common size exclusion limit (SEL) of 8–10 kDa observed for PDs at the youngest interfaces of tomato and poplar which may mediate diffusive exchange of developmental signals of equal molecular size. The regular growth patterns in internodal cambial zones of poplar and tomato result from synchronized cell division activity of neighbouring initials. A. thaliana hypocotyls have an irregular mode of secondary growth. Here, signalling through PDs in misaligned radial walls between non-homologous derivatives may control tissue development. The observed organizational differences between the cambia cast doubts on the suitability of A. thaliana as a model plant for cambial research.     

Integration of Grafted Neural Progenitor Cells in a Host Hippocampal Circuitry after Ischemic Injury

The induction of development of neurons and glial cells from neural progenitor cells (NPCs) is at present considered a promising strategy for recoverу after ischemic insult-evoked damage to the brain. To estimate whether grafted NPCs can develop morphological properties of the mature neurons and become functionally integrated within a host hippocampal circuitry, immunohistochemical approaches at the light and electron microscopy levels have been used. Ischemic insult in FVB-strain mice was evoked by 20-min-long occlusion of both carotid arteries. One day after occlusion, NPCs from GFP-transgenic fetuses were suboccipitally transplanted into the ischemic brain. We found that 44.7 ± 3.8% (mean ± s.e.m) of the grafted GFP-positive cells differentiated 3 months after transplantation into cells demonstrating morphological features of hippocampal pyramidal neurons. Moreover, grafted cells demonstrated manifestations of rather intense formation of the synapses between host and donor neural cells. Thus, our observations show that the NPC-based transplantation approach may be promising in the treatmen t of ischemic insult.     

Response of plasmodesmata formation in leaves of C4 grasses to growth irradiance

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cell interface in C₄ leaves is a key requirement for C₄ photosynthesis and occurs via plasmodesmata (PD). Here, we investigated how growth irradiance affects PD density between M and BS cells and between M cells in two C₄ species using our PD quantification method, which combines three‐dimensional laser confocal fluorescence microscopy and scanning electron microscopy. The response of leaf anatomy and physiology of NADP‐ME species, Setaria viridis and Zea mays to growth under different irradiances, low light (100 μmol m^?2 s^?1), and high light (1,000 μmol m^?2 s^?1), was observed both at seedling and established growth stages. We found that the effect of growth irradiance on C₄ leaf PD density depended on plant age and species. The high light treatment resulted in two to four‐fold greater PD density per unit leaf area than at low light, due to greater area of PD clusters and greater PD size in high light plants. These results along with our finding that the effect of light on M‐BS PD density was not tightly linked to photosynthetic capacity suggest a complex mechanism underlying the dynamic response of C₄ leaf PD formation to growth irradiance.     

Double ring structure around the constricting neck of dividing plastids ofAvena sativa

Summary Ultrastructure of the constricting neck of dividing proplastids and young chloroplasts in the first leaves ofAvena sativa was examined by electron microscopy. An electron-dense, double ring structure (plastid-dividing ring doublet; PD ring doublet) with a width of 15–40 nm was revealed around the narrow neck of the constricted and dividing plastids by serial section technique. The inner and outer ring of the doublet coated the inside (stromal side) of the inner envelope membrane and the outside (cytoplasmic side) of the outer envelope membrane, respectively. However, electron-dense materials were not observed within the lumen between the outer and inner envelope membranes.Although the PD ring doublet was commonly observed in the constricted plastids with a 70–140 nm wide neck, they could be scarcely observed in the constricted plastids with a 160 or more nm wide neck. The components of the PD ring were assumed not to be concentrated enough to identify by electron microscopy in the early stage of constriction and the PD ring may be formed and recognized at the final stage.The significance of the formation of the PD ring and its role in plastokinesis (plastid kinesis) were discussed.