scholarly journals Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana

PROTOPLASMA ◽  
2021 ◽  
Author(s):  
Saki Fukushima ◽  
Kae Akita ◽  
Tomoko Takagi ◽  
Keiko Kobayashi ◽  
Nobuko Moritoki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Complex Structure ◽  
Three Dimensional ◽  
Mitochondrial Matrix ◽  
Chemical Fixation ◽  
Giant Mitochondria ◽  
Ultrathin Sections ◽  
Green Fluorescent

AbstractMitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

Visualization of individual astrocytes in three-dimensional cerebellar tissues using green fluorescent protein

1996 ◽  
Vol 54 ◽  
pp. 908-909
Author(s):  
M. D. Andersen ◽  
D. H. Szarowski ◽  
J. N. Turner ◽  
W. Shain
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Complex Structure ◽  
Three Dimensional ◽  
Cellular Interactions ◽  
Organotypic Cultures ◽  
Stable Transfectants ◽  
The Central Nervous System ◽  
Green Fluorescent

The central nervous system is a complex structure traditionally studied through selective stains and two-dimensional images of fixed tissues. A long term goal of this laboratory is the study of dynamic cellular interactions in three dimensional tissues such as organotypic cultures. Due to the large volume of tissue, traditional microscopy methods contain excessive out-of-focus data making it difficult to observe clearly specific cellular interactions. The visualization of morphological changes in specific cells in living, three-dimensional tissues is commonly done with vital dyes. An alternate approach is labeling individual astrocytes with Green Fluorescent Protein (GFP) and injecting them into organotypic cultures.GFP is a naturally fluorescent protein originally isolated from the jellyfish Aequorea victorea. To label individual astrocytes, LRM55 astroglial cells were transfected with GFP using calcium phosphate. The percentage of stable transfectants was low (< 1%) resulting in expression of varying amounts of GFP, as seen in Figs 5 and 6.

scholarly journals The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial Bud

1998 ◽  
Vol 142 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Koji Okamoto ◽  
Philip S. Perlman ◽  
Ronald A. Butow
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Mitochondrial Matrix ◽  
Outer Membranes ◽  
Marker Proteins ◽  
The Matrix ◽  
Green Fluorescent ◽  
Mitochondrial Reticulum

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

scholarly journals Microvascular Sprouting, Extension, and Creation of New Capillary Connections with Adaptation of the Neighboring Astrocytes in Adult Mouse Cortex under Chronic Hypoxia

2013 ◽  
Vol 34 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Kazuto Masamoto ◽  
Hiroyuki Takuwa ◽  
Chie Seki ◽  
Junko Taniguchi ◽  
Yoshiaki Itoh ◽  
...  
Keyword(s):  
Adult Mouse ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Mouse Cortex ◽  
Hypoxia Adaptation ◽  
Green Fluorescent

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14  μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60  μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

Developmental derivation of vocal fold mucosa from human induced pluripotent stem cells

2019 ◽  
Author(s):  
Vlasta Lungova ◽  
Susan Thibeault
Keyword(s):  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Vocal Fold ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Stratified Squamous Epithelium ◽  
Three Dimensional Models ◽  
Induced Pluripotent ◽  
Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. We report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa.

scholarly journals Phosphatidylinositol-4,5-Bisphosphate-Rich Plasma Membrane Patches Organize Active Zones of Endocytosis and Ruffling in Cultured Adipocytes

2004 ◽  
Vol 24 (20) ◽  
pp. 9102-9123 ◽  
Author(s):  
Shaohui Huang ◽  
Larry Lifshitz ◽  
Varsha Patki-Kamath ◽  
Richard Tuft ◽  
Kevin Fogarty ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Actin Dynamics ◽  
Ph Domain ◽  
Membrane Ruffling ◽  
Green Fluorescent

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.

scholarly journals A New Model for Nuclear Envelope Breakdown

2001 ◽  
Vol 12 (2) ◽  
pp. 503-510 ◽  
Author(s):  
Mark Terasaki ◽  
Paul Campagnola ◽  
Melissa M. Rolls ◽  
Pascal A. Stein ◽  
Jan Ellenberg ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Second Phase ◽  
New Model ◽  
Nuclear Envelope Breakdown ◽  
Two Phases ◽  
Green Fluorescent

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

scholarly journals Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix

1998 ◽  
Vol 140 (4) ◽  
pp. 821-829 ◽  
Author(s):  
Arthur Partikian ◽  
Bence Ölveczky ◽  
R. Swaminathan ◽  
Yuxin Li ◽  
A.S. Verkman
Keyword(s):  
Green Fluorescent Protein ◽  
Aqueous Phase ◽  
Correlation Time ◽  
Fluorescent Protein ◽  
Rotational Correlation Time ◽  
Mitochondrial Matrix ◽  
Phase Diffusion ◽  
The Matrix ◽  
Green Fluorescent ◽  
Metabolite Channeling

Abstract. It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100× objective (0.8-μm spot diam) gave half-times for fluorescence recovery of 15–19 ms with &gt;90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2–3 × 10−7 cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid β-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 ± 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, ∼0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.

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