scholarly journals A CLEM approach to access to the ultrastructure at the graft interface in Arabidopsis thaliana

2021 ◽  
Author(s):  
Clément Chambaud ◽  
Sarah Jane Cookson ◽  
Nathalie Ollat ◽  
Emmanuelle M. F. Bayer ◽  
Lysiane Brocard
Keyword(s):  
Electron Microscopy ◽  
Arabidopsis Thaliana ◽  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Cellular Events ◽  
Membrane Tethering ◽  
Secondary Plasmodesmata ◽  
Graft Interface

Despite recent progress in our understanding of the graft union formation, we still know little about the cellular events underlying the grafting process. This is partially due to the difficulty of reliably targeting the graft interface in electron microscopy to study its ultrastructure and three-dimensional architecture. To overcome this technological bottleneck, we developed a correlative light electron microscopy approach (CLEM) to study the graft interface with high ultrastructural resolution. Grafting hypocotyls of Arabidopsis thaliana lines expressing YFP or mRFP in the endoplasmic reticulum allowed the efficient targeting of the grafting interface for under light and electron microscopy. To explore the potential of our method to study sub-cellular events at the graft interface, we focused on the formation of secondary plasmodesmata (PD) between the grafted partners. We showed that 4 classes of PD were formed at the interface and that PD introgression into the call wall was initiated equally by both partners. Moreover, the success of PD formation appeared not systematic with a third of PD not spanning the cell wall entirely. Characterizing the ultrastructural characteristics of these failed PD gives us insights into the process of secondary PD biogenesis. We showed that the thinning of the cell wall and the endoplasmic reticulum-plasma membrane tethering seem to be required for the establishment of symplastic connections between the scion and the rootstock. The resolution reached in this work shows that our CLEM method offer a new scale to the study for biological processes requiring the combination of light and electron microscopy.

Phase-contrast and electron-microscopic observations on a membranous complex in primary spermatocytes of the mouse

1975 ◽  
Vol 18 (1) ◽  
pp. 1-17
Author(s):  
A. Pleshkewych ◽  
L. Levine
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Phase Contrast ◽  
Cultured Cells ◽  
Light And Electron Microscopy ◽  
Cytoplasmic Inclusion ◽  
Electron Microscopic ◽  
Secondary Spermatocyte ◽  
Living Mouse ◽  
Circular Contour

A prominent cytoplasmic inclusion present in living mouse primary spermatocytes has been observed by both light and electron microscopy. It began to form at prometaphase and continued to increase in thickness and length as the cells developed. By metaphase it was a distinct sausage-shaped boundary that enclosed a portion of the cytoplasm between the spindle and the cell membrane. At the end of metaphase, the inclusion reached its maximum length. At telophase, it was divided between the daughter secondaries. The inclusion persisted as a circular contour in the interphase secondary spermatocyte. Electron microscopy of the same cultured cells that were previously observed with light microscopy revealed that the inclusion was a distinctive formation of membranes. It consisted of agranular cisternae and vesicles, and was therefore a membranous complex. Many of the smaller vesicles in the membranous complex resembled those found in the spindle. The cisternae in the membranous complex were identical to the cisternal endoplasmic reticulum of interphase primary spermatocytes. Nevertheless, the organization of vesicles and cisternae into the membranous complex was unique for the primaries in division stages, since such an organization was not present in their interphase stages.

Anatomy of the Unpollinated and Pollinated Watermelon Stigma

1982 ◽  
Vol 54 (1) ◽  
pp. 341-355
Author(s):  
M. SEDGLEY
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Light And Electron Microscopy ◽  
Freeze Fracture ◽  
Pollen Grain ◽  
Acidic Polysaccharides ◽  
Before And After ◽  
Em Autoradiography ◽  
Pollen Grain Wall ◽  
Stigma Secretion

The structure of the watermelon stigma before and after pollination was studied using light and electron microscopy, freeze-fracture and autoradiography. The wall thickenings of the papilla transfer cells contained callose and their presence prior to pollination was confirmed using EM-autoradiography, freeze-fracture and fixation. No further callose thickenings were produced following pollination. Pollination resulted in a rapid increase in aqueous stigma secretion and localized disruption of the cuticle, which appeared to remain on the surface of the secretion. Autolysis of the papilla cells, which had commenced prior to pollination, was accelerated and appeared to take place via cup-shaped vacuoles developed from distended endoplasmic reticulum. The reaction was localized to the papilla cells adjacent to the pollen tube only. Both pollen-grain wall and stigma secretion contained proteins, carbohydrates, acidic polysaccharides, lipids and phenolics.

Formation of chlamydospores in Gilbertella persicaria

1981 ◽  
Vol 59 (5) ◽  
pp. 908-928 ◽  
Author(s):  
Martha J. Powell ◽  
Charles E. Bracker ◽  
David J. Sternshein
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Light And Electron Microscopy ◽  
Multivesicular Bodies ◽  
Marker Enzyme ◽  
Transparent Layer ◽  
Thiamine Pyrophosphatase ◽  
Gilbertella Persicaria

The cytological events involved in the transformation of vegetative hyphae of the zygomycete Gilbertella persicaria (Eddy) Hesseltine into chlamydospores were studied with light and electron microscopy. Thirty hours after sporangiospores were inoculated into YPG broth, swellings appeared along the aseptate hyphae. Later, septa, traversed by plasmodesmata, delimited each end of the hyphal swellings and compartmentalized these hyphal regions as they differentiated into chlamydospores. Nonswollen regions adjacent to chlamydospores remained as isthmuses. Two additional wall layers appeared within the vegetative wall of the developing chlamydospores. An alveolate, electron-dense wall formed first, and then an electron-transparent layer containing concentrically oriented fibers formed between this layer and the plasma membrane. Rather than a mere condensation of cytoplasm, development and maturation of the multinucleate chlamydospores involved extensive cytoplasmic changes such as an increase in reserve products, lipid and glycogen, an increase and then disappearance of vacuoles, and the breakdown of many mitochondria. Underlying the plasma membrane during chlamydospore wall formation were endoplasmic reticulum, multivesicular bodies, vesicles with fibrillar contents, vesicles with electron-transparent contents, and cisternal rings containing the Golgi apparatus marker enzyme, thiamine pyrophosphatase. Acid phosphatase activity was localized cytochemically in a cisterna which enclosed mitochondria and in vacuoles which contained membrane fragments. Tightly packed membrane whorls and single membrane bounded sacs with finely granular matrices surrounding vacuoles were unique during chlamydospore development. Microbodies were rare in the mature chlamydospore, but endoplasmic reticulum was closely associated with lipid globules. As chlamydospores developed, the cytoplasm in the isthmus became highly vacuolated, lipid globules were closely associated with vacuoles, mitochondria were broken down in vacuoles, unusual membrane configurations appeared, and eventually the membranes degenerated. Unlike chlamydospores, walls of the isthmus did not thicken, but irregularly shaped appositions containing numerous channels formed at intervals on the inside of these walls. The pattern of cytoplasmic transformations during chlamydospore development is similar to events leading to the formation of zygospores and sporangiospores.

scholarly journals Ultrastructural Characterization of Flashing Mitochondria

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641880142
Author(s):  
Manon Rosselin ◽  
Paula Nunes-Hasler ◽  
Nicolas Demaurex
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Volume Ratio ◽  
Tubular Structures ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ∼1,200 slices of 10 nm thickness from a 28 µm × 15 µm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

scholarly journals Serial Block-Face Scanning Electron Microscopy Reveals That Intercellular Nuclear Migration Occurs in Most Normal Tobacco Male Meiocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Sergey Mursalimov ◽  
Nobuhiko Ohno ◽  
Mami Matsumoto ◽  
Sergey Bayborodin ◽  
Elena Deineko
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cell Wall ◽  
Three Dimensional ◽  
Nuclear Migration ◽  
Male Meiosis ◽  
Face Scanning ◽  
Block Face ◽  
Plant Meiosis ◽  
Scanning Electron

Serial block-face scanning electron microscopy (SBF-SEM) was used here to study tobacco male meiosis. Three-dimensional ultrastructural analyses revealed that intercellular nuclear migration (INM) occurs in 90–100% of tobacco meiocytes. At the very beginning of meiosis, every meiocyte connected with neighboring cells by more than 100 channels was capable of INM. At leptotene and zygotene, the nucleus in most tobacco meiocytes approached the cell wall and formed nuclear protuberances (NPs) that crossed the cell wall through the channels and extended into the cytoplasm of a neighboring cell. The separation of NPs from the migrating nuclei and micronuclei formation were not observed. In some cases, the NPs and nuclei of neighboring cells appeared apposed to each other, and the gap between their nuclear membranes became invisible. At pachytene, NPs retracted into their own cells. After that, the INM stopped. We consider INM a normal part of tobacco meiosis, but the reason for such behavior of nuclei is unclear. The results obtained by SBF-SEM suggest that there are still many unexplored features of plant meiosis hidden by limitations of common types of microscopy and that SBF-SEM can turn over a new leaf in plant meiosis research.

scholarly journals Dynamics of in vivo ASC speck formation

2017 ◽  
Vol 216 (9) ◽  
pp. 2891-2909 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Endogenous Gene ◽  
Pyrin Domain ◽  
Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

Ultrastructural Observations of Cell Wall Regeneration Around Isolated Tobacco Protoplasts

1974 ◽  
Vol 14 (2) ◽  
pp. 439-449
Author(s):  
J. BURGESS ◽  
E. N. FLEMING
Keyword(s):  
Electron Microscopy ◽  
Heavy Metal ◽  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Cell Wall Regeneration ◽  
Membrane Systems ◽  
Tobacco Protoplasts ◽  
Wall Formation ◽  
Wall Regeneration ◽  
Wall Growth

The process of cell wall regeneration around cultured protoplasts isolated from tobacco mesophyll has been examined by electron microscopy. The initially formed wall contains 2 components which stain with conventional heavy metal stains. The first consists of un-branched fibres, at first oriented at right angles to the plasmalemma surface. As wall growth proceeds the fibres lengthen and assume an orientation parallel to the plasmalemma. It seems probable that this component is cellulose. The second component of the wall is more amorphous and more densely stained. It is most frequently seen in situations where leaching of materials into the medium would be expected to be minimal. The endoplasmic reticulum and the plasmalemma are the only membrane systems which appear to contribute towards wall formation. No pattern of structure has been detected to explain the orientation or method of synthesis of the microfibrillar part of the wall.

scholarly journals LOCALIZATION OF PEROXIDASE ACTIVITY IN MICROBODIES OF FETAL MOUSE LIVER

1969 ◽  
Vol 17 (7) ◽  
pp. 454-466 ◽  
Author(s):  
EDWARD ESSNER
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Peroxidase Activity ◽  
Mouse Liver ◽  
Fetal Liver ◽  
Light And Electron Microscopy ◽  
Small Population ◽  
Fetal Mouse ◽  
Fetal Mouse Liver

The peroxidase activity of microbodies in fetal mouse liver was studied by light and electron microscopy. Two types of microbodies were present; a small population of bodies that lacked a nucleoid, predominant on the 16th day of gestation, and a larger population of nucleoid-bearing microbodies, predominant on the 19th day, in association with the rough endoplasmic reticulum from which they probably originate. Both types of bodies were visualized when incubated for peroxidase activity but were negative (19th day) for acid phosphatase activity. The findings suggest that the anucleoid- and nucleoid-bearing organelles together constitute the microbody population of the fetal liver.

Initial stages of cell wall formation in the dinoflagellate Peridinium trochoideum

1975 ◽  
Vol 53 (5) ◽  
pp. 483-494 ◽  
Author(s):  
John P. Kalley ◽  
Thana Bisalputra
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Osmotic Pressure ◽  
Fibrous Material ◽  
Light And Electron Microscopy ◽  
Cell Wall Formation ◽  
Vast Number ◽  
Wall Formation ◽  
Marine Dinoflagellate ◽  
Interphase Cells

The formation of the cell wall in the marine dinoflagellate Peridinium trochoideum was studied using light and electron microscopy. In mature, interphase cells, densely staining inclusions termed ‘prothecal bodies’ were found distributed throughout the cytoplasm. Before ecdysis each amorphous prothecal body developed into many vesicles, each of which contained fibrous material in an electron-transparent matrix. The vast number of vesicles so formed may have increased the cell's osmotic pressure enough to initiate ecdysis. At ecdysis the thecal plates and overlying membranes were lost and a new wall was formed by deposition of intact prothecal vesicles at the protoplast surface. The newly formed wall was continuous over the protoplast and no plates existed as such

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