Single-Pixel Densitometry Revealed the Presence of Peptidoglycan in the Intermembrane Space of the Moss Chloroplast Envelope in Conventional Electron Micrographs

Using a glycerination procedure designed to avoid excessive plasmolysis or disruption of the ectoplasm, microfilaments in bundles at the ectoplasm-endoplasm interface of Nitella internode cell segments were found to bind rabbit heavy meromyosin (HMM) in situ. All HMM arrowheads in a bundle seem to have the same polarity and many lie in register as judged from the electron micrographs; the arrowhead periodicity is approximately 380 . The decorated microfilaments are thus similar to those seen in negatively stained cytoplasmic suspensions of internode cells. In glycerinated material, as well as in suspensions, the microfilaments are closely associated with chloroplasts. The microfilaments lie adjacent to or are attached to the chloroplast envelope. The results provide further evidence that the microfilaments thought to play a role in cytoplasmic streaming in vivo in Nitella consist of actin and suggest that they may be anchored to the chloroplasts.

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Two plastid POLLUX ion channel-like proteins are required for stress-triggered stromal Ca2+ release

Plant Physiology ◽

10.1093/plphys/kiab424 ◽

2021 ◽

Author(s):

Carsten Völkner ◽

Lorenz Josef Holzner ◽

Philip M Day ◽

Amra Dhabalia Ashok ◽

Jan de Vries ◽

...

Keyword(s):

Ion Channels ◽

Ion Channel ◽

Chloroplast Envelope ◽

Mycorrhizal Symbiosis ◽

Intermembrane Space ◽

Envelope Membrane ◽

Loss Of Function ◽

Chloroplast Envelope Membrane ◽

Envelope Membranes ◽

Yeast Mutants

Abstract Two decades ago, large cation currents were discovered in the envelope membranes of Pisum sativum L. (pea) chloroplasts. The deduced K+-permeable channel was coined fast-activating chloroplast cation (FACC) channel but its molecular identity remained elusive. To reveal candidates, we mined proteomic datasets of isolated pea envelopes. Our search uncovered distant members of the nuclear POLLUX ion channel family. Since pea is not amenable to molecular genetics, we used Arabidopsis thaliana to characterize the two gene homologs. Using several independent approaches, we show that both candidates localize to the chloroplast envelope membrane. The proteins, designated PLASTID ENVELOPE ION CHANNELS (PEC1/2), form oligomers with regulator of K+ conductance (RCK) domains protruding into the intermembrane space. Heterologous expression of PEC1/2 rescues yeast mutants deficient in K+ uptake. Nuclear POLLUX ion channels cofunction with Ca2+ channels to generate Ca2+ signals, critical for establishing mycorrhizal symbiosis and root development. Chloroplasts also exhibit Ca2+ transients in the stroma, probably to relay abiotic and biotic cues between plastids and the nucleus via the cytosol. Our results show that pec1pec2 loss-of-function double mutants fail to trigger the characteristic stromal Ca2+ release observed in wild-type plants exposed to external stress stimuli. Besides this molecular abnormality, pec1pec2 double mutants do not show obvious phenotypes. Future studies of PEC proteins will help to decipher the plant’s stress-related Ca2+ signaling network and the role of plastids. More importantly, the discovery of PECs in the envelope membrane is another critical step towards completing the chloroplast ion transport protein inventory.

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Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

The Journal of Cell Biology ◽

10.1083/jcb.143.4.991 ◽

1998 ◽

Vol 143 (4) ◽

pp. 991-1002 ◽

Cited By ~ 158

Author(s):

Andrei Kouranov ◽

Xuejun Chen ◽

Bruno Fuks ◽

Danny J. Schnell

Keyword(s):

Protein Import ◽

Intermediate Stage ◽

Chloroplast Envelope ◽

Intermembrane Space ◽

Outer Face ◽

Envelope Membrane ◽

Stromal Compartment ◽

Inner Membrane ◽

General Protein ◽

Inner Envelope

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

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Utilizing Dark Field Electron Microscopy to Produce Lantern Slides

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050597 ◽

1974 ◽

Vol 32 ◽

pp. 116-117

Author(s):

J. N. Meador ◽

C. N. Sun ◽

H. J. White

Keyword(s):

Electron Microscope ◽

Electron Micrographs ◽

Dark Field ◽

Limiting Factor ◽

Clinical Laboratories ◽

Field Electron ◽

Time Element ◽

Field Electron Microscopy ◽

Dark Field Electron ◽

Dark Field Micrographs

The electron microscope is being utilized more and more in clinical laboratories for pathologic diagnosis. One of the major problems in the utilization of the electron microscope for diagnostic purposes is the time element involved. Recent experimentation with rapid embedding has shown that this long phase of the process can be greatly shortened. In rush cases the making of projection slides can be eliminated by taking dark field electron micrographs which show up as a positive ready for use. The major limiting factor for use of dark field micrographs is resolution. However, for conference purposes electron micrographs are usually taken at 2.500X to 8.000X. At these low magnifications the resolution obtained is quite acceptable.

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Light Optical Diffraction Studies of Macromolecular Ultrastructure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010006828x ◽

1970 ◽

Vol 28 ◽

pp. 258-259

Author(s):

Glen B. Haydon

Keyword(s):

High Resolution ◽

Diffraction Analysis ◽

Diffraction Pattern ◽

Electron Micrographs ◽

Optical Diffraction ◽

Electron Micrograph ◽

Its Analysis ◽

Resolution Electron ◽

Diffraction Patterns

Analysis of light optical diffraction patterns produced by electron micrographs can easily lead to much nonsense. Such diffraction patterns are referred to as optical transforms and are compared with transforms produced by a variety of mathematical manipulations. In the use of light optical diffraction patterns to study periodicities in macromolecular ultrastructures, a number of potential pitfalls have been rediscovered. The limitations apply to the formation of the electron micrograph as well as its analysis.(1) The high resolution electron micrograph is itself a complex diffraction pattern resulting from the specimen, its stain, and its supporting substrate. Cowley and Moodie (Proc. Phys. Soc. B, LXX 497, 1957) demonstrated changing image patterns with changes in focus. Similar defocus images have been subjected to further light optical diffraction analysis.

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On an Application of the Metallurgical Stage to Chromosome Morphology

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060155 ◽

1967 ◽

Vol 25 ◽

pp. 28-29

Author(s):

Godfrey C. Hoskins

Keyword(s):

Human Chromosome ◽

Electron Micrographs ◽

Chromosome Morphology ◽

Electron Optics ◽

Electron Micrograph ◽

Photographic Evidence ◽

Sophisticated Equipment ◽

Periodic Arrangement

The first serious electron microscooic studies of chromosomes accompanied by pictures were by I. Elvers in 1941 and 1943. His prodigious study, from the manufacture of micronets to the development of procedures for interpreting electron micrographs has gone all but unnoticed. The application of todays sophisticated equipment confirms many of the findings he gleaned from interpretation of images distorted by the electron optics of that time. In his figure 18 he notes periodic arrangement of pepsin sensitive “prickles” now called secondary fibers. In his figure 66 precise regularity of arrangement of these fibers can be seen. In his figure 22 he reproduces Siegbahn's first stereoscopic electron micrograph of chromosomes.The two stereoscopic pairs of electron micrographs of a human chromosome presented here were taken with a metallurgical stage on a Phillips EM200. These views are interpreted as providing photographic evidence that primary fibers (1°F) about 1,200Å thick are surrounded by secondary fibers (2°F) arranged in regular intervals of about 2,800Å in this metanhase human chromosome. At the telomere the primary fibers bend back on themselves and entwine through the center of each of each chromatid. The secondary fibers are seen to continue to surround primary fibers at telomeres. Thus at telomeres, secondary fibers present a surface not unlike that of the side of the chromosome, and no more susceptible to the addition of broken elements from other chromosomes.

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Criteria and Methods for Reliable Three-Dimensional Image Reconstruction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051669 ◽

1974 ◽

Vol 32 ◽

pp. 330-331

Author(s):

R. A. Crowther

Keyword(s):

Image Reconstruction ◽

Finite Number ◽

Density Distribution ◽

Electron Micrographs ◽

Mathematical Problem ◽

Three Dimensional ◽

Ideal Case ◽

Dimensional Image ◽

General Object ◽

The Ideal

The reconstruction of a three-dimensional image of a specimen from a set of electron micrographs reduces, under certain assumptions about the imaging process in the microscope, to the mathematical problem of reconstructing a density distribution from a set of its plane projections.In the absence of noise we can formulate a purely geometrical criterion, which, for a general object, fixes the resolution attainable from a given finite number of views in terms of the size of the object. For simplicity we take the ideal case of projections collected by a series of m equally spaced tilts about a single axis.

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Some Structural Features of Metaphase Chromosomes of Mice and Men

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060969 ◽

1967 ◽

Vol 25 ◽

pp. 190-191

Author(s):

Godfrey C. Hoskins ◽

Betty B. Hoskins

Keyword(s):

Electron Microscopy ◽

Electron Micrographs ◽

Structural Features ◽

Metaphase Chromosomes ◽

The Future ◽

Of Mice And Men ◽

Mouse Cells ◽

Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

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The structure of pre-mRNA and mRNA from erythroid cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082030 ◽

1978 ◽

Vol 36 (2) ◽

pp. 234-235

Author(s):

A.-M. Ladhoff ◽

B.J. Thiele ◽

Ch. Coutelle ◽

S. Rosenthal

Keyword(s):

Bone Marrow ◽

Structural Transformation ◽

Electron Micrographs ◽

Ammonium Acetate ◽

Bone Marrow Cells ◽

Erythroid Cells ◽

Globin Mrna ◽

Ordered Structures ◽

Rabbit Bone ◽

Rabbit Reticulocytes

The suggested precursor-product relationship between the nuclear pre-mRNA and the cytoplasmic mRNA has created increased interest also in the structure of these RNA species. Previously we have been published electron micrographs of individual pre-mRNA molecules from erythroid cells. An intersting observation was the appearance of a contour, probably corresponding to higher ordered structures, on one end of 10 % of the pre-mRNA molecules from erythroid rabbit bone marrow cells (Fig. 1A). A virtual similar contour was observed in molecules of 9S globin mRNA from rabbit reticulocytes (Fig. 1B). A structural transformation in a linear contour occurs if the RNA is heated for 10 min to 90°C in the presence of 80 % formamide. This structural transformation is reversible when the denatured RNA is precipitated and redissolved in 0.2 M ammonium acetate.

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High Resolution Stereography of Complementary Freeze-Fracture Replicas Reveals the Presence of Depressions Opposite Membrane Associated Particles of Split Membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066267 ◽

1971 ◽

Vol 29 ◽

pp. 442-443

Author(s):

Russell L. Steere

Keyword(s):

High Resolution ◽

Cardiac Muscle ◽

Electron Micrographs ◽

Chromic Acid ◽

Freeze Fracture ◽

Distilled Water ◽

Fine Scale ◽

Acid Cleaning ◽

Cleaning Solution

Complementary replicas have revealed the fact that the two common faces observed in electron micrographs of freeze-fracture and freeze-etch specimens are complementary to each other and are thus the new faces of a split membrane rather than the original inner and outer surfaces (1, 2 and personal observations). The big question raised by published electron micrographs is why do we not see depressions in the complementary face opposite membrane-associated particles? Reports have appeared indicating that some depressions do appear but complementarity on such a fine scale has yet to be shown.Dog cardiac muscle was perfused with glutaraldehyde, washed in distilled water, then transferred to 30% glycerol (material furnished by Dr. Joaquim Sommer, Duke Univ., and VA Hospital, Durham, N.C.). Small strips were freeze-fractured in a Denton Vacuum DFE-2 Freeze-Etch Unit with complementary replica tooling. Replicas were cleaned in chromic acid cleaning solution, then washed in 4 changes of distilled water and mounted on opposite sides of the center wire of a Formvar-coated grid.

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