scholarly journals Expansion Microscopy of Lipid Membranes

2019 ◽  
Author(s):  
Emmanouil D. Karagiannis ◽  
Jeong Seuk Kang ◽  
Tay Won Shin ◽  
Amauche Emenari ◽  
Shoh Asano ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Lipid Membranes ◽  
Intracellular Transport ◽  
Building Blocks ◽  
Chemical Tag ◽  
Resolution Imaging ◽  
Antibody Labeling ◽  
Microscopy Techniques

AbstractLipids are fundamental building blocks of cells and their organelles, yet nanoscale resolution imaging of lipids has been largely limited to electron microscopy techniques. We introduce and validate a chemical tag that enables lipid membranes to be imaged optically at nanoscale resolution via a lipid-optimized form of expansion microscopy, which we call membrane expansion microscopy (mExM). mExM, via a novel post-expansion antibody labeling protocol, enables protein-lipid relationships to be imaged in organelles such as mitochondria, the endoplasmic reticulum, the nuclear membrane, and the Golgi apparatus. mExM may be of use in a variety of biological contexts, including the study of cell-cell interactions, intracellular transport, and neural connectomics.

scholarly journals SYNTHESIS, MIGRATION, AND RELEASE OF PRECURSOR COLLAGEN BY ODONTOBLASTS AS VISUALIZED BY RADIOAUTOGRAPHY AFTER [3H]PROLINE ADMINISTRATION

1974 ◽  
Vol 60 (1) ◽  
pp. 92-127 ◽  
Author(s):  
Melvyn Weinstock ◽  
C. P. Leblond
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Phosphotungstic Acid ◽  
Secretory Granules ◽  
Low Ph ◽  
Collagen Fibrils ◽  
Dentin Collagen ◽  
Odontoblast Process ◽  
High Radioactivity

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.

Altered labelling of the cell surface and intracellular organelles with [3H]mannose in enucleated amoebae

1984 ◽  
Vol 68 (1) ◽  
pp. 83-94
Author(s):  
C.J. Flickinger
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Cell Organelles ◽  
Intracellular Organelles ◽  
And Control ◽  
Kinetics Of ◽  
Heavy Labelling

The production, transport, and disposition of material labelled with [3H]mannose were studied in microsurgically enucleated and control amoebae. Cells were injected with the precursor and samples were prepared for electron-microscope radioautography at intervals, up to 24 h later. Control cells showed heavy labelling of the rough endoplasmic reticulum and the Golgi apparatus at early intervals after injection. Later, labelling of groups of small vesicles increased, and the percentage of grains over the cell surface peaked 12 h after administration of the precursor. Two major changes were detected in enucleate amoebae. First, the kinetics of labelling of cell organelles with [3H]mannose were altered in the absence of the nucleus. The Golgi apparatus and cell surface both displayed maximal labelling at later intervals in enucleates, and the percentage of grains over the rough endoplasmic reticulum varied less with time in enucleated than in control cells. Second, the distribution of radioactivity was altered. A greater percentage of grains was associated with lysosomes in enucleates than in control cells. The change in the kinetics of labelling of the endoplasmic reticulum, Golgi apparatus and cell surface indicates that intracellular transport of surface material was slower in the absence of the nucleus. It is suggested that this is related to the decreased motility of enucleate cells.

scholarly journals Light and dark cells in the lumbar sensory ganglia of pre and post-hatching domestic fowl

1988 ◽  
Vol 46 (1) ◽  
pp. 3-5
Author(s):  
Claudio A. Ferraz de Carvalho ◽  
Ciro F. da Silva
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Satellite Cell ◽  
Satellite Cells ◽  
Domestic Fowl ◽  
Sensory Ganglia ◽  
Dark Cells ◽  
Junctional Complexes ◽  
Functional Features

Clear and dark satellite cell classes were identified by electron microscopy in the lumbar sensory ganglia of domestic fowl in 8 pre and 4 post-hatching stages of development. Some cytologic differences found between the two classes relating to the rough-endoplasmic reticulum, ribosomes, Golgi apparatus and junctional complexes suggest the existence of distinct functional features for both types of satellite cells.

scholarly journals Is cytochrome P-450 transported from the endoplasmic reticulum to the Golgi apparatus in rat hepatocytes?

1985 ◽  
Vol 101 (5) ◽  
pp. 1733-1740 ◽  
Author(s):  
A Yamamoto ◽  
R Masaki ◽  
Y Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Intracellular Transport ◽  
Subcellular Fraction ◽  
Plasma Membranes ◽  
Rat Hepatocytes ◽  
Cytochrome P 450 ◽  
Microscopic Techniques ◽  
Lysosomal Proteins

The Golgi apparatus mediates intracellular transport of not only secretory and lysosomal proteins but also membrane proteins. As a typical marker membrane protein for endoplasmic reticulum (ER) of rat hepatocytes, we have selected phenobarbital (PB)-inducible cytochrome P-450 (P-450[PB]) and investigated whether P-450(PB) is transported to the Golgi apparatus or not by combining biochemical and quantitative ferritin immunoelectron microscopic techniques. We found that P-450(PB) was not detectable on the membrane of Golgi cisternae either when P-450 was maximally induced by phenobarbital treatment or when P-450 content in the microsomes rapidly decreased after cessation of the treatment. The P-450 detected biochemically in the Golgi subcellular fraction can be explained by the contamination of the microsomal vesicles derived from fragmented ER membranes to the Golgi fraction. We conclude that when the transfer vesicles are formed by budding on the transitional elements of ER, P-450 is completely excluded from such regions and is not transported to the Golgi apparatus, and only the membrane proteins destined for the Golgi apparatus, plasma membranes, or lysosomes are selectively collected and transported.

Development of the lateral palatal brush in larvae of Aedes aegypti (L.) (Diptera: Culicidae)

1990 ◽  
Vol 68 (7) ◽  
pp. 1454-1467 ◽  
Author(s):  
K. M. Fry ◽  
S. B. McIver
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Aedes Aegypti ◽  
Rough Endoplasmic Reticulum ◽  
Light And Electron Microscopy ◽  
Fourth Instar ◽  
Muscle Fibres ◽  
Final Length

Light and electron microscopy were used to observe development of the lateral palatal brush in Aedes aegypti (L.) larvae. Development was sampled at 4-h intervals from second- to third-instar ecdyses. Immediately after second-instar ecdysis, the epidermis apolyses from newly deposited cuticle in the lateral palatal pennicular area to form an extensive extracellular cavity into which the fourth-instar lateral palatal brush filaments grow as cytoplasmic extensions. On reaching their final length, the filaments deposit cuticulin, inner epicuticle, and procuticle sequentially on their outer surfaces. The lateral palatal crossbars, on which the lateral palatal brush filaments insert, form after filament development is complete. At the beginning of development, the organelles involved in plasma membrane and cuticle production are located at the base and middle of the cells. As the filament rudiments grow, most rough endoplasmic reticulum, mitochondria, and Golgi apparatus move to the apex of the epidermal cells and into the filament rudiments. After formation of the lateral palatal brush filaments and lateral palatal crossbars, extensive organelle breakdown occurs. Lateral palatal brush formation is unusual in that no digestion and resorption of old endocuticle occurs prior to deposition of new cuticle. No mucopolysaccharide secretion by the lateral palatal brush epidermis was observed, nor were muscle fibres observed to attach to the lateral palatal crossbars, as has been suggested by other workers.

scholarly journals FINE STRUCTURE AND ORGANELLE ASSOCIATIONS IN BROWN ALGAE

1965 ◽  
Vol 26 (2) ◽  
pp. 523-537 ◽  
Author(s):  
G. Benjamin Bouck
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Fine Structure ◽  
Golgi Apparatus ◽  
Nuclear Envelope ◽  
Brown Algae ◽  
Algal Cell ◽  
Plant Cells ◽  
Membrane Systems ◽  
Two Envelopes

The structural interrelationships among several membrane systems in the cells of brown algae have been examined by electron microscopy. In the brown algae the chloroplasts are surrounded by two envelopes, the outer of which in some cases is continuous with the nuclear envelope. The pyrenoid, when present, protrudes from the chloroplast, is also surrounded by the two chloroplast envelopes, and, in addition, is capped by a third dilated envelope or "pyrenoid sac." The regular apposition of the membranes around the pyrenoid contrasts with their looser appearance over the remainder of the chloroplast. The Golgi apparatus is closely associated with the nuclear envelope in all brown algae examined, but in the Fucales this association may extend to portions of the cytoplasmic endoplasmic reticulum as well. Evidence is presented for the derivation of vesicles, characteristic of those found in the formative region of the Golgi apparatus, from portions of the underlying nuclear envelope. The possibility that a structural channeling system for carbohydrate reserves and secretory precursors may be present in brown algae is considered. Other features of the brown algal cell, such as crystal-containing bodies, the variety of darkly staining vacuoles, centrioles, and mitochondria, are examined briefly, and compared with similar structures in other plant cells.

Evidence for Golgi Origin of Melanosomes

1968 ◽  
Vol 26 ◽  
pp. 148-149
Author(s):  
Gerd G. Maul
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Golgi Complex ◽  
Serial Sections ◽  
Human Malignant Melanoma ◽  
Close Proximity ◽  
Membrane Bound ◽  
Tubular Endoplasmic Reticulum ◽  
Internal Architecture

Electron microscopy has provided evidence that the melanosome evolves as a membrane bound structure with a highly complex internal architecture. The premelanosomes are found in close proximity to the golgi apparatus. Therefore, it was generally agreed that the melanosomes originate from the golgi apparatus.Vesicles have been described to pinch off the cysternae of the golgi apparatus. The vesicles would then grow and acquire a dense material. This material is aggregating to form the characteristic helical strands onto which melanin is deposited. Cloned human malignant melanoma lines were used to reinvestigate the problem of melanosome formation. The reconstruction of serial sections revealed the arrangement of premelanosomes and melanosomes in relation to the golgi complex. This study demonstrated that premelanosomes and melanosomes are continuous with the golgi complex by a smooth-surfaced tubular endoplasmic reticulum (SER) (Fig. la-d). The continuity of membranes of the SER and the premelanosome is depicted in Fig. 2. In this early premelanosome the protein strands have not yet coiled up into a helix. Rough-surfaced endoplasmic reticulum (RER) was also observed to be continuous with the golgi apparatus and melanosomes. After melanogenesis has started (Fig. 3) small vesicles appear inside the premelanosomes.

Immunolocalization of the intracellular sites of accumulation of mutant influenza hemagglutinins by Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 968-969
Author(s):  
K. McCammon ◽  
M. Segal ◽  
J. Sambrook ◽  
M. J. Gething ◽  
A. McDowall
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Cysteine Residue ◽  
Homologous Sequence ◽  
Golgi Compartment ◽  
Golgi Network

The hemagglutinin (HA) of influenza virus has been used as a model system to study the biosynthesis and intracellular transport of integral membrane proteins in mammalian cells. To investigate the role of protein structure in facilitating transport along the secretory pathway, we have examined the expression in monkey CV-1 cells of a large number of mutant HA molecules. The majority of the HA mutants do not progress along the secretory pathway and accumulate in the endoplasmic reticulum (ER), and we have shown that assembly of newly-synthesized HA monomers into correctly folded trimeric structures is required for transport of the protein to the Golgi apparatus. By contrast, only one HA mutant has beegn characterized whose transport is blocked at a post-Golgi stage of the pathway and thus little is known about the factors involved in the sorting of the HA molecule from the Golgi apparatus to the plasma membrane (PM). In this study we are using electron microscopy to precisely define the intracellular site of accumulation of two mutant HAs whose transport is blocked at different stages of the secretory pathway. In mutant HAJS67, a cysteine residue (cys67) involved in a key disulfide bond has been substituted by a serine residue. In mutant HA164, the 10 amino acid cytoplasmic tail of the wild-type HA has been replaced by a non-homologous sequence of 16 amino acids. Biochemical and immunof1uoresence analyses have indicated that HAJS67 molecules remain in the ER compartment while HA164 is largely confined to a post-Golgi compartment, possibly the trans Golgi network (TGN).

The Electron Microscopy of the ‘Golgi Apparatus’in the Purkinje Cells of Owls

1960 ◽  
Vol s3-101 (56) ◽  
pp. 389-394
Author(s):  
S. K. MALHOTRA ◽  
G. A. MEEK
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Purkinje Cells ◽  
Electron Micrographs ◽  
Optical Microscope

The classical site of the ‘Golgi apparatus’, the Purkinje cells of the cerebellum of owls, has been examined by electron microscopy. The greater part of the cytoplasm consists of aggregates of closely packed granular membranes ofendoplasmic reticulum. The objects described by Dalton and Felix in electron micrographs and called by them the Golgi apparatus are rarely seen in these preparations. It seems likely that the ‘Golgi apparatus’ of this cell as seen in the optical microscope is formed by the deposition of silver or osmium on the membranes of the endoplasmic reticulum, which form a network throughout the cytoplasm of this cell.

scholarly journals The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1647-1655 ◽  
Author(s):  
T J Stoller ◽  
D Shields
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Gh3 Cells ◽  
Alpha Globin ◽  
Regulated Secretory Pathway

We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min. Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16 degrees C suggesting proteolysis occurred during transport to the cis-Golgi apparatus. In contrast to the control globin, approximately 30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed. Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 microM chloroquine. 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" alpha-globin. Most significantly, approximately 50% of processed alpha-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP. We conclude that the somatostatin propeptide mediated transport of alpha-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of alpha-globin into vesicles whose secretion was stimulated by cAMP.

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