Serial section analysis of coated pits and coated vesicles in soybean protoplasts

Divergent viewpoints have been expressed regarding the existence of free coated vesicles in animal cells; this question has not been carefully addressed with plant tissues. Soybean suspension culture protoplasts were exposed to cationized ferritin (CF) for short times to label coated pits and coated vesicles. A serial section analysis did not reveal deep coated pits with long necks as reported in animal cells. Serial sections clearly demonstrated CF-labelled coated vesicles to be separate organelles and is consistent with the idea that they transfer CF from coated pits to other cytoplasmic organelles during endocytosis.     

Preparation of a coated vesicle-enriched fraction from plant cells

A fraction rich in coated vesicles has been prepared from suspension-cultured cells of tobacco (Nicotiana tabacum L.) by sucrose gradient centrifugation. Isolated, negatively-stained plant coated vesicles are approx. 100 nm in diameter, and show the characteristic basket-like structure of the clathrin coat previously reported for both plant [2–5] and animal [1, 6–9] coated vesicles. Analysis of the various plant subcellular fractions by SDS polyacrylamide gel electrophoresis demonstrates that a polypeptide of 190 000 D is enriched in parallel with the morphologically identifiable coated vesicles. It is postulated that this polypeptide is plant clathrin with a molecular weight about 10 000 D greater than that previously reported for animal clathrin [1, 6].     

The isolation and enrichment of coated vesicles from suspension-cultured carrot cells

Summary A method has been developed to isolate and purify coated vesicles from suspension cultured carrot (Daucus carota L.) cells. It incorporates features of centrifugation methods (sucrose step gradient; Ficoll/D₂O gradient) previously employed in the isolation of coated vesicles from mammalian brain tissue. Most important is the treatment of the crude coated vesicle fraction (postmicrosomal supernatant) with ribonuclease to remove ribosomes which are a serious source of contamination in such fractions. The fraction finally obtained is contaminated to the extent of 30% of total observed particles in negatively stained preparations with naked vesicles whose diameter are smaller than those of the coated vesicles. These vesicles are interpreted as being coated vesicles which have been stripped of their coats. SDS-PAGE of coated vesicle fractions purified by this method reveal significant differences in the polypeptide patterns obtained from plant and animal systems.     

Homology between antigenic determinants of bovine clathrin and clathrin from the brown alga Laminaria digitata

Coated vesicles, essential organelles of intracellular membrane traffic, have been extensively studied in animal and higher plant cells. In the algae, cytological studies only have been performed which demonstrate the presence of such coated vesicles with their surrounding clathrin lattice. The present work has been carried out on coated vesicles isolated for the first time from the brown algae Laminaria digitata. For comparison of the antigenic characteristics of clathrin prepared from the Bovine brain or adrenocortical cells and the clathrin prepared from algae, polyclonal antibodies have been raised to a purified Bovine brain clathrin in Goat and to Bovine adrenocortical clathrin in Rabbit. The positive immunological responses of the coated vesicles and the clathrin from Algae to these antibodies, evidence an homology between antigenic determinants of clathrin from animal and vegetal cells.     

Recent advances in the study of plant coated vesicles

Coated vesicles are organelles common in plant cells. They are associated with the plasma membrane, Golgi apparatus, partially coated reticulum and multivesiclular bodies. In this paper we discuss recent developments in the use of immunochemistry and cytochemistry to investigate the structure and function of plant coated vesicles.     

Plant coated vesicles

Abstract. Coated vesicles are organelles frequently encountered in many plant cell types often in association with the plasma membrane, Golgi apparatus, partially coated reticulum and multivesicular bodies. They are readily identified by a characteristic cage or basket composed of interlocking triskelions of the protein clathrin which are bound to the surface of the vesicle membrane. Although their transport function has been well studied and characterized in mammalian systems, the possible importance of coated vesicles as transport organelles in plant cells is only just beginning to be explored. In this review, the authors describe the structure of higher plant coated vesicles and discuss their possible involvement in the endocytosis of marcromolecules, in exocytosis and in the intracellular transport of material between cytoplasmic compartments. Their possible role in maintaining the macromolecular composition of the plasma membrane whilst allowing recycling of excess lipid bilayer and their potential application as vehicles for the introduction of foreign macromolecules into plant cells are discussed.     

Coated Vesicles From Locust Oocytes: Isolation and Characterization

Summary

This report demonstrates for the first time the isolation of coated vesicles from insect oocytes. Coated vesicles were purified from oocytes of Locusta migratoria by differential centrifugation and sucrose density centrifugation. The coated vesicles were characterized by electron microscopy, SDS-PAGE and scanning densitometry. Like coated vesicles isolated from pig brain and chicken oocytes, the coated vesicles from locust oocytes contained clathrin as the major protein component. Apart from clathrin, another major protein characteristic of coated vesicles had a molecular weight of about 115,000, and in addition, several minor unidentified bands were identified.     

Preparation of a homogeneous coated vesicle fraction from bean leaves

Summary Using a procedure previously developed for suspension-cultured carrot cells, we have been able to isolate two different coated vesicle-containing fractions from green bean leaves (Vicia faba). The two fractions differ in their isopycnic densities in D₂O-Ficoll as well as in their diameters. One of the fractions (the less dense of the two) is almost 100% pure as judged by negative staining. Because of this the polypeptide pattern obtained from SDS-PAGE is most clear and has enabled a clear recognition of clathrin light chains, in addition to the 190 kDa heavy chain coat component. Significantly the 100k Da and 50k Da polypeptides typical of brain coated vesicles are absent from bean leaf coated vesicles. Due to a) the high degree of vacuolation b) the presence of large amounts of ribulose bisphosphate carboxylase in the postmicrosomal supernatant, the yield of coated vesicles from bean leaves, as compared to nongreen plant cells, or to bovine brain tissue is extremely low (1 mg coated vesicles from 2.4 kg leaf tissue).Abbreviations D₂O deuterium oxide - EGTA ethylene glycol-bis ( amino ethyl ether) N,N,N,N tetraacetic acid - MES, 2 (N-morpholino)-ethanesulfonic acid - PMSF phenyl methylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TRIS Tris-hydroxy methyl amino methane     

Presence of a partially-coated reticulum in angiosperms

Summary The form and distribution of partially-coated membrane systems and coated vesicles in the leaf glands ofPhaseolus and the root cortical cells ofZea were investigated. Partially-coated membranes exist as a reticulum which is either sparsely branched or extensively anastomosed. Coated vesicles and coated membrane regions may occur at all points that are in the vicinity of such a reticulum. Golgi stack membranes, though associated with the partially-coated reticulum in many cases, show no consistent orientation to it. Occasionally, the reticulum and the associated coated vesicles are located away from any Golgi stack. We examined other plant tissues from such species asAllium cepa, Beta vulgaris andNicotiana tabacum and the partially-coated reticulum was observed in all material. We suggest that membrane flow may be occurring from the partially-coated reticulum to either the Golgi stack or to other intracellular compartments.     

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.     

LDL receptors in coated vesicles isolated from bovine adrenal cortex: binding sites unmasked by detergent treatment

Coated vesicles isolated from bovine adrenal cortex contain specific binding sites that recognize 125I-labeled human low density lipoprotein (LDL). These sites share the properties of the functional LDL receptors previously demonstrated on the surface of adrenal cells and in unfractionated adrenal membranes. Approximately 90% of the LDL receptors of the isolated coated vesicles were initially masked. Binding of 125I-LDL increased 10 fold after the vesicles were disrupted with the detergent octylglucoside. The LDL receptors of intact coated vesicles were also shielded from destruction by pronase; proteolytic destruction occurred only after the vesicles had been disrupted with octylglucoside. The adrenal coated vesicles measured 60 nm in diameter, suggesting that they were derived from the Golgi apparatus. Like the previously studied coated vesicles from brain and other tissues, the coated vesicles from adrenal cortex contained clathrin as the major protein component. In contrast to the coated vesicles of adrenal cortex, however, the brain coated vesicles failed to reveal masked LDL receptor activity when treated with octylglucoside. The current data indicate that isolated coated vesicles from the adrenal cortex contain LDL receptors and that these receptors exist in a masked form, apparently because their binding sites face the interior of the vesicle.     

Morphometric analysis of coated pits and vesicles in the proximal and distal caput epididymidis

A morphometric analysis of coated and uncoated structures found in the apical portion of principal cells from both the proximal and distal caput epididymidis has been carried out. Almost all endocytic, coated vesicles are found within 1 micron of the luminal surface of principal cells and the volume fraction of these and of uncoated vesicles is much greater in the proximal caput epididymidis. A serial section analysis indicated that many coated "vesicles" are tangentially sectioned coated pits and that a complex network of interconnected vesicular and tubular structures exists in the apical cytoplasm. Efferent duct ligation has no effect on the number of size of large coated and uncoated vesicles in either the proximal or distal caput epididymidis, indicating that substances delivered to principal cells from the lumen are not required to maintain the endocytic machinery. However, this treatment does result in a considerable increase in the number of large coated vesicles associated with the basal surface of principal cells from the proximal but not the distal caput epididymidis. The volume fraction of small, presumably exocytic, coated vesicles is significantly greater in the apical cytoplasm of cells from the distal caput epididymidis in control animals. Efferent duct ligation results in a significant increase in the volume fraction of these vesicles in the proximal but not distal caput epididymidis. These results show that there are marked differences in structure among principal cells from these two regions of the epididymis and that this may reflect differences in control and function.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Purification and Characterization of Clathrin-Coated Vesicles from Chlamydomonas

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas rein-hardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter±SD of 95±17 nm, n=300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.     

Two polypeptides (30 and 38kDa) in plant coated vesicles with clathrin light chain properties

Summary Coated vesicles have been isolated from bovine brain and etiolated zucchini hypocotyls by centrifugal methods. By putting to use two properties of the light chain polypeptides of brain coated vesicles (calcium binding, heat stability) we have been able to demonstrate the presence of two similar polypeptides with apparent molecular masses of 30 and 38 kDa in plant coated vesicles.Abbreviations CV coated vesicle - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - MES 2-(N-Morpholino)-ethanesulfonic acid - Tris tris(hydroxymethyl) aminomethane     

Purification and characterization of clathrin-coated vesicles from Chlamydomonas

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.     

Endocytosis of 1,3-β-glucans by broad bean cells at the penetration site of the cowpea rust fungus (haploid stage)

Te penetration hypha of basidiospore-derived infection structures of the cowpea rust fungus (Uromyces vignae Barclay) in epidermal cells of the nonhost, broad bean (Vicia faba L.), was studied with the electron microscope after high-pressure freezing and freeze substitution. After fungal invasion of the epidermis, a plug in the penetration hypha separated the infection structures on the cuticle from the intraepidermal vesicle of the fungus. The plug and the fungal cell wall reacted with a polyclonal 1,3-β-glucan antibody. The plug in the haploid stage seems to have a task similar to the septum formed in the diploid stage of the fungus. Around the penetration hypha, the plant wall stained darkly and a papilla was deposited by the plant. In the papilla, 1,3-β-glucans were labelled by a monoclonal and a polyclonal antibody. In the infected epidermal cell, clathrin-coated pits, coated vesicles, partially coated reticula and multivesicular bodies were found. The contents of the coated pits, coated vesicles, partially coated reticula and multivesicular bodies bound to monoclonal and polyclonal 1,3-β-glucan antibodies. Accumulation and uptake of this paramural material into the plant cell by endocytosis is concentrated at the fungal penetration site. It may influence the host-parasite interaction.     

The isolation of coated vesicles from protoplasts of soybean

Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant clathrin, had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.NRCC No. 23142     

Plasma membrane coat and a coated vesicle-associated reticulum of membranes: their structure and possible interrelationship in Chara corallina

Primary fixation with buffered glutaraldehyde plus 2.0 mM CaCl2 and 0.1% tannic acid results in the preservation of certain portions of the plasma membrane coat of Chara when seen with the electron microscope. Such a coat is not observable after fixation with glutaraldehyde alone. The coat appears to be present on all the above ground, vegetative cells of the male plant. Within complex invaginations of the plasma membrane, which are known as charasomes, the coat has two structural components, a central core that is either tubular or solid and a fibrous or granular peripheral region that surrounds the core. The coat material appears to be at least partially derived, via exocytosis, from the contents of single membrane-bound organelles known as glycosomes. Glycosomes seem to originate from within an assemblage of membranes and coated vesicles that can be described, in purely structural terms, as a partially coated reticulum. Such a reticulum is distinguishable from Golgi stacks because the reticulum (a) is not composed of stacked membranes, (b) is extensively involved with large, clearly detailed coated vesicles and coated invaginations, (c) is closely associated with glycosomes, and (d) is only slightly stained by the zinc-iodide- osmium tetraoxide reagent.     

Coated vesicle isolation by immunoadsorption on Staphylococcus aureus cells

Porcine brain coated vesicles were isolated from crude fractions of tissue homogenates by affinity separation using anticlathrin-coated STaphylococcus aureus (Staph A) cells as a solid-phase immunoadsorbent. The specificity of the immunoadsorption was monitored by SDS PAGE analysis and by competitive ELISA assays. SDS PAGE of the material immunoadsorbed from a fraction of porcine bran smooth microsomes showed a selective enrichment in a 180,000 mol wt protein. In an ELISA assay, this protein competed effectively--in binding anticlathrin--with clathrin extracted from a coated vesicle preparation. When the immunoadsorbed fraction was examined by electron microscopy, coated vesicles and vesicle-free cages were found forming a quasicontinuous monolayer on the surface of the Staph A cells. Other particles were not adsorbed, and the controls were free of either clathrin cages or coated vesicles. Upon extensive dialysis (against MES buffer, pH 6.5), similar cages appeared on the surface of anticlathrin-coated Staph A cells reacted with extracted clathrin. This study demonstrates that anticlathrin-coated Staph A cells can be used for the isolation and purification of a homogeneous population of coated vesicles. In addition, the ability of extracted clathrin to bind and to polymerize onto the Staph A cells raises the possibility of using this technique to further explore the conditions required for cage and/or vesicle reconstitution.