Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).     

Identification of free coated pinocytic vesicles in Swiss 3T3 cells.

Whether or not free coated vesicles are involved during internalization of ligands bound to the receptors of coated pits is controversial. Free coated vesicles cannot be identified with certainty in random individual thin sections - reconstructions based on consecutive thin sections are required. The thickness of the sections determines the reliability of such reconstructions. In the present study, serial section electron microscopy was applied to Swiss 3T3 cells and the topographical resolution yielded by 80 nm and 20 nm sections was compared. Swiss 3T3 cells in monolayer at 37 degrees C were exposed for 5 min to cationized ferritin (CF) which is a marker of pinocytic vesicles. Subsequently the cells were fixed, pelleted and further processed for electron microscopy. The results showed that reconstructions of coated CF-labeled structures based on consecutive sections of an average thickness of approximately 80 nm could not be performed with certainty. A substantial fraction (25%) of the examined profiles appeared to be free vesicles, but narrow surface connections could easily have been missed in these thick sections. The series of the much thinner 20 nm sections provided a better resolution allowing the narrowest surface connections to be identified. Accordingly, the number of truly free, coated vesicles was much lower than the number of apparently free vesicles in the thick sections. However, free coated vesicles labeled with CF were identified in the consecutive 20 nm sections (4% of the examined profiles).     

Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts

Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 125I-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 125I-EGF both bound preferentially to coated pits at 4 degrees C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 125I-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but 125I-EGF bound to coated pits and was internalized just as in normal fibroblasts.     

Independent variation in the number of coated pits and of coated vesicles in cultured fibroblasts

When tissue culture cells were maintained at 37 degrees C in a serum-free medium for 4 hr no change in the number of coated pits could be detected using ultrastructural techniques. However, the number of coated vesicles was highly significantly increased, being 179% more than in the control cultures. If the cells were put back into a medium supplemented with 5% calf serum, the number of coated pits was unchanged, but the number of coated vesicles decreased and returned to the control level within a few minutes. The same results were obtained when using ligands such as Low Density Lipoprotein or alpha-2-macroglobulin which are known to be internalized via coated structures. It is concluded that coated pits appear and disappear at equal rates and that coated vesicles can accumulate independently. It is suggested that this could be due to the presence of a large reserve of soluble clathrin. This pool would have a low turnover rate because cycloheximide did not block coated vesicle accumulation over the period studied.     

Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices

Previous studies have shown that when human fibroblasts are depleted of intracellular K+, coated pits disappear from the cell surface and the receptor-mediated endocytosis of low density lipoprotein (LDL) is inhibited. We have now used the K+ depletion protocol to study several aspects of coated pit function. First, since coated pits rapidly form when K+-depleted fibroblasts are incubated in the presence of 10 mM KCl, we studied the sequence of assembly of coated pits as visualized in carbon-platinum replicas of inner membrane surfaces from cells that had been incubated in the presence of K+ for various times. New coated pits initially appeared as planar clathrin lattices that increased in size by the formation of polygons at the margin of the lattice. Once the lattice reached a critical size it invaginated to form coated vesicles. Second, we determined that LDL-ferritin can induce clustering of LDL receptors over noncoated membrane on the surface of K+-depleted fibroblasts; however, when these cells are subsequently incubated in the presence of K+, these clusters become associated with newly formed coated pits and are internalized. Finally, we determined that K+ depletion inhibits the assembly of coated pits, but that existing coated pits in K+-depleted cells are able to internalize LDL. These results suggest that the clathrin lattice of coated pits is actively involved in membrane shape change during endocytosis and that the structural proteins of the lattice are cyclically assembled and disassembled in the process.     

Formation of coated vesicles from coated pits in broken A431 cells

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti- transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.     

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.     

Induction of mutant dynamin specifically blocks endocytic coated vesicle formation

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline- inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.     

Acidification of the cytosol inhibits endocytosis from coated pits

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.     

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.     

Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Immunogold visualization of actin near the coated pits at the apical surface of human mammary epithelial cells.

This ultrastructural study of both the normal human breast tissue and differentiated mammary carcinoma (NOS) epithelial cells has revealed pictures demonstrating luminal receptor-mediated endocytosis. By application of immunogold anti-actin labeling, actin surrounding the fusion ring of coated pits was visualized. However, the coated membrane was not actin labeled. We suggest that association of the actin with coated pits may evidence for its participation in pinching off of the coated vesicles.     

The relationship of echinate inclusions and coated vesicles on wound healing inNitella flexilis (Characeae)

Summary Wound healing in internodal cells of the freshwater algaNitella flexilis (Characeae) was studied in the light and electron microscope. Immediately after punctation of the cell wall a wound plug is formed which stops outflow of cytoplasm. The plug consists of echinate inclusions which are normally located in the central vacuole. A wound wall consisting of pectin and cellulose microfibrils is formed beneath the plug within one to several hours. During that time the wound shows intensive fluorescence when treated with chlorotetracycline indicating transmembrane Ca²⁺ fluxes. Numerous coated pits and vesicles are found at the plasmalemma. The glycosomes undergo pronounced structural changes. Neither plug nor wound wall formation depend on actin filaments or microtubules as shown by inhibitor experiments with cytochalasin and amiprophos-methyl. The function of the coated vesicles and their interrelationship with other cell organelles is discussed.     

Tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen

The fine structure of plasmalemmal tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen has been demonstrated by scanning and transmission electron microscopy. In addition, the three-dimensional structure of the tubular invagination has been revealed by computer-aided reconstruction. The tubular invaginations of the plasma membrane plunged into the cytoplasm everywhere from the apical, lateral, and basal surfaces of the plasma membrane. The invaginations were tubular and branched away, and their plasma membranes were reinvaginated to form numerous caveolae and occasional coated pits. Numerous caveolae were found in clusters that looked similar to a bunch of grapes and the coated pits were present at the base of the clusters. The caveolae and coated pits derived from the tubular invaginations were almost ultrastructurally identical to those derived from the surface plasma membrane. From examination of the fractured surfaces of the endothelial cells treated with the aldehyde prefix osmium-dimethyl sulfoxide-osmium method and of ultrathin sections of those infiltrated by lanthanum nitrate, the tubular invaginations were found to not penetrate any endothelial cells. A computer-aided reconstruction revealed that the caveolae derived from the tubular invaginations were in close apposition to the surface-connected canaliculi. The reaction product of Concanavalin A conjugated to horseradish peroxidase was present on the outer leaflet of the membranes of the coated pits and coated vesicles and also in the contents of the endosomes, but it was absent from any caveolae. Based on our observations, the functional significance of the tubular invaginations in sinus endothelial cells is discussed. Accepted: 13 September 1999     

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.     

Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein

Filipin has been widely used as an electron microscopic probe to detect 3-beta-hydroxysterols, principally cholesterol, in cellular membranes. When it complexes with sterol, it forms globular deposits that disrupt the planar organization of the membrane. Previous studies have shown that coated pits and coated vesicles, specialized membranes involved in receptor-mediated endocytosis, do not appear to bind filipin. This has led to the suggestion that these membranes are low in cholesterol compared with the remainder of the plasma membrane. Since coated endocytic vesicles become uncoated vesicles during the transport of internalized ligands to the lysosome, we have carried out studies to determine whether or not the membranes that surround these transport vesicles are unable to bind filipin and therefore, are also low in cholesterol. Cells were incubated with ferritin-conjugated ligands that bind to low density lipoprotein (LDL) receptors in coated pits. After allowing internalization of the conjugates, we fixed the cells in either the presence or absence of filipin. This permitted us to identify all of the vesicles involved in the transport of LDL to the lysosome and to determine whether the membranes of these vesicles were able to bind filipin. We found that, coordinate with the dissociation of the clathrin coat from the endocytic vesicles, the membranes became sensitive to the formation of filipin-sterol complexes. Furthermore, all of the uncoated endocytic vesicle membranes, as well as the lysosomal membranes, bound filipin. This suggests either that coated membrane contains normal cholesterol levels, which is not easily detected with filipin, or that cholesterol rapidly moves into endocytic vesicles after the clathrin coat dissociates from the membrane.     

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.     

Coat proteins isolated from clathrin coated vesicles can assemble into coated pits

Isolated human fibroblast plasma membranes that were attached by their extracellular surface to a solid substratum contained numerous clathrin coated pits that could be removed with a high pH buffer (Moore, M.S., D.T. Mahaffey, F.M. Brodsky, and R.G.W. Anderson. 1987. Science [Wash. DC]. 236:558-563). When these membranes were incubated with coat proteins extracted from purified bovine coated vesicles, new coated pits formed that were indistinguishable from native coated pits. Assembly was dependent on the concentration of coat protein with half maximal assembly occurring at 7 micrograms/ml. Assembly was only slightly affected by the presence of divalent cations. Whereas normal appearing lattices formed in a low ionic strength buffer, when assembly was carried out in a low pH buffer, few coated pits were evident but numerous small clathrin cages decorated the membrane. Coated pits did not form randomly on the surface; instead, they assembled at differentiated regions of membrane that could be distinguished in carbon/platinum replicas of frozen and etched membranes by the presence of numerous particles clustered into patches the size and shape of a coated pit.     

Ferromagnetic isolation of endosomes: a novel method for subcellular fractionation of Xenopus oocytes

A novel method has been developed using ferric particles to label endosomes, and to achieve magnetic sorting of the various endocytic compartments involved in lipoprotein uptake into cells. Ferric particles conjugated to a receptor-recognized ligand are bound to coated membrane pits and become internalized into the cytoplasm inside coated vesicles. After apparent fusion of the vesicles to tubular endosomes, the conjugates accumulate and finally discharge into multivesicular endosomes. Pulse-chase experiments elucidate the pathway of internalized conjugates and allow both early compartments (pinosomes and tubular endosomes) and late compartments (multivesicular endosomes and storage organelles) to be selectively labelled. After ferroloading of the various transport compartments, the cells are homogenized and subcellularly fractionated. Sorting of labelled endosomes is performed by a specially designed "free-flow" magnetic chamber. Prophase I-arrested oocytes of the toad Xenopus laevis are used as a model system for studying the transport pathway and the conversion of the yolk precursor vitellogenin. It is possible to follow the route of internalization of vitellogenin-iron conjugates via coated pits, coated vesicles, uncoated vesicles, tubular endosomes, multivesicular endosomes, and light primordial yolk platelets. These endosomes shuttle the ferric particles together with the vitellogenin from oolemma to performed heavy yolk organelles which are still growing. In addition, these various compartments can be isolated according to their function and subjected to electron microscopy and to gel electrophoresis for detailed characterization of their limiting membranes as well as their contents.     

Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.