Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.     

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).     

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.     

alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.     

A tissue-engineered human dermal construct utilizing fibroblasts and transforming growth factor β1 to promote elastogenesis

Numerous studies have shown that extracellular matrix (ECM)-based scaffolds are suitable for dermal constructs for the differentiation of various cell types in vitro and for constructive tissue remodeling after implantation in vivo. However, a shortcoming of these ECM materials is its limited elastogenesis. Elastic fibers constitute an essential component of mammalian connective tissue and the presence of elastic fibers is crucial for the proper function of the cardiovascular, pulmonary, and intestinal systems. Since it is still largely unknown how cells coordinate the molecular events of elastic-fiber assembly, understanding the ability to regenerate elastic fibers in tissues remains a significant challenge. For this reason, human neonatal dermal fibroblasts (HDFneo) were analyzed for their potential to serve as a cell culture model for elastic fiber assembly. Using optical technologies such as multiphoton laser-scanning microscopy (MPSLM) we demonstrate that HDFneo stimulated with transforming growth factor β1 (TGF-β1) are able to produce a distinct and complex elastic fiber system in vitro. As shown by the desmosine and isodesmosine content, crosslinked elastic fibers were formed within the 3D ECM-based scaffold. This tissue-engineered dermal construct may prove to be an effective template for the development of medicinal approaches in regenerative soft skin tissue reconstruction through TGF-β1 induction.     

Distribution and organization of the elastic system fibres in healthy human gingiva

Summary The ultrastructural distribution and organization of the elastic system fibres, i.e. oxytalan, elaunin and elastic fibres, were studied by transmission electron microscopy and by an immunohistochemical method for the detection of elastin in healthy human gingiva. The morphological distribution of these fibres was characterized by the presence of oxytalan, elaunin and elastic fibres, respectively, in the upper, medium, and deep layers of gingival connective tissue. Anti-elastin antibody reacted with microfibrils and amorphous material of the elastic system fibres throughout the gingival connective tissue. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin at their surface.     

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.     

Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.     

Uptake of ferritin by the mosaic egg surface of Brachydanio

The internalization of membrane from the mosaic egg surface of the zebra fish, Brachydanio, was investigated using anionic ferritin and transmission electron microscopy. The cortical cytoplasm of the 5-min activated egg showed numerous membrane-bound vesicles not found in the unactivated egg cortex. Two types of vesicles were identified: uncoated (smooth) and coated. Coated vesicles measured about 0.7 to 0.9 micrometer in diameter. Coated pits, considered to be precursors to the formation of coated vesicles, were frequently observed at the base of membrane-lined cortical granule crypts. Anionic ferritin was localized over coated pits and in both smooth and coated vesicles. The absence of any morphological evidence of a surface origin for smooth vesicles suggested these ferritin-labeled organelles might be formed by coated vesicle fusion. Our results indicate that the plasma membrane redundancy created by the exocytosis of cortical granules in Brachydanio appears to be resolved in part by the internalization of membrane through endocytosis.     

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.     

Microinjection of anticlathrin antibodies into fibroblasts does not interfere with the receptor-mediated endocytosis of alpha2-macroglobulin

Affinity-purified antibodies prepared against the major coat protein of brain coated vesicles, clathrin, were microinjected into cultured fibroblasts, and their intracellular distribution was followed by immunofluorescence microscopy and ultrastructural immunocytochemistry. Microinjected anticlathrin antibodies were concentrated on coated regions of the plasma membrane and the GERL apparatus. When an excess of anticlathrin antibodies was injected into the cytosol, coated pits on the plasma membrane were covered by anticlathrin antibody but still functioned to cluster an internalize alpha2-macroglobulin. These results are discussed in terms of the role of clathrin in the pathway of receptor-mediated endocytosis. Our data indicate that in cultured fibroblasts coated pits are stable elements permanently attached to the plasma membrane.     

Localization of a renal kallikrein immunoreactive-like substance in rat ureter

An antibody against rat kallikrein was produced in rabbits and its localization was studied in various organs of the rat to confirm its specificity. The distribution of immunoreactive kallikrein was studied in rat ureter by use of immunochemical techniques. Ureteral tissue was fixed in Zamboni's-glutaraldehyde fixative and immunostained with indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) method for light and electron microscopy. Preabsorption of the primary polyclonal antiserum with purified rat urinary kallikrein and substitution with normal serum were used as controls. By light microscopy, kallikrein was localized in the lamina propria and in the adventitial connective tissue surrounding the entire ureter. Immunoelectron microscopy confirmed this immunolocalization. Immunoreactive kallikrein was concentrated in fibroblasts of connective tissue and was not present in collagen fibers. Immunoreactivity was associated with the Golgi complex, free polyribosomes, and rough endoplasmic reticulum. No immunostaining was observed in other subcellular components of fibroblasts.     

Organelle relationships in cultured 3T3-L1 preadipocytes

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha- naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol.     

Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells

Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.     

Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as “coated pits” and “coated plaques.” Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane—classical coated pits and coated plaques—and that these two assemblies interact quite differently with other intracellular structures.     

The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts

It has previously been shown by immunofluorescence experiments that the cross-linking of HLA antigens into patches (by antibody reagents directed to human β₂-microglobulin) on the surfaces of cultured human fibroblasts leads to the lining up of the patches over the actomyosin-containing stress fibers lying immediately under the surface membrane. These experiments have now been extended to the resolution of the electron microscope by the use of ferritin-conjugated antibody. The results show that a substantial part of the HLA surface clusters that form by 5 min after the addition of the antibody reagents is found in small uncoated surface invaginations which are subsequently endocytosed and ultimately fuse with lysosomal bodies. At no stage in this process is there any indication that coated pits or coated vesicles participate. These and other results suggest, therefore, that there are at least two distinct mechanisms for the ligand-induced endocytosis and lysosomal processing of membrane components, one involving coated pits and the other the noncoated invaginations described in this paper. Transmembrane associations of clusters with intracellular actomyosin-containing structures may have a role in the endocytosis of these noncoated invaginations.     

Surface interactions and intracellular localization of cationic ferritin: similarity to 125I-insulin in 3T3-L1 adipocytes

We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.     

Fibroblast spreading induced by connective tissue stretch involves intracellular redistribution of α- and β-actin

Mechanical stretching of connective tissue occurs with normal movement and postural changes, as well as treatments including physical therapy, massage and acupuncture. Connective tissue fibroblasts were recently shown to respond actively to short-term mechanical stretch (minutes to hours) with reversible cytoskeletal remodeling, characterized by extensive cell spreading and lamellipodia formation. In this study, we have examined the effect of tissue stretch on the distribution of α- and β-actin in subcutaneous tissue fibroblasts ex vivo. Normal fibroblasts uniformly exhibited α-smooth muscle actin (α-SMA) immunoreactivity. Unlike cultured fibroblasts and smooth muscle cells, α-SMA in these fibroblasts was not in F-actin form (indicated by lack of phalloidin co-localization) nor was it organized into distinct stress fibers. The lack of stress fibers and fibronexus was confirmed by electron microscopy, indicating that these cells were not myofibroblasts. In unstretched tissue, the pattern of α-actin was diffuse and granular. With tissue stretch (30 min), α-actin formed a star-shaped pattern centered on the nucleus, while β-actin extended throughout the cytoplasm including lamellipodia and cell cortex. This dual response pattern of α- and β-actin may be an important component of cellular mechanotransduction mechanisms relevant to physiologic and therapeutic mechanical forces applied to connective tissue.     

Fibronectin Receptor Internalization and AP-2 Complex Reorganization in Potassium-Depleted Fibroblasts

Potassium-depleted fibroblasts are unable to develop polarized morphology and lack coated pits. Experiments were carried out to measure internalization of fibronectin receptors (FNR) in potassium-depleted cells and possible association of FNR with AP-2 complexes after adding potassium back to the cells, which restores cell polarization. AP-2 complexes are the cell surface component of coated pits that contain both clathrin and membrane receptor binding domains. Potassium-depleted fibroblasts endocytosed antibody-tagged FNR and also internalized fluorescent fibronectin that previously had been adsorbed to the substratum. During cell polarization, antibody-tagged FNR reorganized into fibrillar structures along stress fibers beginning from nucleation sites at cell margins. Plasma membrane AP-2 complexes, which were undetectable in potassium-depleted cells, reappeared at the cell surface above the nucleus and then spread toward the cell margins. The results show that endocytosis of FNR can occur at least partially by a coated pit-independent mechanism.     

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.