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Biomedical subjects

M C Willingham

Publications and source records attributed to M C Willingham.

At least 235 records · Page 13Linked to original sources
PubMed1981

Morphologic study of the internalization of a lysosomal enzyme by the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells.

The binding and internalization of a model lysosomal enzyme, beta-galactosidase, was visualized by use of rabbit anti-beta-galactosidase and goat anti-rabbit IgG; the second antibody was labeled with rhodamine or fluorescein (for detection by fluorescence) or with horseradish peroxidase (for electron microscopy). Chinese hamster ovary cells were incubated with beta-galactosidase at 4 degrees C, and then were washed and sequentially incubated in the cold with the two antibodies. The beta-galactosidase was found primarily in coated pits. The binding of the enzyme was completely inhibited by 5 mM mannose 6-phosphate. After the reaction with enzyme and antibodies, the cells were warmed to 37 degrees C; within 1 minute, the beta-galactosidase--antibody complex had begun to move to uncoated vesicles (receptosomes). After 8 min, the beta-galactosidase--antibody complex was seen in receptosomes near tubular elements in the Golgi/GERL area, within such tubular elements and at times, in vesicular elements that may correspond to coated structures of the GERL system. After 15 min, the enzyme--antibody complex was found in lysosomes near the Golgi/GERL are and a half-hour later it was in lysosomes distributed throughout the cytoplasm. Double-label experiments using beta-galactosidase and gold/alpha 2-macroglobulin showed the presence of the two ligands in the same coated pits and receptosomes. Thus, the pathway for internalization of beta-galactosidase via the mannose 6-phosphate receptor is similar to the pathway established for other ligands such as low density lipoprotein and alpha 2-macroglobulin.

Animals↗
PubMed1981

alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis.

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.

Animals↗
PubMed1981

An immunoperoxidase study of senile cerebral amyloidosis with pathogenetic considerations.

Samples of human cerebral cortex were obtained from twelve autopsied patients with Alzheimer's disease or "normal" aging. Rabbit or goat anti-human antisera to the following plasma proteins: IgG, F(ab')2, Fc, kappa and lambda light chains, IgM, IgA, fibrinogen, albumin, C3, lysozyme, haptoglobin, macroglobulin, and microglobulin; antibodies to the following intracellular proteins: glial fibrillary acidic (GFA) protein, filamin, actin, non-muscle myosin, tubulin, cholinergic vesicle proteins, and neurofilament (NF) proteins were utilized in the immunoglobulin peroxidase bridge. Amyloid cores of classical or perivascular plaques and dyshoric angiopathy exhibited a strong reaction for intact IgG and for both of its light chains, moderate reactions for lysozyme, fibrinogen, albumin and IgA, and weak reactions for IgM, C3, Fc, F(ab')2, haptoglobin, macroglobulin and microglobulin. Antibodies to all three NF proteins, individually and pooled, stained dyshoric and plaque amyloid, while antibodies to other intracellular proteins did not. The coronae of classical plaques and many primitive plaques stained for GFA, but inconsistently for IgG, both light chains, lysozyme, actin, tubulin, and NF proteins. Affected vessels of three patients with Congophilic angiopathy were reactive for all plasma proteins (especially IgG, fibrinogen, and albumin) and for NF proteins. NF staining in Congophilic blood vessels, although variable, revealed a peripheral or adventitial distribution, whereas plasma proteins tended to be localized in the media of the vessel wall. The distributions of Congo red and NF positivity were often identical. Both NF and Congo red staining was sensitive to oxidation. Isolated NF proteins were Congophilic and capable of displaying apple-green birefringence. A hypothesis concerning the role of NF proteins in senile cerebral amyloid is presented.

Amyloid↗
PubMed1981

Intracellular localization of actin in cultured fibroblasts by electron microscopic immunocytochemistry.

Antibodies to skeletal muscle actin were produced in rabbits and purified by affinity chromatography. Direct labeling of SDS-PAGE gels of whole cell homogenates from mouse fibroblast cells showed that actin was the only protein detected by these antibodies. Using this immunospecific reagent, we localized actin in cultured fibroblasts using the EGS fixation-permeabilization procedure with the ferritin bridge labeling technique. Swiss 3T3-4 mouse fibroblasts were chosen as an example of highly adherent untransformed cells with prominent microfilament bundles, and L929 mouse fibroblasts were chosen as an example of poorly adherent, rounded, transformed cells with prominent microvilli. Using these two cell types, we have characterized the intracellular distribution of action. Actin was only detected in locations in which morphologically recognizable 60 A microfilaments were found. By both fluorescence and electron microscopy, actin was found in surface ruffles, microvilli, microfilament bundles, the microfilament mat, and the leading lamellae of Swiss 3T3 and L929 cells. In addition, actin was found surrounding micropinosomes and macropinosomes. On the other hand, there was no actin detected around the base of coated pits. Morphometric quantitation showed that almost all the actin was localized in microfilamentous structures. Our results suggest that actin has an important role in cell motility and adhesion, and in the endocytosis of pinosomes, but that actin may not be involved in intracellular processes such as saltatory motion of intracellular organelles.

Actins↗
PubMed1981

Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells.

Nonmuscle myosin in the cytoplasm of cultured fibroblastic cells has been localized using light and electron microscopic immunocytochemistry. Antibodies to purified fibroblast myosin were produced in goat and rabbit and purified by affinity chromatography. Light microscopic immunofluorescence localization showed patterns similar to those previously published. Electron microscopic localization using the ethyldimethyl aminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation-permeabilization procedure and the ferritin bridge localization method produced quantifiable localization in intracellular sites with well-preserved ultrastructural morphology. Myosin was found to be a major component of the cytosol. It was distributed diffusely with no preferential localization on membranous organelles. Myosin was found to be slightly concentrated on the surface of microfilament-containing structures, including the subplasmalemmal microfilament mat and stress fibers, occasionally with an interrupted periodicity. However, no myosin was found in surface ruffles or microvilli. Morphometric quantitation showed that the majority of the cell's myosin was in the cytosol. This location is compatible with myosin being a component of the microtrabecular lattice of the cytoplasmic ground substance. The concentration of myosin in association with microfilaments was only twice that of the cytosol. This interpretation must be somewhat tempered by the possibility that some myosin bound to tightly packed actin may be inaccessible. The significance of this distribution of myosin in cell function is discussed.

Actins↗
PubMed1981

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.

Animals↗
PubMed1980

Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells.

The binding and internalization of 125I-epidermal growth factor (125I-EGF) was studied in cultures of BALB 3T3 cells using a novel method that involved removal of cell-surface hormone by treatment with acetic acid under conditions that did not remove internalized hormone. In control cultures, 125I-EGF initially bound to its receptor on the plasma membrane and then was rapidly internalized. After 30 min, only 15% of the cell-bound hormone remained on the surface. In contrast, cultures treated with dansylcadaverine retained 82% of the cell-bound hormone on the cell surface. We propose that dansylcadaverine inhibits EGF internalization by preventing it from clustering in clathrin-coated pits.

Animals↗
PubMed1980

Electron microscopic immunocytochemical localization of intracellular antigens in cultured cells: the EGS and ferritin bridge procedures.

A new primary fixative, ethyldimethylaminopropyl carbodi-imide-glutaraldehyde-Tris, has been combined with the use of saponin for membrane permeabilization to yield a procedure which preserves ultrastructural morphology, yet retains a cytoplasmic matrix permeable to globulin molecules. This allows pre-embedding localization of intracellular protein antigens in cultured cells by fluorescence or electron microscopy. A further combination of these methods with the 'ferritin bridge' techniqe has allowed discrete localization which is quantifiable. Together, these methods yield an overall technique which provides high quality ultrastructural morphological preservation and precise antigen localization. Examples of the localization of alpha 2-macroglobulin, actin, SV40 T-antigen, tubulin and p60src are demonstrated. Extension of these methods from cultured cells to intact tissue should be possible without major changes.

Actins↗
PubMed1980

Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry.

Using electron microscopic immunocytochemistry, we have investigated the intracellular location of the src protein (p21) in cells transformed by the Harvey strain of Murine Sarcoma Virus (Ha-MSV). Antibodies to p21 were derived from tumor-bearing rats inoculated with Ha-NRK cells. The distribution of p21 in intracellular sites in MDCK dog cells transformed by Ha-MSV was examined and quantified using a recently developed immunocytochemical technique. More than 95% of p21 was localized to the inner surface of the plasma membrane in these Ha-MSV-transformed cells; p21 was not exposed on the outside surface of the plasma membrane. A similar location was observed by immunofluorescence in other Ha-MSV-transformed cell lines, including cells derived from rat, mouse and mink. This finding, and the previous demonstration that p60src of avian sarcoma virus is concentrated on the inner surface of the plasma membrane, suggests that the plasma membrane is a major site of action for transforming proteins.

Animals↗
PubMed1980

The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts.

Receptor-mediated endocytosis of specific ligands is mediated through clustering of receptor-ligand complexes in coated pits on the cell surface. Following this clustering event, the ligand is internalized into a noncoated intracellular vesicle, the receptosome, which selectively avoids fusion with lysosomes and moves toward the Golgi region of the cell by saltatory motion. Using alpha 2-macroglobulin as the ligand and electron microscopic cytochemical methods, we have shown the unusual appearance of this previously undescribed organelle and have followed the labeled ligand in these vesicles in the cytoplasm. To accomplish this, cells were incubated with immunolabeled alpha 2-macroglobulin at 4C under conditions where ligand-receptor complexes cluster into coated pits on the cell surface. Formation of the receptosome occurs between 2 and 5 min after raising the temperature of cells to 37C. These labeled receptosomes were seen to associate with many small vesicular elements in the cytoplasm, and were often found near the Golgi or GERL region after 15 min. Between 15 and 30 min a significant transfer of labeled ligand occurred from the receptosomal population to a population of small uniform lysosomes. By 60 min, all of the label was contained in these small lysosomes. Immunocytochemical studies showed that the receptosomes were not associated with clathrin, actin, myosin or tubulin. This unique, short-lived, specialized organelle selectively delivers the products of receptor-mediated endocytosis to intracellular sites.

Animals↗
PubMed1980

Receptor-mediated uptake of 3,3',5-triiodo-L-thyronine by cultured fibroblasts.

Using video intensification fluorescence microscopy and tetramethylrhodamine (Rho)-labeled 3,3',5-triiodo-L-thyronine (T3), we studied the uptake of T3 by cultured mouse fibroblasts. After incubation of cells with Rho-T3 for 30 min at 37 degrees C the fluorescent hormone was concentrated in many small bright accumulations. With a 1000-fold excess of unlabeled T3, only weak background fluorescence was seen. Furthermore, when cells were incubated with Rho or Rho-thyronine only background fluorescence was detected. These results indicate that the cellular uptake of Rho-T3 occurred through a T3-specific receptor-mediated process. Most of these accumulations underwent saltatory motion in living cells, indicating that the T3 was contained within endocytic vesicles. When cultured cells were incubated with Rho-T3 for 60 min at 4 degrees C, only diffuse fluorescence was observed, Rho-T3 became concentrated in vesicles upon warming of the cells to either 23 degrees C or 37 degrees C. Simultaneous incubation of cells with fluorescein-labeled alpha 2-macroglobulin and Rho-T3 showed that Rho-T3 was internalized in the same vesicles as alpha 2-macroglobulin. Furthermore, as previously reported for alpha 2-macroglobulin in the presence of methylamine, dansylcadaverine, or bacitracin, clustering and internalization were inhibited but the overall fluorescence intensity of the cells did not appear to be affected. Because it has been previously shown that receptor-mediated endocytosis of alpha 2-macroglobulin occurs through clustering of ligands in coated pits on the cell surface, these results indicate that Rho-T3 follows the same pathway. Thus it has now been demonstrated that a low-molecular weight hormone enters cells by this pathway.

Animals↗
PubMed1980

Ultrastructural localization of tubulin in cultured fibroblasts.

Antibodies to barin tubulin were created in rabbits and used to localize tubulin in cultured fibroblasts. Cells were fixed and permeabilized using the EGS procedure (Willingham et al: J Cell Biol 79:256a, 1978). Tubulin was localized and quantified using a ferritin-bridge labeling method. Antibody localization was generally confined to morphologically identifiable microtubules, with a smaller concentration in a diffuse cytoplasmic distribution. Following treatment with colchicine, morphologically identifiable microtubules disappeared and the amount of diffuse localization in the cytosol increased. However, a significant concentration of tubulin was found associated with the microfilament mat under the plasma membrane after colchicine treatment, whereas in untreated cells, very little tubulin was found in the microfilament mat. These results demonstrate that tubulin is a component of microtubules and that colchicine treatment results in an association of this tubulin with microfilaments.

Animals↗
PubMed1980

Ultrastructural localization to 10 nm filaments of an insoluble 58K protein in cultured fibroblasts.

Using a simple procedure, we have isolated a Triton X-100 insoluble protein (58,000 mol wt) from cultured Chinese hamster ovary (CHO) cells, a fibroblastic cell line. The isolated protein, judged homogeneous by two-dimensional gel electrophoresis, was used to elicit antibodies in rabbits. The antiserum obtained is specific for the 58K protein as determined by immunoprecipitation from detergent solubilized 35S-labeled CHO cells and by antibody labeling of SDS solubilized Swiss 3T3 cell extracts separated on a 10% polyacrylamide gel. Indirect immunofluorescence localization with this antiserum in fixed permeabilized Swiss 3T3 cells, whose flat morphology makes them superior for localization studies, reveals filaments that radiate from the perinuclear region to the cell periphery. These filaments were identified as 10 nm filaments by electron microscopic localization using the EGS and ferritin bridge procedures. We conclude that the 58K protein is a major constituent of fibroblast 10 nm filaments. Other intracellular structures were not labeled by this technique.

Animals↗
PubMed1980

Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts.

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.

Animals↗