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M C Willingham

Publications and source records attributed to M C Willingham.

At least 253 records · Page 14Linked to original sources
PubMed1980

Intracellular localization of fibronectin by immunoelectron microscopy.

We have localized fibronectin, a major extracellular glycoprotein of cultured fibroblasts, in chick embryo fibroblasts at the ultrastructural level using affinity-purified antibodies to fibronectin. The use of a ferritin bridge procedure permitted quantitation of localization in various organelles. These results provide the first intracellular ultrastructural localization of fibronectin. Extracellular labeling was confined to aggregates and fibrils, with little or no labeling of the plasma membrane. The principal sites of intracellular localization were the rough endoplasmic reticulum and the Golgi apparatus. Treatment of cells with the protein synthesis inhibitors cycloheximide and pactamycin reduced fibronectin localization in the endoplasmic reticulum to 50% of normal levels. Removal of cycloheximide permitted recovery of labeling to 85% of control levels in the endoplasmic reticulum. Similar, but much reduced, changes also occurred in the Golgi apparatus.

Animals↗
PubMed1979

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

The cellular location of the src gene product (p60src) of the Schmidt-Ruppin strain of avian sarcoma virus has been determined by electron microscopic immunocytochemistry in Schmidt-Ruppin ASV-transformed NRK cells, and the amount of the protein in different regions of the cell has been quantified. The protein is concentrated on the inner surface of the plasma membrane, particularly under ruffles, and it is highly concentrated on the inner surface of the membrane near junctions connecting adjacent cells. Small amounts of p60src were detected in the cytoplasm and in the perinuclear Golgi region of the cell. No significant localization was detected in control NRK cells or in NRK cells transformed by the Kirsten strain of murine sarcoma virus. The presence of p60src on the inner surface of the plasma membrane indicates that the changes in cell growth, cell shape and cell membrane structure noted in ASV-transformed cells are due to an initial action of p60src at the cell membrane.

Alpharetrovirus↗
PubMed1979

Epidermal growth factor stimulation of DNA synthesis is potentiated by compounds that inhibit its clustering in coated pits.

We have used inhibitors of receptor-mediated endocytosis to investigate the mechanism and function of epidermal growth factor uptake by cultured cells. When rhodamine-labeled epidermal growth factor is bound to cell surface receptors on confluent monolayers of BALB/c 3T3 cells, it rapidly collects in cell surface clusters and is internalized. The clustering of occupied receptors requires Ca(2+) and is inhibited by primary alkylamines; both of these properties are shared by the enzyme transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13). In Chinese hamster ovary cell extracts, methylamine inhibits 25-50% of the transglutaminase activity with a K(i) of 0.2 mM, and it inhibits the remaining transglutaminase activity with a K(i) of 20 mM. Clustering is almost completely inhibited by 10 mM methylamine. The polypeptide antibiotic bacitracin inhibits clustering of rhodamine-labeled epidermal growth factor or alpha(2)-macroglobulin at 0.7 mM, and it inhibits approximately 40% of the transglutaminase activity in Chinese hamster ovary cells with a K(i) of 0.03 mM. Fluorescent ligands bound to cell surface receptors in the presence of bacitracin form clusters within 30 min after bacitracin is removed from the culture medium. These results indicate that a transglutaminase-like enzyme may be required for the clustering and subsequent internalization of occupied receptors. The effects of 10 mM methylamine and 0.7 mM bacitracin on epidermal growth factor stimulation of DNA synthesis were examined. The stimulation of DNA synthesis by epidermal growth factor was increased 2- to 7-fold in the presence of methylamine or bacitracin. Alone, methylamine or bacitracin increased DNA synthesis 1.1- to 3-fold. The stimulation of DNA synthesis resulting from the simultaneous presence of the hormone and the clustering inhibitor was always greater than the sum of the stimulations produced by the hormone and the clustering inhibitors alone. The potentiation of epidermal growth factor activity by clustering inhibitors suggests that the hormone acts at the cell surface. We propose that rapid internalization of occupied receptors via coated pits may be a mechanism to limit the response to hormones.

Animals↗
PubMed1979

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.

Animals↗
PubMed1979

Development of a new primary fixative for electron microscopic immunocytochemical localization of intracellular antigens in cultured cells.

We have developed a new primary fixative that permits the localization of intracellular antigens with well preserved ultrastructural morphology. This primary fixation method employs a mixture of a water soluble carbodiimide with glutaraldehyde, and preserves morphology, yet produces a permeable cytosol matrix so that antibodies can gain access to fixed proteins. Cultured cells were primarily fixed, treated with detergent to permeabilize their membranes, reacted with peroxidase labeled antibodies, secondarily fixed, and embedded in situ. The variations in morphology and accessibility of intracellular antigens were evaluated for a variety of fixatives. Concanavalin A and alpha 2 macroglobulin were chosen as examples of intracellular protein antigens to evaluate these fixation methods. Both of the proteins were localized in intracellular vesicles.

Aldehydes↗
PubMed1978

Filamin-actin interaction. Dissociation of binding from gelation by Ca2+-activated proteolysis.

Chicken gizzard filamin has been digested with purified Ca2+-activated protease. The subunits of (Mr = 250,000) of the protein are cleaved asymmetrically into two fragments, heavy merofilamin, Mr = 240,000, and light merofilamin, Mr = 9,500. Digestion is complete at substrate to enzyme ratios of 100:1 and requires Ca2+ concentrations in excess of 0.3 mM. Heavy merofilamin binds to F-actin as evidenced by cosedimentation with F-actin, by direct observation under the electron microscope, and by its ability to inhibit actin activation of heavy meromyosin ATPase. Heavy merofilamin does not form a gel when mixed with actin, except at very low concentrations of KCl. Thus, actin binding and gelation are separable activities of filamin. We speculate that Ca2+-stimulated proteolysis may play a role in the regulation of actin-filamin interactions.

Actins↗
PubMed1978

The visualization of fluorescent proteins in living cells by video intensification microscopy (VIM).

A highly sensitive television camera (silicon intensifier target) has been combined with fluorescence microscopy to examine living cultured cells. This system is termed Video Intensification Microscopy (VIM). By using very small amounts of excitation light, one limits the damage to living cells from excessive illumination and is able to visualize fluorescence probes for periods up to 24 hr without bleaching. With VIM, the cellular uptake and fate of two rhodamine-labeled proteins, concanavalin A and alpha2 macroglobulin, have been followed for up to 24 hr. These proteins were first located in endocytic vesicles with a low phase density. Later, at 24 hr, alpha2 macroglobulin was located in phase-dense structures, probably secondary lysosomes. Both the fluorescent endocytic vesicles and lysosomes were observed to undergo saltatory motion. VIM combined with fluorescence promises to have a widespread application in the study of the behavior of living cells.

Cell Line↗
PubMed1978

Collection of insulin, EGF and alpha2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization.

We have used video intensification microscopy to observe fluorescent derivatives of insulin, epidermal growth factor and alpha2-macroglobulin added to Swiss 3T3-4 cells. At 4 degrees C, each of these polypeptides binds diffusely to specific receptors on the cell surface. When the cells are warmed to 23 or 37 degrees C, the bound insulin epidermal growth factor or alpha2-macroglobulin rapidly forms patches on the cell surface and is internalized. Using fluorescein-labeled alpha2-macroglobulin and rhodamine-labeled derivatives of insulin and epidermal growth factor, we show that all three polypeptides are internalized within the same vesicles by a common pathway. The mechanism for the internalization of these molecules is discussed.

Cell Line↗
PubMed1978

Quantitative determination of the lateral diffusion coefficients of the hormone-receptor complexes of insulin and epidermal growth factor on the plasma membrane of cultured fibroblasts.

Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are mobile on the cell surface, with similar diffusion coefficients, D approximately (3--5) x 10(-10) cm2/sec at 23 degrees C. Increasing the temperature to 37 degrees C results in rapid receptor immobilization. The immobilization is attributed to aggregation of hormone-receptor complexes, their internalization, or a combination of both processes.

Azides↗
PubMed1978

Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells.

We have studied in detail the binding of fluorescent derivatives of insulin and epidermal growth factor to 3T3 fibroblasts. We have used two types of fluorescent analogues of insulin and epidermal growth factor: highly fluorescent derivatives which have seven to eight rhodamine molecules or fluorescent derivatives which have a single rhodamine molecule per one molecule of insulin or epidermal growth factor. Both types of analogue retained substantial binding affinity as determined by radioreceptor assays and biological activity. The cells labeled with the fluorescent analogues were visualized with a sensitive video intensification microscopic system that enabled us to directly observe the location of the fluorescent hormone on the surface and within the living fibroblasts. We found that both insulin and epidermal growth factor initially bound diffusely to the cell surface and, at 4 degrees , remained dispersed. Within a few minutes at 23 degrees or 37 degrees the hormone-receptor complexes aggregated into patches that could be readily removed by trypsin but not by excess native hormone. The hormone-receptor complexes, which were initially mobile in the plane of the membrane, become immobilized later as the consequence of the receptor aggregation or internalization. Within approximately 30 min at 37 degrees , much of the labeled hormone was found within the cell in endocytic vesicles that moved about in the cytoplasm in a saltatory manner. The aggregation and immobilization of the hormone-receptor complexes could be due to either hormone-hormone interactions on the cell membrane or a hormone-induced conformational change in the hormone-receptor complex. Aggregation and internalization of hormone-receptor complexes could be associated with certain aspects of hormone action, hormone degradation, down regulation of receptors, or negative cooperativity of hormone binding.

Cell Line↗
PubMed1978

Ultrastructural antibody localization of alpha2-macroglobulin in membrane-limited vesicles in cultured cells.

We have been developing a procedure for localizing intracellular antigens in cultured cells, by using peroxidase-labeled antibodies, that allows good morphologic preservation. Although useful, our previous technique did not preserve the morphology of membranes, and the location of the peroxidase reaction product was difficult to establish. In this paper, we report major improvements on the basic technique that markedly enhance the quality of localization and of morphology. Saponin is used to permeabilize membranes without destroying their morphology. The amount of reaction product is enhanced with a peroxidase-antiperoxidase label. The clarity of morphologic detail and contrast of reaction product density are increased by using postsectioning staining with the osmium/thiocarbohydrazide/osmium and uranyl acetate/lead citrate procedures. We have applied this technique to the ultrastructural localization of alpha2-macroglobulin and demonstrated that it is localized in membrane-limited vesicles. We have also used this method to improve the preservation of structures for localization by fluorescence microscopy.

Cell Line↗
PubMed1978

A mechanism for the destruction of pinosomes in cultured fibroblasts. Piranhalysis.

The destruction of large pinosomes was examined with phase-contrast microscopy in cultured mouse fibroblasts. In areas of rapid pinosome breakdown, lysosomes were observed to repeatedly collide with pinosomes without fusing, tearing off small pieces until the pinosomes became smaller and denser. This segmentation of pinosomes by lysosomal collision has been named "piranhalysis."

Acid Phosphatase↗
PubMed1977

Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts.

The distribution of microfilament bundles in cells was examined using antibodies to fibroblast myosin and indirect immunofluorescence microscopy. There is no correlation between the presence of bundles of microfilaments and normal growth control. A normal cell line (Balb/c 3T3) cultured on a poorly adhesive substratum showed no microfilament bundles. Similarly, a mutant cell line (AD6) with normal growth, but a rounded shape due to defective adhesiveness to substratum, showed no bundle formation. On the other hand, two transformed cell lines with a flat morphology (Swiss SV3T3 and Balb MSV-85) showed extensive bundle formation. When a transformed cell line with poor adhesiveness (MC5-5) was treated with CSP (a major surface glycoprotein of normal cells) which increases adhesiveness to substratum, the cells formed extensive microfilament bundles without any decrease in growth. We conclude that the distribution of microfilament bundles is related to adhesiveness to substratum and cell shape but not to growth properties.

Acetylglucosamine↗
PubMed1975

Cyclic AMP modulates microvillus formation and agglutinability in transformed and normal mouse fibroblasts.

We have utilized dark field microscopy to observe the surface microstructure of living cultured cells. Using this method, we have found that dibutyryl cAMP treatment causes regression of the numerous, long cell surface microvilli present on L929 cells. Thirty minutes after removal of dibutyryl cAMP, microvilli reappear. An inhibitor of phosphodiesterase (methylisobutylxanthine) and a stimulator of adenylate cyclase (prostaglandin E1), both of which raise cAMP levels, cause regression of microvilli in 15 min. Untransformed 3T3 cells show very few microvilli when viewed still attached to their substratum or after removal with EDTA. Treatment of these cells with trypsin causes the formation of numerous microvilli on their surface. When clumps of cells agglutinated by concanavalin A are examined by thin section electron microscopy, the cells are seen to be held together by a "forest" of interdigitating microvilli and only rarely is there apposition of the areas of membrane between microvilli. At the same time the distribution of surface-bound concanavalin A was examined using immunofluorescent light microscopy, and concanavalin A was found to be uniformly distributed over the cell surface. We propose that agglutinability of mouse and rat fibroblasts is regulated through the modulation of cell surface microvilli by cAMP, and that transformed cells are highly agglutinable because their low cAMP levels result in the formation of numerous surface microvilli.

Agglutination↗