Isolation of microvillar microfilaments and associated transmembrane complex from ascites tumor cell microvilli

Carraway, Coralie A. Carothers; Jung, Goeh; Hinkley, Robert E.; Carraway, Kermit L.

doi:10.1016/0014-4827(85)90153-3

Cited by 25 publications

(19 citation statements)

References 26 publications

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Section: Association Of Signal Transduction Pathway Components With Smentioning

confidence: 99%

“…Extraction under mild conditions (isotonic buffer, pH 6.8) and differential centrifugation yields a relatively intact microfilament core that sediments at low speed (61,72). Centrifugation of the supernatant from this sedimentation at 100 kg yields the TMC containing actin but no microfilaments (61,72). A microfilament-depleted high speed pellet called the cytoskeletal TMC, which is enriched in membrane skeletal proteins (63,64,72), was made by Triton extraction of microvilli at pH 9.5 and low ionic strength to depolymerize microfilaments.…”

Section: Association Of Signal Transduction Pathway Components With Smentioning

confidence: 99%

“…Preparation of microvillar microfilament core, membrane, and TMC-enriched fractions-Microfilament cores, microvillar membranes, and TMC-enriched microvillar fractions were prepared as previously reported (61,63,64,71,72). Microfilament cores were prepared by extraction of microvilli in an isotonic microfilament-stabilizing buffer (TPK buffer; 0.5% Triton, 100 mM KCl, 2 mM MgCl 2 , 0.1 mM phenylmethylsulfonyl fluoride, and 20 mM PIPES, pH 6.8) for 15 min and either differential centrifugation (61) or phalloidin shift velocity sedimentation, a diagnostic procedure for analysis of microfilament-associated proteins (71).…”

Section: Passaging Of Ascites Tumor Cells and Preparation Of Microvilmentioning

confidence: 99%

“…63 and 64) containing a protease inhibitor mixture. Low speed centrifugation removed undissociated filaments, and high speed centrifugation yielded a cytoskeletal TMC (cTMC) preparation containing small amounts of microfilament-associated proteins such as ␣-actinin and ezrin (63,64,72). A more highly purified transmembrane complex was prepared by nonionic detergent solubilization of microvillar membranes, followed by fractionation by velocity sedimentation or gel filtration (62).…”

Section: Passaging Of Ascites Tumor Cells and Preparation Of Microvilmentioning

confidence: 99%

“…62 and 73). The lysate was centrifuged at 15,000 ϫ g for 15 min at 4°C, and the supernatant enriched in a cytoskeletal transmembrane complex preparation (63,64,72) was used for immunoprecipitations with antibodies to c-Neu, Raf, and MAPK. Mouse monoclonal anti-MAPK (extracellular signal-regulated kinases 1 and 2), mouse monoclonal anti-c-Neu, rabbit anti-human Raf-1, and appropriate nonimmune control antisera were incubated with protein A-agarose (Sigma).…”

Section: Immunoprecipitations Of Transmembrane Complex-containing Lowmentioning

confidence: 99%

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Association of the Ras to Mitogen-activated Protein Kinase Signal Transduction Pathway with Microfilaments

Carraway¹,

Carvajal²,

Carraway³

1999

Journal of Biological Chemistry

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Microvilli of the aggressive 13762 ascites mammary adenocarcinoma contain a large, microfilament-associated signal transduction particle whose scaffolding is a stable glycoprotein complex (Li, Y., Hua, F., Carraway, K. L., and Carraway, C. A. C. (1999) J. Biol. Chem. 274, 25651-25658) associated with the growth factor receptor p185neu . The receptor is constitutively tyrosine-phosphorylated in the cells and microvilli, predicting that it should recruit mitogenic pathway components to this membrane-microfilament interaction site. Immunoprecipitation of cell lysates with anti-phosphotyrosine and immunoblotting showed phosphorylated forms of the mitogenic pathway proteins Shc and MAPK in addition to p185 neu , suggesting that the Ras to MAPK mitogenic pathway is activated. Immunoblotting of p185 neu -containing microvillar fractions revealed the presence in each of stably associated Shc, Grb-2, Sos, Ras, Raf, mitogen-activated protein kinase kinase, and mitogenactivated protein kinase/extracellular signal-regulated kinase, as well as the transcription factor-phosphorylating kinase Rsk. All of these pathway components coimmunoprecipitated with p185neu from cleared lysates of microvilli solubilized under microfilament-depolymerizing conditions. The recruitment of constitutively phosphorylated p185 neu and the activated mitogenic pathway proteins to this membrane-microfilament interaction site provides a physical model for integrating the assembly of the mitogenic pathway with the transmission of growth factor signal to the cytoskeleton. This linkage is probably a requisite step in the global cytoskeleton remodeling accompanying mitogenesis.Growth factors trigger both mitogenesis and changes in cell morphology and microfilament organization (1). Since both of these effects result from the binding of the factors to a single type of receptor, their pathways must somehow be integrated. One suggestion for this integration is that the receptors and components of the signaling pathways are localized to sites where microfilaments interact with membranes (2). The EGF 1 receptor (3) and the EGF receptor family member p185 neu (ErbB2/HER2) (4) have been shown to be associated with microfilaments. EGF receptor binds directly to actin through a motif similar to the actin-binding motif of profilin (5). The mechanism for the association of p185 neu /ErbB2 with microfilaments is presently unclear. Interestingly, p185neu /ErbB2, which has been implicated in tumor cell metastasis, is proposed to play a role in the regulation of cell structure and motility (6). The receptor is concentrated on microvilli in SK-BR-3 breast cancer cells. Activation of its phosphorylation leads to cell spreading, enlargement of microvilli, formation of pseudopodia, and aggregation of the p185 at the plasma membrane protrusions and pseudopodia (6). A specific role for p185 neu /ErbB2 in these microfilament-dependent processes known to enhance metastatic capability of tumor cells was shown in p185 neuoverexpressing transfectants of NCI-H460 lung carcinoma ce...

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Section: Association Of Signal Transduction Pathway Components With Smentioning

confidence: 99%

Section: Association Of Signal Transduction Pathway Components With Smentioning

confidence: 99%

Section: Passaging Of Ascites Tumor Cells and Preparation Of Microvilmentioning

confidence: 99%

Section: Passaging Of Ascites Tumor Cells and Preparation Of Microvilmentioning

confidence: 99%

Section: Immunoprecipitations Of Transmembrane Complex-containing Lowmentioning

confidence: 99%

See 3 more Smart Citations

Association of the Ras to Mitogen-activated Protein Kinase Signal Transduction Pathway with Microfilaments

Carraway¹,

Carvajal²,

Carraway³

1999

Journal of Biological Chemistry

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Immunohistochemical study of MUC1 mucin in premalignant oral lesions and oral squamous cell carcinoma

Nitta

Sugihara

Tsuyama

et al. 2000

Cancer

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Cytoskeletal features of the syncytial epidermis of the tapeworm Hymenolepis diminuta

Holy

Oaks

1989

Cell Motil. Cytoskeleton

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A hallmark feature of parasitic platyhelminths is a cytoarchitecturally unusual syncytial epidermis composed of a peripheral layer of continuous cytoplasm (the ectocytoplasm) connected to underlying nucleated cell bodies by small cytoplasmic bridges. The helminth epidermis, or tegument, plays important roles in protection and nutrient acquisition; cestodes, in fact, completely lack a gastrointestinal tract and absorb all nutritive material through the tegument. Perhaps not surprisingly, the cestode tegument bears certain resemblances to the mucosal epithelium of the vertebrate small intestine, including the possession of a microvillous brush border upon the surface of the ectocytoplasm. In contrast to the intestinal epithelial cell, however, very little is known concerning the nature and organization of the cytoskeleton within the helminth epidermis. Therefore, a number of different microscopical preparative techniques were used to examine the tegument of the tapeworm Hymenolepis diminuta for the presence and distribution of microfilaments, intermediate filaments, and microtubules. It was found that both actin-containing microfilaments and intermediate-sized filaments are present but are restricted to specific locations along the plasmalemmae of the ectocytoplasm. In contrast, microtubules are found throughout the tegument, and are concentrated in the supranuclear regions of the perikarya and in the cytoplasmic bridges interconnecting the perikarya and ectocytoplasm. Unlike brush borders of most other epithelia, the cestode epidermal brush border lacks a filamentous terminal web and is instead associated with microtubules. A network of fine filaments, 5-8 nm in diameter but distinct from actin-containing microfilaments, runs throughout the ectocytoplasm and appears to interlink tegumental vesicles. These fine filaments may represent the primary "skeletal" system responsible for maintaining the structure of the tegumental cytoplasm.

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Isolation of microvillar microfilaments and associated transmembrane complex from ascites tumor cell microvilli

Cited by 25 publications

References 26 publications

Association of the Ras to Mitogen-activated Protein Kinase Signal Transduction Pathway with Microfilaments

Association of the Ras to Mitogen-activated Protein Kinase Signal Transduction Pathway with Microfilaments

Immunohistochemical study of MUC1 mucin in premalignant oral lesions and oral squamous cell carcinoma

Cytoskeletal features of the syncytial epidermis of the tapeworm Hymenolepis diminuta

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