Plasma membrane‐microfilament interaction in animal cells

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Isolated microvilli of the MAT-C1 subline of the 13762 rat mammary adenocarcinoma contain a transmembrane complex composed of a cell surface, cytoskeleton-associated glycoprotein (CAG), actin, and a 58,000-dalton polypeptide (58K). The behavior of CAG has been studied by differential centrifugation and velocity sedimentation gradient centrifugation of detergent extracts of microvilli. CAG can be pelleted along with a fraction of the microvillar actin even in the presence of ionic detergents and under microfilament-depolymerizing conditions. By velocity sedimentation analysis CAG in Triton/PBS extracts sediments as a large, heterogeneous species (sedimentation coefficient greater than 25S). In Sarkosyl and sodium dodecyl sulfate (SDS) the size and heterogeneity are somewhat reduced. In SDS CAG sediments as a 20S species in the absence of mercaptoethanol and as a 5S species in the presence of mercaptoethanol. These results indicate that CAG is a disulfide-linked multimer in the microvillus membrane. We suggest that the stable multimeric structure of CAG permits it to act as the membrane association site for several microfilaments and plays an important role in the formation and stabilization of the microvillus structure.

Actin‐associated cell‐surface glycoprotein from ascites cell microvilli: A disulfide‐linked multimer

Jung

Andrews

Carraway

et al. 1985

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Isolation of a calcium‐sensitive, 35,000‐dalton microfilament‐ and liposome‐binding protein from ascites tumor cell microvilli: Identification as monomeric calpactin

Liu

Brew

Carraway

et al. 1987

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Microvilli isolated from the MAT-C1 ascites subline of the 13762 rat mammary adenocarcinoma contain a major calcium-sensitive microfilament-binding protein, AMV-p35 (ascites microvillar p35). Association of AMV-p35 with microfilament cores during Triton X-100 extraction of the microvilli is half-maximal at 0.1-0.2 mM calcium. The protein, which comprises 6% of the total microvillar protein, can be isolated from microfilament cores prepared in the presence of calcium by extraction with EGTA and purification by ion-exchange chromatography. Alternatively, the protein can be isolated from Triton extracts of microvilli prepared in the absence of calcium by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on an ion-exchange column. AMV-p35 binds to phosphatidylserine liposomes and F-actin with half-maximal calcium concentrations of about 10 microM and 0.2 mM, respectively. Treatment of AMV-p35 with chymotrypsin yields a 33,000-dalton fragment, behavior similar to the tyrosine kinase substrates calpactins I and II and lipocortins I and II. Immunoblot analyses using antibodies directed against calpactin I, lipocortin I, and lipocortin II showed strong reactivity of AMV-p35 with anti-calpactin I and anti-lipocortin II, but little reactivity toward anti-lipocortin I. The close relationship between AMV-p35 and calpactin I was verified by amino acid sequence analyses of peptides isolated from cyanogen bromide digests of AMV-p35. By gel filtration and velocity sedimentation analyses purified AMV-p35 is a 35,000-dalton monomer. Moreover, AMV-p35 extracted directly from microvilli in Triton/EGTA also behaves as a 35,000-dalton menomer. These findings indicate that AMV-p35 is closely related to the pp60src kinase substrate calpactin I (p36). However, AMV-p35 occurs in the microvilli as a monomer rather than as the heterotetrameric calpactin found in several other cell types.

Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli

Sheng

Vanderpuye

Hull

et al. 1989

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Membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the associated concanavalin A (Con A)-binding glycoprotein ASGP-2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP-1 from the microvilli and increases the association of ASGP-2 with the Triton-insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP-2 in the membrane and its association with the microfilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X-100 for the preparation of microfilament cores and Triton-soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L-phytohemagglutinin, and immunoblotting with anti-ASGP-2. The earliest major proteolysis product from this procedure was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa Triton-soluble fragments, and 25-30 kDa membrane- and microfilament-associated fragments were observed. Phalloidin shift analysis of microfilament-associated proteins on velocity sedimentation gradients indicated that the 25-30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP-2 has a site for indirect association with the microfilament core near the membrane on a 15-20 kDa segment.