Microfilament Core Research Articles

Perspectives on low voltage transmission electron microscopy as applied to cell biology.

Low voltage transmission electron microscopy (LVTEM) with accelerating voltages as low as 5 kV was applied to cell biology. To take advantage of the increased contrast given by LVTEM, tissue preparation was modified omitting all heavy metals such as osmium, uranium, and lead from the fixation, on block staining and counterstaining. Nonstained ultra-thin tissue sections (40 nm thick) generated highly contrasted images. While the aspect of the cells remains similar to that obtained by conventional TEM, some new substructures were revealed. The pancreatic acinar cells granules present a heterogeneous matrix with partitions corresponding to segregation of their different secretory proteins. Microvilli display their core of microfilaments anchored to the dense top membrane. Mitochondria revealed the presence of distinct particles along their cristea membranes that may correspond to the ATP synthase complexes or oxysomes. The dense nuclear chromatin displays a honey-comb appearance while distinct beads aligned along thin threads were seen in the dispersed chromatin. These new features revealed by LVTEM correlate with structures described or predicted through other approaches. Masking effects due to thickness of the tissue sections and to the presence of heavy metals must have prevented their observation by conventional TEM. Furthermore, the immunogold was adapted to LVTEM revealing nuclear lamin-A at the edge of the dense chromatin ribbons. Combining cytochemistry with LVTEM brings additional advantages to this new approach in cell biology.

Cellular dynamics of epithelial clefting during branching morphogenesis of the mouse submandibular gland

We cultured the rudimental submandibular gland (SMG) of mice with a non-cell-permeable fluorescent tracer, and observed cell behavior during epithelial branching morphogenesis using confocal time-lapse microscopy. We traced movements of individual cells as shadowgraph movies. Individual epithelial cells migrated dynamically but erratically. The epithelial cleft extended by wiggling and separated a cluster of cells into two buds during branching. We examined the ultrastructure of the clefts in SMG rudiments treated with the laminin peptide A5G77f, which induces epithelial clefting. A short cytoplasmic shelf with a core of microfilaments was found at the deep end of the cleft. We propose that epithelial clefting involves a dynamic movement of cells at the base of the cleft, and the formation of a shelf within a cleft cell. The shelf might form a matrix attachment point at the base of the cleft with a core of microfilaments driving cleft elongation.

Processus and recessus adhaerentes: giant adherens cell junction systems connect and attract human mesenchymal stem cells

Substrate-adherent cultured cells derived from human bone marrow or umbilical cord blood ("mesenchymal stem cells") are of special interest for regenerative medicine. We report that such cells, which can display considerable heterogeneity with respect to their cytoskeletal protein complement, are often interconnected by special tentacle-like cell processes contacting one or several other cells. These processus adhaerentes, studded with many (usually small) puncta adhaerentia and varying greatly in length (up to more than 400 microm long), either contact each other in the intercellular space ("ET touches") or insert in a tight-fitting manner into deep plasma membrane invaginations (recessus adhaerentes), thus forming a novel kind of long (up to 50 microm) continuous cuff-like junction (manubria adhaerentia). The cell processes contain an actin microfilament core that is stabilized with ezrin, alpha-actinin, and myosin and accompanied by microtubules, and their adhering junctions are characterized by a molecular complement comprising the transmembrane glycoproteins N-cadherin and cadherin-11, in combination with the cytoplasmic plaque proteins alpha- and beta-catenin, together with p120(ctn), plakoglobin, and afadin. The processes are also highly dynamic and rapidly foreshorten as cell colonies approach a denser state of cell packing. These structures are obviously able to establish cell-cell connections, even over long distances, and can form deep-rooted and tight cell-cell adhesions. The possible relationship to similar cell processes in the embryonic primary mesenchyme and their potential in cell sorting and tissue formation processes in the body are discussed.

Aurigamonas solis n. gen., n. sp., a Soil-Dwelling Predator with unusual Helioflagellate Organisation and Belonging to a Novel Clade within the Cercozoa

A flagellate predator, Aurigamonas solis n. gen., n. sp., with numerous radiating axopodia-like appendages, has been isolated in culture from soils. Despite its heliozoan-like appearance, Aurigamonas is not a sit-and-wait predator but a mobile hunter and its stiff appendages are not microtubule-supported axopodia but elongate haptopodia, each supported by a cylindrical core of microfilaments and bearing at its capitate tip a single extrusome-like body (haptosome). Prey flagellates are trapped on the sticky tips of the haptopodia and a large funnel-shaped pseudopodium then emerges to engulf the prey or suck out part of it for internal digestion. Pseudopodial contact is accompanied by killing, possibly as a result of the injection of spicules by the predator. Cytoplasmic haptosomes appear to induce formation of a haptopodium on making contact with the plasma membrane. Propulsion of the organism along the substratum is effected by beating of a long trailing flagellum, the short and inconspicuous second flagellum lacks motility. Small subunit rDNA sequencing shows that Aurigamonas arose within the Cercozoa. Its closest relatives are Cercobodo agilis and several flagellates currently known only as environmental sequences. This conclusion is supported further by the presence of only a single amino acid insertion in the polyubiquitin sequence of Aurigamonas solis.

Renal ischemia induces tropomyosin dissociation-destabilizing microvilli microfilaments.

Ischemic-induced cell injury results in rapid duration-dependent actin-depolymerizing factor (ADF)/cofilin-mediated disruption of the apical microvilli microfilament cores. Because intestinal microvillar microfilaments are bound and stabilized in the terminal web by the actin-binding protein tropomyosin, we questioned whether a protective effect of tropomyosin localization to the terminal web of the proximal tubule microfilament cores is disrupted during ischemic injury. With tropomyosin-specific antibodies, we examined rat cortical sections under physiological conditions and following ischemic injury by confocal microscopy. In addition, Western blot analysis of cortical extracts and urine was undertaken. Our studies demonstrated the presence of tropomyosin isoforms in the proximal tubule microvillar terminal web under physiological conditions and their dissociation in response to 25 min of ischemic injury. This correlated with the excretion of tropomyosin-containing plasma membrane vesicles in urine from ischemic rats. In addition, we noted increased tropomyosin Triton X-100 solubility following ischemia in cortical extracts. These studies suggest tropomyosin binds to and stabilizes the microvillar microfilament core in the terminal web under physiological conditions. With the onset of ischemic injury, we propose that tropomyosin dissociates from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.

Cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli.

The interaction of weak electromagnetic fields (EMF) with living cells is a most important but still unresolved biophysical problem. For this interaction, thermal and other types of noise appear to cause severe restrictions in the action of weak signals on relevant components of the cell. A recently presented general concept of regulation of ion and substrate pathways through microvilli provides a possible theoretical basis for the comprehension of physiological effects of even extremely low magnetic fields. The actin-based core of microfilaments in microvilli is proposed to represent a cellular interaction site for magnetic fields. Both the central role of F-actin in Ca2+ signaling and its polyelectrolyte nature eliciting specific ion conduction properties render the microvillar actin filament bundle an ideal interaction site for magnetic and electric fields. Ion channels at the tip of microvilli are connected with the cytoplasm by a bundle of microfilaments forming a diffusion barrier system. Because of its polyelectrolyte nature, the microfilament core of microvilli allows Ca2+ entry into the cytoplasm via nonlinear cable-like cation conduction through arrays of condensed ion clouds. The interaction of ion clouds with periodically applied EMFs and field-induced cation pumping through the cascade of potential barriers on the F-actin polyelectrolyte follows well-known physical principles of ion-magnetic field (MF) interaction and signal discrimination as described by the stochastic resonance and Brownian motor hypotheses. The proposed interaction mechanism is in accord with our present knowledge about Ca2+ signaling as the biological main target of MFs and the postulated extreme sensitivity for coherent excitation by very low field energies within specific amplitude and frequency windows. Microvillar F-actin bundles shielded by a lipid membrane appear to function like electronic integration devices for signal-to-noise enhancement; the influence of coherent signals on cation transduction is amplified, whereas that of random noise is reduced.

Ischemic injury induces ADF relocalization to the apical domain of rat proximal tubule cells.

Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.

The p185neu-containing Glycoprotein Complex of a Microfilament-associated Signal Transduction Particle: PURIFICATION, RECONSTITUTION, AND MOLECULAR ASSOCIATIONS WITH p58gag AND ACTIN

Microfilaments associate with the microvillar membrane of 13762 ascites mammary adenocarcinoma cells via a large transmembrane complex (TMC) comprising the major glycoproteins TMC-gp120, -110, -80, -65, and -55, the receptor kinase p185(neu), and the cytoplasmic proteins actin and p58(gag), linking the receptor with microfilaments in a signal transduction particle. Immunoblot screening with polyclonal antisera to TMC glycoproteins showed selective epithelial expression in normal rat tissues and epithelially derived tumor cells. The TMC glycoproteins were isolated by solubilization of microfilament core preparations in SDS, dilution, and separation on a concanavalin A-agarose affinity column. The large p185(neu)-containing complex was reconstituted from the column eluate after displacement of SDS with nonionic detergent, demonstrated by gel filtration and co-immunoprecipitation of the glycoproteins with anti-gp55 or anti-p185(neu). Exhaustive biotinylation of the glycoproteins gave a stoichiometry of gp120:gp110:gp80:gp65:gp55 of approximately 1:1:1:0.5:1. Overlay blots with biotinylated actin and in vitro translated, [(35)S]methionine-labeled p58(gag), respectively, showed specific interactions of actin with gp55 and gp120 and of p58(gag) with gp65 and gp55. These results provide evidence for a specific complex of microfilament-associated glycoproteins containing p185(neu) and p58(gag) and suggest a role for the complex in signal transduction scaffolding.

Dynamics of the oocyte cortical cytoskeleton during oogenesis in Rhodnius prolixus

The oocyte cortex undergoes dramatic changes during oogenesis in Rhodnius prolixus. Despite numerous studies examining oogenesis in the telotrophic ovariole, none has investigated the ultrastructural details of the oocyte cortex, in particular, the lateral cortical cytoskeleton. Indirect immunofluorescent staining of sections, rhodamine phalloidin staining of whole mounts and scanning and transmission EM of permeabilized and unpermeabilized preparations revealed the dynamic changes of the oocyte cortex from early previtellogenesis through to late vitellogenesis. During early previtellogenesis, oocytes 50–150 μm in length have a smooth oolemma, with no discernible cortical cytoskeleton. During mid to late previtellogenesis (oocytes 150–350 μm in length) a tightly woven network of microfilaments and microtubules forms, excluding mitochondria and Golgi complexes from the lateral cortex. At the onset of vitellogenesis, the follicuiar epithelium becomes patent, and there is an increase in microvilli covering the lateral oocyte surface. The microfilament cores form a discrete pattern that corresponds to the imprint of the follicle cells on the oocyte surface. While the lateral microfilament cytoskeleton becomes more elaborate, the lateral microtubule cytoskeleton diminishes, remaining sparse throughout vitellogenesis. The oocyte cortical cytoskeleton undergoes dramatic changes during oogenesis. These cortical dynamics are intricately related to the cellular and molecular processes that occur during oogenesis.

Association of p185neu with microfilaments via a large glycoprotein complex in mammary carcinoma microvilli. Evidence for a microfilament-associated signal transduction particle.

The protein product of the neu (proto)oncogene p185neu is an analog of the epidermal growth factor receptor. Immunoblot analyses of cell surface fractions (microvilli) from the ascites 13762 rat mammary adenocarcinoma indicate that these cells contain p185neu but not epidermal growth factor receptor. Phalloidin shift velocity sedimentation analysis indicated that essentially all of the microvillar p185 co-migrated with microvillar microfilament cores when extractions were performed under microfilament-stabilizing conditions. Fractionation studies of these microvilli indicated that the association of p185 with the microfilament core is mediated by its stable interaction with a previously described transmembrane complex (TMC), composed minimally of at least four glycoproteins, a 58-kDa cytoplasmic membrane protein, and actin (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434). A fraction of the p185 co-purifies with a large (> 2 x 10(6) kDa) complex of the TMC glycoproteins on gel filtration of microvilli, microfilament cores, or microvillar membranes in buffer containing 1 M KCl at pH 9.5, which are conditions required for the dissociation of actin from the complex. We propose that p185-containing TMC serves as a signal transduction particle at the surface of the 13762 cells and may be a prototype for similar microfilament-associated signal-transducing complexes in other cells.

Expression of tetrahymena actin in mammalian cells

We previously revealed that Tetrahymena actin can copolymerize with rabbit skeletal muscle actin whereas it has a very divergent primary structure and some unusual properties. To investigate the effects of coexistence of this unusual Tetrahymena actin in mammalian cells, we here transfected Tetrahymena actin gene on an expression vector into COS-1 cells. From the results of immunofluorescence microscopy, it was found that Tetrahymena actin expressed in COS-1 cells copolymerized with intrinsic actin, and it was conspicuously localized to the tips of microfilament core bundles in microspikes. On the other hand, increase in cell number tended to cease temporarily about 24 hr after transfection with Tetrahymena actin gene, implying the inhibition of cytokinesis by Tetrahymena actin coexistence.

Membrane-microfilament interactions in ascites tumor cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex

[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.

On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos

The efficiency of isolation and the characteristics of embryo-derived cell lines from murine, porcine, and ovine embryos cultured on STO feeders or homologous embryonic fibroblasts (HEF) feeders were compared. While murine isolated ICM or intact embryos plated on STO or HEF feeders gave rise to cell lines with embryonic stem cell-like (ES-like) morphology, ovine embryos did not. Cell lines with ES-like morphology were isolated from porcine intact embryos and isolated ICM when plated on STO feeders but not when plated on HEF. Neither murine nor porcine ES-like cell lines expressed cytokeratin 18 or vimentin. Unlike murine ES-like cell lines, porcine ES-like cells did not undergo observable differentiation in vitro or in vivo. Cell lines with epithelial-like morphology were isolated from porcine and ovine embryos. Both porcine and ovine epithelial-like cell kines expressed cytokeratin 18. When induced to differentiate in vitro, porcine and ovine epithelial-like cell lines formed vesicular structures. Electron microscopy revealed that the porcine vesicles were composed of polarized epithelial cells, each with a basally-located nucleus and an apical border containing numerous microvilli with a well organized microfilament core. The results of this study show that conditions which allow isolation of ES cells from murine embryos allow the isolation of porcine embryo-derived cell lines sharing some, but not all, the characteristics of murine ES cells.

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

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.

Influence of cumulus cell processes on oolemma permeability and lethality of isolated mouse oocytes cultured in Ca2+-free medium.

Cumulus cell processes remaining in the zona pellucida of mouse oocytes mechanically isolated from the ovary have been indirectly visualized by labeling their actin microfilament core with rhodaminyl-phalloidin. If the isolation of the oocytes is performed in Ca2+-free medium, the presence of such processes allows the entry into the cell of low molecular weight molecules (such as 5-6 carboxyfluorescein) and contributes to the death of the cell in such experimental conditions. Following dissolution of the zona pellucida (by enzymatic or acidic treatment) the oocyte is no longer permeable to small molecules and becomes resistant to Ca2+-free medium, probably as a consequence of the collapse of cumulus cell processes. The role of cumulus cell processes and gap junctions in the permeability of mechanically isolated ovarian oocytes is discussed.

Strong association of bovine IgM with microvilli and their microfilament core from 13762 ascites tumor cells.

Microfilament cores, obtained by extracting 13762 mammary ascites tumor cell microvilli with Triton X-100, contain a major glycoprotein migrating at an apparent molecular weight of 80 kDa by dodecyl sulfate-polyacrylamide gel electrophoresis. The 80-kDa component is a disulfide-linked multimer, as demonstrated by velocity sedimentation and agarose-acrylamide gel electrophoresis analyses under nonreducing conditions. This 80-kDa species is not metabolically labeled, as is a minor 80-kDa glycoprotein found in the cores, membranes, and an isolated transmembrane complex with actin. Antibodies prepared against the 80-kDa glycoprotein react strongly with bovine IgM and more weakly with rat IgM. These antibodies were used to demonstrate that the 80-kDa component is present in microvilli, microvillar microfilament cores, and microvillar membranes only if the microvilli are prepared in the presence of calf serum. The 80-kDa component, purified by velocity sedimentation in dodecyl sulfate, reacts with anti-rat IgM by immunoblot analyses. Moreover, immunoprecipitation of detergent extracts of microvilli with anti-rat IgM specifically sediments the 80-kDa component. The 80-kDa glycoprotein fractionates with the actin-containing transmembrane complex prepared by gel filtration of Triton-solubilized microvillar membranes. These results indicate that the disulfide-linked, multi-meric 80-kDa component is bovine IgM, which binds strongly to a cell-surface component of the microvilli, and is indirectly associated with the microfilament cores. Thus, the IgM provides a marker by which the transmembrane complexes to the microfilaments can be identified.

Isolation and Characterization of a 58-kDa Membrane- and Microfilament-associated Protein from Ascites Tumor Cell Microvilli

A 58-kDa protein is found in microvilli and in actin-containing transmembrane complexes of 13762 ascites tumor cells with immobile surface receptors; it is absent from sublines with mobile receptors. 58-kDa protein has been proposed to stabilize microvilli and restrict receptor mobility by stabilizing membrane-microfilament interactions. Antibodies against 58-kDa protein were blot-purified from antisera of rabbits injected with crude transmembrane complex and were used to monitor purification of the protein. 58-kDa protein was extracted from EDTA/EGTA-stripped microvillar microfilament cores with 1 M NaCl. A single depolymerization-polymerization cycle of the microfilaments, followed by solubilization of 58-kDa protein in 1 M NaCl and chromatography on hydroxyapatite-Sephadex G-150, purified the protein to greater than 95% homogeneity. The native molecular weight and frictional coefficient indicated a monomeric, asymmetric structure. 58-kDa protein bound F-actin in pelleting assays and inhibited polymerization of pyrenyl-actin. It also bound phosphatidylserine, phosphatidylinositol, and phosphatidylcholine vesicles in pelleting studies. Immunoblot analyses of endogenously and exogenously proteolyzed microvilli and their membranes and microfilament cores showed specific membrane and microfilament binding fragments of 28-30 kDa. The microfilament- and phospholipid-binding properties of 58-kDa protein and the localization of its proteolysis products are consistent with its proposed role in stabilizing membrane-microfilament interactions in the ascites cell microvilli.

Microfilament association of asgp-2, the concanavalin A-binding glycoprotein of the cell-surface sialomucin complex of 13762 rat mammary ascites tumor cells

Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. “Phalloidin shift” analyses on velocity sedimentation gradients of Triton X-100 extracts of [ 3H]-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. An increase was also observed with sialidase treatment of the microvilli, suggesting that negative charges, probably present on the highly sialated sialomucin ASGP-1 of the ASGP-1/ASGP-2 sialomucin complex, reduce ASGP-2 association with the microfilament core. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Purified ASGP-2 does not bind to microfilaments in sedimentation assays. Since the Triton-insoluble membrane residue is enriched in an actin-containing transmembrane complex, which contains a different glycoprotein, we suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.

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

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.

Phenothiazine binding by a homolog of calpactin, the pp60src tyrosine kinase substrate.

Microvilli isolated from 13762 mammary ascites tumor cells contain a major calcium-sensitive protein (AMV-p35) that can be isolated with microvillar microfilament cores prepared by Triton X-100 extraction in the presence but not absence of calcium. AMV-p35 can be readily purified from ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid extracts of the microfilament cores by chromatography on an anion exchange column, to which it does not bind. Immunoblot analysis indicates that AMV-p35 is related to calpactin I, the pp60src tyrosine kinase substrate. In the presence of calcium, AMV-p35 binds approximately 4 mol of chlorpromazine per mole of protein in a binding process showing apparent positive cooperativity, similar to calmodulin; however, in contrast to calmodulin, AMV-p35 also binds phenothiazine in the absence of calcium.