Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts

The gelation induced by warming (to 25 degrees C) the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 was studied in the presence of myosin and heavy meromyosin (HMM). Myosin mixed with extract induces shrinkage of the gel, but jelled extract or myosin alone does not shrink. In the concentration range, 0.14-1.04 mg/ml of myosin, the degree of shrinkage is roughly proportional to the concentration of myosin. Supplementa MgCl2 also promotes shrinkage. HMM (0.4-0.8 mg/ml) can inhibit gel formation by extract in tubes or floated on a sucrose cushion. Gel electrophoresis of gels shrunken by added myosin or electrophoresis of the proteins which can be sedimented from extract after incubation in the presence of HMM indicate that both myosin and HMM interfere with the changes in sedimentability of the high molecular weight protein (HMWP) thought to participate (together with actin) in gel formation in HeLa cell extracts (R. R. Weihing, 1976. J. Cell Biol. 71:303-307). These results, together with previous results showing that actin is present and that HMWP is enriched in the plasma membrane fraction of HeLa cells (R. R. Weihing, 1976. Cold Spring Harbor Conf. Cell Proliferation. 3:671-684), point to the possibility of dynamic changes in the interactions of HMWP or myosin with actin in processes of movement occurring at the cell surface.     

Purification of a HeLa cell high molecular weight action binding protein and its identification in HeLa cell plasma membrane ghosts and intact HeLa cells

The high molecular weight protein (HMWP) which was previously observed to be a major component of the actin based gels formed by incubating cytoplasmic extracts of HeLa cells at 25 degrees C [Weihing, R. R. (1977) J. Cell Biol. 75, 95-103] has now been purified by gel filtration of 0.6 M KCl extracts of precipitated gels. A few hundred micrograms of HMWP, which is about 90% pure, can be isolated from 4 X 10(9) cells. HMWP can gel muscle actin and cross-link it into filament bundles. Its subunit molecular weight is 250 0000, its Stokes radius is 125 A, and its sedimentation coefficient is 9 S. A native molecular weight of 480 000 was calculated by using the latter two parameters, and therefore the native molecule is a dimer. Its amino acid analysis is nearly indistinguishable from that of macrophage actin binding protein and of mammalian and avian filamins. All of these findings indicate that HMWP is homologous to the latter proteins. However, HeLa cell HMWP and avian filamin must differ in their primary sequences because their partial peptide maps are distinct and because an antiserum against HMWP reacts only weakly with filamin. For studies on the intracellular location of HMWP, a goat antiserum against purified HMWP was prepared and characterized and then used to localize HMWP in suspension grown cells. The technique of immunoblotting revealed that the antiserum reacted virtually exclusively with the high molecular weight polypeptide that comigrates with HMWP in cell lysates and in ZnCl2-stabilized plasma membrane ghosts prepared from HeLa cells [Gruenstein, E., Rich, A., & Weihing, R. R. (1975) J. Cell Biol. 64, 223-234] and that it did not react with rabbit myosin heavy chain, microtubule proteins (MAPS and tubulin) from HeLa cells and calf brain, or the proteins of human erythrocyte ghosts including spectrin. Suspension-grown cells which were stained with the antiserum by the technique of indirect immunofluorescence showed bright fluorescence at the rim of the cells and less intense generalized fluorescence. If preimmune serum or immune serum treated with HMWP was substituted for the immune serum, then staining at the rim was not observed, but the generalized fluorescence was only slightly reduced; unpermeabilized cells were not stained. These results indicate that HMWP is a component of the cortical cytoplasm of HeLa cells. Possible functions of cortical HMWP are discussed briefly.     

Gelation and fodrin purification from rat brain extracts

Extracts from rat brain tissue have been shown to give rise to a gel which exhibits the following features. It is mainly enriched in actin and in a high-molecular-weight protein with polypeptide chains of 235 and 240 kDa, which we identified as fodrin. Tubulin is also a major component of the gel but it appears to be trapped non-specifically during the gelation process. Gelation is pH-, ionic strength- and Ca2+-concentration-dependent, and is optimal under the conditions which promote the interaction between polymerized actin and fodrin. In a similar way to that described for the purification of rat brain actin (Levilliers, N., Péron-Renner, M., Coffe, G. and Pudles, J. (1984) Biochimie 66, 531-537), we used the gelation system as a selective means of recovering fodrin from the mixture of a low-ionic-strength extract from whole rat brain and a high-ionic-strength extract of the particulate fraction. From this gel, fodrin was purified with a good yield by a simple procedure involving gel dissociation in 0.5 M KCl and depolymerization in 0.7 M KI, Bio-Gel A-15m chromatography, followed by ammonium sulfate precipitation.     

The effects of insulin,cycloheximide and phalloidin on the content of actin and p35 in extracts prepared from the nuclear fraction of Krebs II ascites cells

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton × 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50–205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton × 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.     

Actin polymerization and interaction with other proteins in temperature- induced gelation of sea urchin egg extracts

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 22,000 mol wt. When combined in 0.1 M KCl, even at 0 degrees C, these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm. This association of F-actin with the other proteins to form a gel is very likely the last step fo the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous F-actin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.     

Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium

A low-salt extract prepared from human erythrocyte membranes forms a solid gel when purified rabbit muscle G- or F-actin is added to it to give a concentration of approximately 1 mg/ml. This extract contains spectrin, actin, band 4.1, band 4.9, hemoglobin, and several minor components. Pellets obtained by centrifugation of the gelled material at 43,000 g for 10 min contain spectrin, actin, band 4.1, and band 4.9. Although extracts that are diluted severalfold do not gel when actin is added to them, the viscosity of the mixtures increases dramatically over that of G-actin alone, extract alone, or F-actin alone at equivalent concentrations. Heat-denatured extract is completely inactive. Under conditions of physiological ionic strength and pH, information of this supramolecular structure is inhibited by raising the free calcium ion concentration to micromolar levels. Low-salt extracts prepared by initial extraction at 37 degrees C (and stored at 0 degree C) gel after actin is added to them only when warmed, whereas extracts prepared by extraction at 0 degree C are active on ice as well as after warming. Preincubation of the 37 degrees C low-salt extract under conditions that favor conversion of spectrin dimer to tetramer greatly enhances gelation activity at 0 degree C. Conversely, preincubation of the 0 degree C low-salt extract under conditions that favor conversion of spectrin tetramer to dimer greatly diminishes gelation activity at 0 degree C. Spectrin dimers or tetramers are purified from the 37 dgrees or 0 degree C low-salt extract by gel filtration at 4 degrees C over Sepharose 4B. The addition of actin to either purified spectrin dimer (at 32 degrees C) or tetramer (at 0 degree C or 32 degrees C) results in relatively small increases in viscosity, whereas the addition of actin to a high-molecular-weight complex (HMW complex) containing spectrin, actin, band 4.1, and band 4.9 results in dramatic, calcium-sensitive increases in viscosity. These viscosities are comparable to those obtained with the 37 degrees or 0 degree C low-salt extracts. The addition of purified band 4.1 to either purified spectrin dimer (at 32 degrees C) or purified spectrin tetramer (at 0 degree C) plus actin results in large increases in viscosity similar to those observed for the HMW complex and the crude extract, which is in agreement with a recent report by E. Ungewickell, P. M. Bennett, R. Calvert, V. Ohanian, and W. B. Gratzer. 1979 Nature (Lond.) 280:811-814. We suggest that this spectrin-actin-band 4.1 gel represents a major structural component of the erythrocyte cytoskeleton.     

Induction of either contractile or structural actin-based gels in sea urchin egg cytoplasmic extract

The gel formed by warming the 100,000 g supernate of isotonic extracts of sea urchin eggs to 40 degrees C is made up of actin and two additional proteins of mol wt of 58,000 and 220,000. Actin and 58,000 form a characteristic structural unit which has now been identified in the microvilli of the urchin egg and in the filopods of urchin coelomocytes. However, egg extract gels did not contract as those from other cell types do, and the aim of these experiments was to determine the reason for this lack of contraction. Although the extracts are dialyzed to a low ionic strength, myosin is present in soluble form and makes up approximately 1% of the protein of the extract. It becomes insoluble in the presence of high ATP concentrations at 0 degrees C, and the precipitate formed under these conditions consists almost entirely of myosin. This procedure provides a simple method of isolating relatively pure myosin without affecting other extract components and functions. Contraction will follow gelation in these extracts if the temperature and time of incubation used to induce actin polymerization are reduced to minimize myosin inactivation. At the optimal ATP and KCl concentration for contraction, the contracted material has an additional 250,000 component and contains very little 58,000. The conditions found to provide maximum gel yields favor the formation of the actin-58,000-220,000 structural gel, while reduced temperature and increase in KCl concentration results in a contractile gel whose composition is similar to those reported from amoeboid cell types. Both the structural protein cores found in the egg microvilli and a gel contraction related to the amoeboid motion which is seen in later urchin embryonic development can thus be induced in vitro in the same extract.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.     

Actin and myosin in oxyntic cell : Gelation and contraction of crude extracts in vitro

Extracts prepared from purified toad oxyntic cells undergo temperature-dependent, cytochalasin B (CB)-sensitive gelation. In the presence of adenosine triphosphatase (ATP), the interaction between gelled actin and myosin produces a contracted gel. Such association ceases spontaneously after shrinkage is completed. A stable myosin-actin interaction takes place in the absence of ATP, but no contraction of the gel is observed. A correlation between this actomyosin system present in oxyntic cells and the high mobility of the secretory pole, resulting in the changes in shape observed during the onset of HCl secretion, is proposed.     

Purification from Acanthamoeba castellanii of proteins that induce gelation and syneresis of F-actin.

From Acanthamoeba castellanii, we have purified four proteins each of which alone causes a solution of F-actin to gel. The four active proteins have subunit molecular weights of about 23,000, 28,000, 32,000 and 38,000, respectively; the last three may be dimers in their native proteins. Together, these four proteins account for about 97% of the gelation activity of the whole extract; not more than about 3% of the total activity of the unfractionated extract can be due to a 250,000-dalton polypeptide. Another protein fraction, purified by agarose chromatography, induces shrinking (syneresis) of gels formed from F-actin and any of the gelation factors. That fraction contains a high Ca2+-, low (K+,EDTA)-ATPase and a major polypeptide of 170,000 daltons both of which bind to actin in the shrunken gel pellet. The active fraction does not contain the previously described Acanthamoeba myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4682-4690).     

Gelation of sarcoma 180 extracts : Membrane involvement and the elimination of the temperature dependence by proteolysis

The gelation reaction of 100 000 g supernatants of sarcoma 180 homogenates has been investigated in an attempt to delineate the factors which are important in the reaction. Gel filtration and sucrose density gradient centrifugation indicate that actin-binding protein is in the form of a membrane-bound complex in the 100 000 g supernatants prior to gelation. When the sarcoma 100 000 g supernatants are warmed to room temperature, gelation occurs. Three major proteins are concentrated in the gel: actin, ABP and a component (E) which barely penetrates dodecyl sulfate polyacrylamide gels. Proteolysis of the 100 000 g supernatants enhances the rate of gelation and eliminates the temperature dependence. At 4 °C the enhancement of gelation by protease occurs without substantial cleavage of ABP or actin. Proteolysis does not enhance actin polymerization under the same conditions. The combined results of these experiments suggest that the temperature and proteolysis effects do not occur directly on the gelation reaction, but rather on factors controlling the ABP or actin interactions necessary for gelation.     

Changes in actin-related gelation of crude cell extracts during differentiation of myeloid leukemia cells

Gelation of extracts of a myeloid leukemia cell line (Ml) was compared before and after differentiation induced with conditioned medium (CM) from rat embryo cells. Although an extract of Mml cells, a macrophage line derived from Ml line, gelled when warmed in the presence of 2 mM MgCl2, undifferentiated Ml cells gelled only after dialysis and a supplement of exogenous actin. After differentiation had been induced, an addition of exogenous actin, but not dialysis, was needed for gelation. Small amounts of KCl always inhibited the gelation of the control Ml cell extracts, but they promoted gelation of the CM-treated Ml and Mml cell extracts. Thus, the dialysis required for gelation of the control Ml cell extract appears to be necessary for the exclusion of endogenous KCl. Several possible mechanisms for the KCl control of gelation, as well as different requirements of exogenous actin needed for gelation are discussed based on the results of our experiments.     

The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba

The temperature-dependent assembly and the interaction of Acanthamoeba contractile proteins have been studied in a crude extract. A cold extract of soluble proteins from Acanthamoeba castellanii is prepared by homogenizing the cells in a sucrose-ATP-ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid buffer and centrifuging at 136,000 g for 1 h. When this supernate of soluble proteins is warmed to room temperature, it forms a solid gel. Upon standing at room temperature, the gel slowly contracts and squeezes out soluble components. The rates of gelation and contraction are both highly temperature dependent, with activation energies of about 20 kcal per mol. Gel formation is dependent upon the presence of ATP and Mg++. Low concentrations of Ca++ accelerate the contractile phase of this phenomenon. The major protein component of the gel is actin. It is associated with myosin, cofactor, a high molecular weight protein tentatively identfied as actin-binding protein, and several other unidentified proteins. Actin has been purified from these gels and was found to be capable of forming a solid gel when polymerized in the presence of ATP, MgCl3, and KCL. The rate of purified actin polymerication is very temperature dependent and is accelerated by the addition of fragments of muscle actin filaments. These data suggest that Acanthamoeba contractile proteins have a dual role in the cell; they may generate the forces for cellular movements and also act as cytoskeletal elements by controlling the consistency of the cytoplasm.     

Interaction of filamentous actin with isolated liver plasma membranes

Actin-membrane interactions have been studied using purified liver plasma membranes and muscular filamentous actin. Despite the large quantity of endogenous actin present in membranes, exogenous muscular filamentous actin cosediments with membranes after a 30 min centrifugation at 30 000 g. The cosedimentation process is time-dependent and exhibits a complex relationship with actin concentration. The cosedimentation of actin with membranes can be partly explained by gelation as shown by low-shear viscosity and electron microscopy. The characterization of the gelation phenomenon as a function of time, actin and membrane concentrations, ionic strength, temperature and Ca2+ concentration is also presented. Gelation alone cannot however account for the overall cosedimentation data, and a more direct mode of association between actin and the membrane must be envisaged. The analogy that exists between the results obtained with liver plasma membranes and those obtained with other membrane systems suggests that a general mechanism may be involved in the interaction of actin with plasma membranes.     

Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.     

The contractile basis of ameboid movement. VI. The solation-contraction coupling hypothesis

The contracted pellets derived from a high-speed supernate of Dictyostelium discoideum (S3) were investigated to determine the functional activity associated with this specific subset of the cellular motile apparatus. A partially purified model system of gelation and contraction (S6) was prepared from the contracted pellets, and the presence of calcium- and pH-sensitive gelation and contraction in this model demonstrated that a functional cytoskeletal-contratile complex remained at least partially associated with the actin and myosin during contraction. Semi-quantitative assays of gelation and solation in the myosin-free preparation S6 included measurements of turbidity, relative viscosity, and strain birefringence. The extent of gelation was optimal at pH 6.8 and a free calcium ion concentration of approximately 3.0 x 10(-8) M. Solation was favored when the free calcium ion concentration was greater than 7.6 x 10(-7) M or when the pH was increased or decreased from pH 6.8. Gelation was reversibly inhibited by increasing the free calcium ion concentration to approxomately 4.6 x 10(-6) M at pH 6.8. The solation-gelation process of this model has been interpreted to involve the reversible cross-linking of actin filaments. The addition of purified D. discoideum myosin to S6 served to reconstitute calcium- and pH-regulated contraction. The results from this study indicate that contraction is coupled functionally to the local breakdown (solation) of the gel. Therefore, solation has been identified as a structural requirement for extensive shortening during contraction. We have called this concept the solation-contraction coupling hypothesis. Fractionation of a preparation derived from the contracted pellets yielded a fraction consisting of actin and a 95,000-dalton polypeptide that exhibited calcium-sensitive gelation at 28 degrees C and a fraction composed of actin and 30,000- and 18,000-dalton polypeptides that demonstrated calcium-sensitive genlation at 0 degrees C.     

A nuclear cap binding protein from HeLa cells.

We have identified a cap binding protein in a HeLa nuclear extract using a gel mobility shift assay probed with capped RNA. Subcellular fractionation of HeLa cells revealed that the majority (about 70%) of the cap binding activity is present in the nuclear extract, about 20% is in the cytoplasmic S100 fraction, and almost none in the ribosome-high salt wash fraction, indicating that the protein in active form localizes mainly in the nuclei. Competition experiments with various cap analogues showed that the G(5')ppp(5')N-blocking structure as well as the methyl residue at the N7 position of the blocking guanosine is important for the binding of this protein, and that the trimethylguanosine cap structure which exists at the 5' termini of many snRNAs is not recognized by this protein. Immunoprecipitation experiments using various anti-snRNP antibodies suggested that this protein is partially associated with U2 snRNP. We purified this protein to near homogeneity from a HeLa nuclear extract by several chromatographic procedures including capped RNA-Sepharose chromatography. The purified protein shows molecular weight of 80 kilodaltons, as judged by SDS gel electrophoresis, and binds specifically to the cap structure.     

Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts

Raspberry extracts enriched in polyphenols, but devoid of organic acids, sugars and vitamin C, were prepared by sorption to C18 solid phase extraction matrices and tested for their ability to inhibit the proliferation of human cervical cancer (HeLa) cells in vitro. The raspberry extract reduced proliferation in a dose-dependent manner whether this was judged by cell number or measurements of cell viability. However, measurements based on cell viability were more accurate and gave an EC(50) value of 17.5 microg/ml gallic acid equivalents (GAE) at day 4 of culture. Raspberry extracts were fractionated by sorption to Sephadex LH-20 into an unbound fraction, which was obviously enriched in anthocyanins, and a bound fraction. The unbound anthocyanin-enriched fraction was much less effective in reducing proliferation then the original extract and gave an EC(50) value estimated at 67 microg/ml. The LH-20 bound fraction was more effective than the original raspberry extract (EC(50)=13 microg/ml) suggesting that the main anti-proliferative agents were retained in the bound fraction. Analysis of the original extract, the unbound and the LH20 bound fractions by LC-MS confirmed that the unbound fraction was enriched in anthocyanins and the bound fraction primarily contained ellagitannins. The ellagitannin-rich bound fraction had the highest antioxidant capacity as measured by the ferric reducing antioxidant potential (FRAP) assay. The mechanism by which the ellagitannins inhibit proliferation of cancer cells is discussed.     

The molecular mobility of alpha-actinin and actin in a reconstituted model of gelation

Dictyostelium discoideum alpha-actinin (D.d. alpha-actinin) is a calcium and pH-regulated actin-binding protein that can cross-link F-actin into a gel at a submicromolar free calcium concentration and a pH less than 7 [Fechheimer, et al., 1982]. We examined mixtures of actin and D.d. alpha-actinin at four pH and calcium concentrations that exhibited various degrees of gelation or solation. The macroscopic viscosities of these mixtures were measured by falling ball viscometry (FBV) and compared to the translational diffusion coefficients measured by gaussian spot and periodic-pattern fluorescence photobleaching recovery (FPR) of both the actin filaments and D.d. alpha-actinin. A homogeneous, macroscopic gel was not composed of a static actin network. Instead, the filament diffusion coefficient decreased to approximately 65% of the control value. If the D.d. alpha-actinin concentration was increased, the solution became inhomogeneous, consisting of domains of higher actin concentration. These domains were often composed of a static actin network. The mobility of D.d. alpha-actinin consisted of a major fraction that freely diffused and a minor fraction that appeared immobile under the conditions employed. This suggested that D.d. alpha-actinin binding to the actin filaments was static over the time course of measurement (approximately 5 sec). Under solation conditions, there was no apparent interaction of actin with D.d. alpha-actinin. These results demonstrate that 1) actin filaments need not be cross-linked into an immobile, static array in order to have macroscopic properties of a gel; 2) interpretation of the rheological properties of actin:alpha-actinin gels are complicated by spatial heterogeneity of the filament concentration and mobility; and 3) a fraction of D.d. alpha-actinin binds statically to actin in undisturbed gels. The implications of these results are discussed in relation to cytoplasmic structure and contractility.