F-actin binding and bundling properties of fimbrin, a major cytoskeletal protein of microvillus core filaments

Fimbrin, previously recognized as a major structural protein of the microfilament core bundles of intestinal epithelial cell microvilli, has been purified to homogeneity and characterized. It is a nearly globular monomeric protein of apparent molecular weight 68,000 and has a single calcium binding site (Kd = 9 microM), for which magnesium ions compete. Fimbrin binds to F-actin and this interaction is characterized in detail. Under our optimal binding of conditions, fimbrin induces tightly packed F-actin bundles, similar to the bundles induced by villin, another microvillus structural protein. The formation of mixed fimbrin-villin-actin bundles provides a further step toward the full in vitro reconstitution of microvillus core filaments from its purified individual components. The reconstituted fimbrin-villin-actin bundles do not display the side arms characteristic of isolated microvillus cores. These results are discussed in terms of our current understanding of the organization of the microvillus core filaments and indicate that this structure contains two bundling proteins, villin and fimbrin. The results complement previous studies and now provide a minimal biochemical characterization of all four major actin-associated structural proteins so far identified in core filaments. Three of these (villin, fimbrin, and calmodulin) are calcium-binding proteins, emphasizing the concept of calcium control over submembranous microfilament organization.     

A 90 000-dalton actin-binding protein from platelets. Comparison with villin and plasma brevin

Affinity chromatography of Ca2+-containing extracts of platelets on DNAase I-Sepharose, using Ca2+-free buffer as eluant, selects a 1:1 complex of a 90 000-dalton protein with actin. The complex shows little interaction with either DNAase or actin unless Ca2+ is present. In the presence of Ca2+, the complex nucleates polymerization of actin, reduces the viscosity attained, and delays filament formation from profilactin with characteristics closely resembling those shown by chicken villin. Proteolysis of the native proteins indicates structural similarity between the platelet protein and villin or villin core; limited proteolytic digestion in the presence of SDS distinguishes the platelet protein from villin but not from the functionally related plasma protein, brevin. The platelet protein is not accessible to enzyme-mediated iodination of surface components on intact cells. The term 'platelet brevin' is proposed for the protein.     

Isolation of actin-binding protein and villin from toad oocytes

Two actin-modulating proteins have been purified from toad oocytes. A high-molecular weight protein, similar in structure and function to macrophage actin-binding protein, accounts for the isotropic actin-crosslinking activity in oocyte homogenates. A calcium-dependent activity in toad oocyte homogenates which shortens actin filaments is accounted for by a 95,000-dalton protein which resembles villin, an actin-severing and -bundling protein of avian epithelial brush borders. In the presence of high (? μM) calcium, this protein shortens actin filaments in a concentration-dependent fashion and stimulates filament assembly when added to monomeric actin. In the absence of calcium the protein promotes the formation of actin filament bundles. Therefore, in the toad oocyte actin can be crosslinked into a network by actin-binding protein. Calcium regulation of the actin network may be mediated by villin. These results are different from those reported in echinoderm eggs.     

Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins

The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.     

The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin

The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 X 10(8) M-1 and 1.2 X 10(4) M-1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-F equilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.     

Selective assay of monomeric and filamentous actin in cell extracts,using inhibition of deoxyribonuclease I

A simple and selective assay for monomeric and filamentous actin is presented, based on the inhibition of DNAase I by actin. In mixtures of monomeric and filamentous actin, only the monomeric form is measured as DNAase inhibitor. The total amount of actin in a sample can be determined after depolymerization of F actin with guanidine hydrochloride. The assay is rapid enough to detect changes in the polymerization state of actin in vitro over time intervals as short as 3 min. Data characterizing unpolymerized and filamentous actin pools in extracts of human platelets, lymphocytes and HeLa cells are presented.     

The phenotype of triparental hepatoma cell hybrids depends on the fusion sequence used to generate them

Villin is a major protein of the microfilament bundle which makes up the core of each microvillus of the brush border of the intestinal epithelial cell. Using antibodies to villin in indirect immunofluorescence microscopy on isolated cells and on frozen tissue sections, the protein is readily detectable in the microvilli of the brush border of both intestinal and renal epithelial cells. However, villin could not be detected in tissue culture cells either by immunofluorescence microscopy or by immune replica procedures. When native villin was microinjected into such cells and its distribution visualized by immunofluorescence microscopy, the protein was found to be associated with microfilamentous structures. Moreover, preferential association of the villin into the microfilaments at the leading edges of the living cell was observed. Since villin behaves in vitro as a calcium-regulated F-actin bundling protein, we discuss the possibility that villin is immunologically distinct but functionally related to putative calcium-regulatory factors assumed to be present in cultured cells.     

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.     

Comparison of Ca++-regulated events in the intestinal brush border

The intestinal epithelial cell and specifically the cytoskeleton of the brush border are thought to be controlled by micromolar levels of free calcium. Calcium-binding proteins of this system include intestinal calcium binding protein (CaBP), calmodulin (CaM), villin, and a 36,000-mol-wt protein substrate of tyrosine kinases. To assess the sequence of events as the intracellular Ca++ level rises, we determined the amount of CaM and CaBP in the intestinal epithelium by western blotting and tested the Ca++ binding of CaM and CaBP by equilibrium dialysis. The Ca++-dependent actin severing activity of villin was analyzed in the presence of physiological CaM levels and increasing calcium concentrations. In addition, we analyzed the Ca++ levels required for interaction between CaM and the microvillus 110,000-mol-wt protein as well as fodrin and the interaction between a polypeptide of 36,000 mol wt (P-36) and actin. The results suggest that CaBP serves as the predominant Ca++ buffer in the cell, but CaM can effectively buffer ionic calcium in the microvillus and thus protect actin from the severing activity of villin. CaM binds to its cytoskeletal receptors, 110,000-mol-wt protein and fodrin differently, governed by the free Ca++ and pH. The interaction between P-36 and actin, however, appears to require an unphysiologically high calcium concentration (10(-4) to 10(-3) M) to be meaningful. The results provide a coherent picture of the different Ca++ regulated events occurring when the free calcium rises into the micromolar level in this unique system. This study would suggest that as the Ca++ rises in the intestinal epithelial cell an ordered sequence of Ca++ saturation of intracellular receptors occurs with the order from the lowest to highest Ca++ requirements being CaBP less than CaM less than villin less than P-36.     

Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth

A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth.     

F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end

We have studied the mechanism of Ca++-dependent restriction of actin filament length by villin, one of the major actin-associated proteins of intestinal microvilli microfilament bundles. Villin acts, even at a ratio of 1 to 1000 with respect to actin, very efficiently as a Ca++-dependent nucleation factor on actin assembly. This gives rise to unidirectional assembly, with the morphologically defined "barbed" end of the resulting filament being capped. Consequently, at steady state treadmilling of actin monomers through the filament is inhibited. Increase of the villin-to-actin ratio enhances the number of nucleated filaments necessarily shorter in length. This results finally in nonsedimentable F actin and a low molecular weight complex of one villin and three monomeric actins, which itself is a potent nucleator. Thus restriction of actin assembly by villin is not due to a direct inhibition of assembly but arises as the consequence of strongly enhanced nucleation followed by unidirectional elongation at the pointed end of the nucleated filaments. In addition, in the presence of Ca++-villin, but not the villin-actin complex, seems able to "break" or "sever" preformed F actin filaments. Thus a variety of cellular phenomena-nucleation, unidirectional assembly, filament end capping, nonpolymerizable actin and F actin bundles-can be observed in vitro in a two-protein component system modulated by the concentration of free Ca++.     

Preparation of immobilized monomeric actin and its use in the isolation of protease-free and ribonuclease-free pancreatic deoxyribonuclease I

A procedure is described for the immobilization of monomeric actin so that about 30% of the immobilized protein is competent to bind the monomeric-actin-binding proteins bovine pancreatic deoxyribonuclease I and chicken villin. The intact tertiary structure of the immobilized actin is required to bind these proteins. Using this resin, a method has been developed for the affinity purification of pancreatic deoxyribonuclease I on a reusable actin column. It involves the binding of deoxyribonuclease I to immobilized actin, extensive washing of the column, followed by elution of the bound deoxyribonuclease I with 10 M formamide. After removal of the formamide, the deoxyribonuclease I has a higher specific activity than the starting material and contained no detectable protease or ribonuclease contamination. This preparation should find considerable application in molecular genetic studies where the enzyme is needed free of these particular contaminants. The affinity column should also be useful for the isolation of other, physiologically relevant, monomeric-actin-binding proteins.     

Differential localization of villin and fimbrin during development of the mouse visceral endoderm and intestinal epithelium

The apical surface of transporting epithelia is specially modified to absorb nutrients efficiently by amplifying its surface area as microvilli. Each microvillus is supported by an underlying core of bundled actin filaments. Villin and fimbrin are two actin-binding proteins that bundle actin filaments in the intestine and kidney brush border epithelium. To better understand their function in the assembly of the cytoskeleton during epithelial differentiation, we examined the pattern of villin and fimbrin expression in the developing mouse using immunofluorescence and immunoelectron microscopy. Villin is first detected at day 5 in the primitive endoderm of the postimplantation embryo and is later restricted to the visceral endoderm. By day 8.5, villin becomes redistributed to the apical surface in the visceral endoderm, appearing in the gut at day 10 and concentrating in the apical cytoplasm of the differentiating intestinal epithelium 2-3 days later. In contrast, fimbrin is found in the oocyte and in all tissues of the early embryo. In both the visceral endoderm and gut epithelium, fimbrin concentrates at the apical surface 2-3 days after villin; this redistribution occurs when the visceral endoderm microvilli first contain organized microfilament bundles and when microvilli first begin to appear in the gut. These results suggest a common mechanism of assembly of the absorptive surface of two different tissues in the embryo and identify villin as a useful marker for the visceral endoderm.     

An actin-binding site containing a conserved motif of charged amino acid residues is essential for the morphogenic effect of villin.

The actin-binding protein villin induces microvillus growth and reorganization of the cytoskeleton in cells that do not normally produce this protein. Transfection of mutagenized villin cDNAs into CV-1 cells was used to show that a conserved, COOH-terminally located cluster of charged amino acid residues (KKEK) is crucial for the morphogenic activity of villin in vivo. In vitro experiments with a 22 amino acid synthetic peptide corresponding to this region of villin provide evidence that this motif is part of an F-actin-binding site that induces G-actin to polymerize. Chemical cross-linking of actin to this peptide, the effects of amino acid substitutions in peptides, and the behavior of villin variants further corroborate the participation of the KKEK sequence in actin contacts.     

Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells

The microvillus cytoskeleton, isolated from chicken intestinal epithelial cell brush borders, is known to contain five major protein components, the 110,000-dalton polypeptide, villin (95,000 daltons), fimbrin (68,000 daltons), actin (43,000 daltons), and calmodulin (17,000 daltons). In this paper we describe our first step in studying the minor components of the isolated core. We have so far identified and purified an 80,000-dalton polypeptide that was present in the isolated structure in approximately 0.7% the molar abundance of actin. Antibodies to the 80,000-dalton component did not react with other microvillus core proteins, and, when used in indirect immunofluorescence microscopy, they stained the microvilli of intestinal epithelial cells fixed in situ. The 80,000-dalton component therefore appears to be a newly-identified, authentic component of intestinal microvilli in vivo and of isolated microvillus cores. Immunological studies demonstrate that the 80,000-dalton component is widely distributed in nonmuscle cells. Indirect immunofluorescence microscopy reveals that it is particularly enriched in surface structures, such as blebs, microvilli, and retraction fibers of cultured cells.     

Actin polymerization induced by pulsed electric stimulation of bone cells in vitro

Electric field pulses, capacitively applied to tissue cultures of embryonic bone cells, were shown to induce changes in the state of cellular actin. Three actin states could be defined by DNAase I inhibition. A rapidly (20-30 s) inhibiting fraction, attributed to monomeric G-actin, amounts to 55% of total actin in nonstimulated cells. An additional fraction of 8% required approx. 20 min to reach full inhibition and was tentatively defined as polymeric 'F'-actin. The remaining 37% could be detected only after treatment of the cells with 0.75 M guanidine hydrochloride, which dissociates actin from all its protein interactions. This fraction, N-actin (network actin) is believed to represent F-actin integrated into some supramolecular structure, where it is not accessible to DNAase I. Upon short electric stimulation the distribution changed to 40% G-actin, 12% F-actin and 48% N-actin. 3-Isobutyl-1-methylxanthine (IBMX; an inhibitor of cAMP phosphodiesterase), depletion of extracellular calcium, and calmodulin inhibitors abolished this field effect.     

Purification of calcium-sensitive regulatory protein of platelets which inhibits the gelation of actin

Ca²⁺-sensitive regulatory protein of human platelets which inhibits the gelation of actin was purified by DEAE-Sepharose and an affinity column using actin as a ligand. The protein was a single polypeptide chain with an average molecular weight of 90,000 and it bound to actin and inhibited its gelation at concentration from 10^?6–10^?7M of free calcium. Since the protein existed in the form of a complex with actin even though at concentration lower than 10^?7M of free calcium, binding and dissociation of actin and the protein appeared to be dependent on the concentration of free calcium, and complete dissociation was not seen.     

The inhibition of bovine and rat parotid deoxyribonuclease I by skeletal muscle actin. A biochemical and immunocytochemical study.

Rat and bovine parotid gland and pancreas contain deoxyribonuclease I (DNAase I) activities in different amounts. The DNAase I activity in tissue homogenates of bovine and rat parotid gland can be inhibited by addition of monomeric actin, as with the enzyme of bovine pancreas. The isolated DNAase I species from bovine and rat parotid gland differ in their molecular weights and also in their affinities for monomeric actin, being lowest for rat parotid DNAase I (5 X 10(6)M(-1). Antibodies raised against rat and bovine parotid and bovine pancreatic DNAase I can be used to study the subcellular localization of DNAase I in these tissues by indirect immunofluorescence. DNAase I was found to be confined solely to the secretory granules of the tissue from which it was isolated.     

A domain of synapsin I involved with actin bundling shares immunologic cross-reactivity with villin

Synapsin I is a neuronal phosphoprotein that can bundle actin filaments in vitro. This activity is under phosphorylation control, and may be related to its putative in vivo role of regulating the clustering and release of small synaptic vesicles. We have compared human and bovine synapsin I by peptide mapping, and have used NTCB (2-nitro-5-thiocyano benzoic acid) cleavage to generate a series of peptide fragments from bovine synapsin I. After chymotryptic digestion, 88% of the tyrosine-containing fragments appear to be structurally identical in human and bovine synapsin I, as judged by their positions on high-resolution two-dimensional peptide maps. The alignment of the NTCB peptides within the parent protein have been determined by peptide mapping, and the ability of these fragments to precipitate with actin bundles has been measured. Only peptides that are derived from regions near the ends of the protein are active. One such 25-kDa peptide which sediments with actin also cross-reacts with antibodies to chicken villin, an actin binding and bundling protein derived from the intestinal microvillus. Since in other respects villin appears to be an unrelated protein, these results suggest the possibility that certain actin binding proteins may show immunologic cross-reactivity due to convergent evolution within the acting binding domain.