Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells

Chicken retinal pigmented epithelial cells have circumferential microfilament bundles (CMBs) at the zonula adherens region. We have isolated these CMBs in intact form and characterized them structurally and biochemically. Pigmented epithelia obtained from 11-d-old chick embryos were treated with glycerol and Triton. Then, the epithelia were homogenized by passing them through syringe needles. Many isolated CMBs were found in the homogenate by phase-contrast microscopy. They formed polygons, mostly pentagons and hexagons, or fragments of polygons. Polygons were filled with meshwork structures, i.e. they were polygonal plates. Upon exposure to Mg-ATP, isolated CMBs showed clear and large contraction. The contraction was inhibited by treatment with N- ethylmaleimide-modified myosin subfragment-1. After purification by centrifugation with the density gradient of Percoll, CMBs were analyzed by SDS PAGE. The electrophoretic pattern gave three major components of 200, 55, and 42 kdaltons and several minor components. Electron microscopy showed that the polygons were composed of thick bundles of actin-containing microfilaments, and the meshworks were composed primarily of intermediate filaments.     

Investigations on circumferential microfilament bundles in rat retinal pigment epithelium

The presence of contractile proteins in normal rat retinal pigment epithelium has been studied using fluorescence and electron microscopy. Investigations using the F-actin binding toxin, phallacidin, coupled to the fluorochrome nitrobenzoxadiazole, revealed a band of fluorescence at or near the cell membrane. Immunofluorescent observations with anti-myosin and anti-alpha-actinin antisera gave similar results. Electron microscopy employing glutaraldehyde-8% tannic acid fixation revealed the presence of a circumferential microfilament band beneath the pigment epithelial apical surface that is closely associated with the plasma membrane and junctional complexes. Freeze-fracture studies confirmed the relationship of this band to the junctional complexes. The microfilament band measures approximately 0.5 micron +/- 0.2 micron in width and is composed of numerous 6 to 7 nm filaments. Some microtubules are seen in regions around the band, but no organelles appear to be associated with this structure. In en face sections through the zonula adherens, the circumferential microfilament band is associated with 30-nm electron-dense particles that are bound to the internal side of the membrane. Morphological evidence suggests that these may serve in anchoring the band to the membrane and assist in aligning the microfilament bands of adjoining cells. In the subapical cytoplasm, a microfilament bundle network was detected that interfaced with the circumferential microfilament band. In some cases, pigment epithelium was incubated in media-199 containing 25 to 50 ng/ml phallacidin prior to fixation. Circumferential microfilament bands of tissues treated in this manner exhibited a striated appearance.     

Thickness distribution of actin bundles in vitro

Bundles of filamentous actin form the primary building blocks of a broad range of cytoskeletal structures, including filopodia, stereocilia and microvilli. In each case, the cell uses specific associated proteins to tailor the dynamics, dimensions and mechanical properties of the bundles to suit a specific cellular function. While the length distribution of actin bundles was extensively studied, almost nothing is known about the thickness distribution. Here, we use high-resolution cryo-TEM to measure the thickness distribution of actin/fascin bundles, in vitro. We find that the thickness distribution has a prominent peak, with an exponential tail, supporting a scenario of an initial fast formation of a disc-like nucleus of short actin filaments, which only later elongates. The bundle thicknesses at steady state are found to follow the distribution of the initial nuclei indicating that no lateral coalescence occurs. Our results show that the distribution of bundles thicknesses can be controlled by monitoring the initial nucleation process. In vivo, this is done by using specific regulatory proteins complexes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of TGF-beta on retinal pigmented epithelium in vitro

Embryonic chick retinal pigmented epithelium (RPE) has been grown on glass derivatized with covalently bound proteins of basement membrane and treated with transforming growth factor-beta (TGF-beta). In the present paper we show that over the concentration range tested (0.1-10 ng/ml) TGF-beta has no effect on RPE cell proliferation either in the presence or the absence of serum, cell motility and the organization of cytoskeleton-extracellular matrix linkage complexes with respect to their structure and presence of actin, vinculin, talin, integrin and fibronectin. The protein profiles of total cell/ECM extracts of cells grown in the presence or the absence of TGF-beta are similar although some stimulation of protein synthesis and of production of fibronectin-containing extracellular matrix has been detected.     

Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo

The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at approximately 1.5 s-1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules.     

Growth conditions control the size and order of actin bundles in vitro.

The bonding rules for actin filament bundles do not lead to a particular packing symmetry, but allow for either regular or disordered filament packing. Indeed, both hexagonal and disordered types of packing are observed in vivo. To investigate factors which control bundle order, as well as size, we examined the effect of protein concentration on the growth of actin-fascin bundles in vitro. We found that bundles require 4-8 d to achieve both maximum size and order. The largest and best ordered bundles were grown at low fascin and high actin concentrations (an initial fascin/actin ratio of 1:200). In contrast, a much larger number of poorly ordered bundles were formed at ratios of 1:25 and 1:50, and most surprisingly, no bundles were formed at 1:300 or 1:400. Based on these observations we propose a two-stage mechanism for bundle growth. The first stage is dominated by nucleation, which requires relatively high concentrations of fascin and which is therefore accompanied by rapid growth. Below some concentration threshold, nucleation ceases and bundles enter the second stage of slow growth, which continues until the supply of fascin is exhausted. By analogy with crystallization, we hypothesize that slower growth produces better order. We are able to use this mechanism to explain our observations as well as previous observations of bundle growth both in vitro and in vivo.     

Ultrastructural and immunocytochemical analysis of the circumferential microfilament bundle in avian retinal pigmented epithelial cells in vitro

Summary The dedifferentiated phenotype of pigmented epithelial cells in vitro is bipotential and is effected by environmental alterations mediated by the cell surface and associated cytoskeleton. We have begun an investigation into the role that contractile microfilaments play in maintaining cell contact and cell shape in retinal pigmented epithelial cells in vitro. In this paper, we report a structural analysis of the intersection of the circumferential microfilament bundle with the cell membrane of cultured pigmented epithelial cells from chick retina. Techniques of electron microscopy, including freezefracturing and deep-etching, reveal that microfilaments of this bundle associate with a junctional complex in the apical cell compartment and with membrane domains which are not components of the junction. Microfilaments link with the cell membrane either at their termini or along the membrane-apposed surface of the circumferential bundle. Furthermore, we report the immunocytochemical localization of filamin (a high molecular weight actin-binding protein, which forms fiber bundles and sheet-like structures when bound with Factin in solution) in the circumferential/microf相似文献   

Peroxynitrite and myocardial contractility: in vivo versus in vitro effects

Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO- had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish beta-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted beta-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3-. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function.     

Persistence length of fascin-cross-linked actin filament bundles in solution and the in vitro motility assay

Background

Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles.

Methods

Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: < cos(θ(0) − θ(s)) > = exp(−s / (2Lp)) where θ(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments.

Results

Bundle-Lp (bundles < 15 μm long) increased from ~ 10 to 150 μm with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 μm), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis.

Conclusions

Fascin–actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle.

General significance

The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin–actin bundles.     

Collagen production in vitro by the retinal pigmented epithelium of the chick embryo

Cloned colonies and explants of embryonic chick retinal pigmented epithelium from donors of various embryonic ages were maintained in culture for different periods and examined by electron microscopy. The cells appeared morphologically differentiated and polarized. Basement-membrane material and striated collagen fibrils were identified as extracellular deposits beneath the basal surfaces of the cells. There appeared to be a distinct spatial and temporal correlation between the production of basement-membrane material and collagen fibrils. Increasing donor age correlated positively with increasing average diameter of the collagenous fibrils produced, as well as a widening of the range of fibril sizes.     

Arp2/3 branched actin network mediates filopodia-like bundles formation in vitro

During cellular migration, regulated actin assembly takes place at the cell leading edge, with continuous disassembly deeper in the cell interior. Actin polymerization at the plasma membrane results in the extension of cellular protrusions in the form of lamellipodia and filopodia. To understand how cells regulate the transformation of lamellipodia into filopodia, and to determine the major factors that control their transition, we studied actin self-assembly in the presence of Arp2/3 complex, WASp-VCA and fascin, the major proteins participating in the assembly of lamellipodia and filopodia. We show that in the early stages of actin polymerization fascin is passive while Arp2/3 mediates the formation of dense and highly branched aster-like networks of actin. Once filaments in the periphery of an aster get long enough, fascin becomes active, linking the filaments into bundles which emanate radially from the aster's surface, resulting in the formation of star-like structures. We show that the number of bundles nucleated per star, as well as their thickness and length, is controlled by the initial concentration of Arp2/3 complex ([Arp2/3]). Specifically, we tested several values of [Arp2/3] and found that for given initial concentrations of actin and fascin, the number of bundles per star, as well as their length and thickness are larger when [Arp2/3] is lower. Our experimental findings can be interpreted and explained using a theoretical scheme which combines Kinetic Monte Carlo simulations for aster growth, with a simple mechanistic model for bundles' formation and growth. According to this model, bundles emerge from the aster's (sparsely branched) surface layer. Bundles begin to form when the bending energy associated with bringing two filaments into contact is compensated by the energetic gain resulting from their fascin linking energy. As time evolves the initially thin and short bundles elongate, thus reducing their bending energy and allowing them to further associate and create thicker bundles, until all actin monomers are consumed. This process is essentially irreversible on the time scale of actin polymerization. Two structural parameters, L, which is proportional to the length of filament tips at the aster periphery and b, the spacing between their origins, dictate the onset of bundling; both depending on [Arp2/3]. Cells may use a similar mechanism to regulate filopodia formation along the cell leading edge. Such a mechanism may allow cells to have control over the localization of filopodia by recruiting specific proteins that regulate filaments length (e.g., Dia2) to specific sites along lamellipodia.     

Multiple-particle tracking measurements of heterogeneities in solutions of actin filaments and actin bundles

One of the central functions of actin cytoskeleton is to provide the mechanical support required for the establishment and maintenance of cell morphology. The mechanical properties of actin filament assemblies are a consequence of both the available polymer concentration and the actin regulatory proteins that direct the formation of higher order structures. By monitoring the displacement of well-dispersed microspheres via fluorescence microscopy, we probe the degree of spatial heterogeneity of F-actin gels and networks in vitro. We compare the distribution of the time-dependent mean-square displacement (MSD) of polystyrene microspheres imbedded in low- and high-concentration F-actin solutions, in the presence and absence of the F-actin-bundling protein fascin. The MSD distribution of a 2. 6-microM F-actin solution is symmetric and its standard deviation is similar to that of a homogeneous solution of glycerol of similar zero-shear viscosity. However, increasing actin concentration renders the MSD distribution wide and asymmetric, an effect enhanced by fascin. Quantitative changes in the shape of the MSD distribution correlate qualitatively with the presence of large heterogeneities in F-actin solutions produced by increased filament concentration and the presence of actin bundles, as detected by confocal microscopy. Multiple-particle tracking offers a new, quantitative method to characterize the organization of biopolymers in solution.     

Nonenzymatic glycosylation of and oxidative damage to actin in vitro and in vivo

A study was made the influence exerted by non-enzymatic glycosylation (glycation) and oxidative destruction on structural and functional parameters of actin (free NH2-groups, advanced glycation end product and bityrosine cross-linking content, DNase inhibition by G-actin and myosin Mg(2+)-ATPase activation by F-actin). The functional properties of actin were shown to change under high molecular weight product formation and oxidative destruction: the extent of DNAase I inhibition decreases (from 70 to 40%) and the extent of myosin Mg(2+)-ATPase decreases (by 40%). Carnosine prevents actin oligomer formation and oxidative destruction which favours preservation of the protein functional properties.     

Helical arrangement of filaments in microvillar actin bundles

Actin filament arrays in in vivo microvillar bundles of rat intestinal enterocyte were re-evaluated using electron tomography (ET). Conventional electron microscope observation of semi-thin cross sections (300nm thick) of high-pressure freeze fixed and resin embedded brush border has shown a whirling pattern in the center of the microvilli instead of hexagonally arranged dots, which strongly suggests that the bundle consists of a non-parallel array of filaments. A depth compensation method for the ET was developed to estimate the actual structure of the actin bundle. Specimen shrinkage by beam irradiation during image acquisition was estimated to be 63%, and we restored the original thickness in the reconstruction. The depth compensated tomogram displayed the individual actin filaments within the bundles and it indicated that the actin filaments do not lie exactly parallel to each other: instead, they are twisted in a clockwise coil with a pitch of ～120°/μm. Furthermore, the lattice of actin filaments was occasionally re-arranged within the bundle. As the microvillar bundle mechanically interacts with the membrane and is thought to be compressed by the membrane's faint tensile force, we removed the shrouding membrane using detergents to eliminate the mechanical interaction. The bared bundles no longer showed the whirling pattern, suggesting that the bundle had released its coiled property. These findings indicate that the bundle has not rigid but elastic properties and a dynamic transformation in its structure caused by a change in the mechanical interaction between the membrane and the bundle.     

Reorganization of actin filament bundles in living fibroblasts

We investigated how actin bundles assemble, disassemble, and reorganize during cell movement. Living chick embryonic fibroblasts were microinjected with actin molecules that had been fluorescently labeled with tetramethylrhodamine. We found that the fluorescent analogue of actin can be used successfully by both existing and newly formed cellular structures. Using time-lapse photography coupled to image- intensified fluorescence microscopy, we were able to detect various patterns of reorganization in motile cells. Assembly of stress fibers occurred near both the leading and the trailing ends of the cell. The initial structure appeared as discrete spots that subsequently extended into stress fibers. The extension occurred unidirectionally. The site of initiation near the leading edge remained stationary relative to the substrate during subsequent cell advancement. However, the orientation of the fiber could change according to the direction of cell movement. In addition, existing stress fibers could merge or fragment. The shortening of stress fibers can occur from either end of the fiber. Shortening from the proximal end (centrifugal shortening) was accompanied by a decrease in fluorescence intensity, as if the bundle were disassembling, and usually led to the total disappearance of the bundle. Shortening from the distal end (centripetal shortening), on the other hand, is usually accompanied by an increase in fluorescence intensity at the distal end of the bundle, as if this end had pulled loose from its attachment and retracted toward the center of the cell. Besides stress fibers, arc-like actin bundles have also been detected in spreading cells. These observations can explain how the organization of actin bundles coordinates with cell movement, and how stress fibers reach a highly regular pattern in static cells.     

In vivo identification of Tetrahymena actin probed by DMSO induction of nuclear bundles

We report the first successful identification of actin, an ubiquitous contractile protein, in Tetrahymena pyriformis (strain W). We employed dimethyl sulfoxide (DMSO) as a probe to induce the formation of actin bundles in the cell nucleus [1, 2] through disruption of cytoplasmic microfilament organization [3, 4]. The cells were incubated for 30 min at 22 °C in the inorganic medium of Prescott & James [5] containing 10% DMSO, and observed under a transmission electron microscope (TEM). Microfilarment bundles were formed in interphase macronuclei, and these microfilaments, approx. 6 nm in diameter, could be decorated by rabbit skeletal muscle heavy meromyosin (HMM) in the glycerinated model. In many cases, the bundles formed closely parallel to natively existing bundles of microtubules. Interestingly, these microtubules had prominent striation with 15–16 nm periodicity. SDS-polyacrylamide gel electrophoresis was designed to show the low actin content of Tetrahymena cells in comparison with that of Dictyostelium. Actin was suggested to comprise less than 1.7% of the total protein in Tetrahymena, whereas as much as 6% was actin in Dictyostelium cells. In assessing the physiological significance of the bundle formation, we further performed HMM and myosin subfragment-1 (S1)-binding studies to clarify the organization process and the polarity of the DMSO-induced nuclear actin filaments by using the tannic acid staining technique [6]. Randomly oriented short filaments appeared in the nucleus treated with 10% DMSO for 10 min. These filaments became elongated and associated with each other to form loose bundles in the following 10 min. With 30-min treatment, the filaments were organized and large bundles with single axes developed. With these well-developed bundles, the Student's t-test was performed on 172 pairs of neighboring filaments and the probability (p) of the deviation from random polarity was 0.08, suggesting that the filaments were organized in an anti-parallel manner. The results show that the DMSO induction of nuclear actin is a powerful tool to demonstrate the existence of cellular actin in vivo and to study the mechanism of microfilament organization in relation to cell physiological activities.     

Demonstration of actin in the protozoon--Gregarina

Actin was identified in the motile trophozoites of Gregarina by indirect immunofluorescence microscopy. Ultrastructurally actin was found by immunogold labelling to be associated with the internal cytomembranes of the ectoplasmic folds and the general ectoplasmic region below the folds. Western blotting with antibodies to actin identified polypeptides with molecular weights around 43kD and 98kD in non-ionic detergent extracted trophozoites. The possibility of two distinct forms of cytoplasmic actin (43kD and 98kD) was considered.