Measuring orientation of actin filaments within a cell: orientation of actin in intestinal microvilli.

Orientational distribution of actin filaments within a cell is an important determinant of cellular shape and motility. To map this distribution we developed a method of measuring local orientation of actin filaments. In this method actin filaments within cells are labeled with fluorescent phalloidin and are viewed at high magnification in a fluorescent microscope. Emitted fluorescence is split by a birefringent crystal giving rise to two images created by light rays polarized orthogonally with respect to each other. The two images are recorded by a high-sensitivity video camera, and polarization of fluorescence at any point is calculated from the relative intensity of both images at this point. From the value of polarization, the orientation of the absorption dipole of the dye, and thus orientation of F-actin, can be calculated. To illustrate the utility of the method, we measured orientation of actin cores in microvilli of chicken intestinal epithelial cells. F-actin in microvillar cores was labeled with rhodamine-phalloidin; measurements showed that the orientation was the same when microvillus formed a part of a brush border and when it was separated from it suggesting that "shaving" of brush borders did not distort microvillar structure. In the absence of nucleotide, polarization of fluorescence of actin cores in isolated microvilli was best fitted by assuming that a majority of fluorophores were arranged with a perfect helical symmetry along the axis of microvillus and that the absorption dipoles of fluorophores were inclined at 52 degrees with respect to the axis. When ATP was added, the shape of isolated microvilli did not change but polarization of fluorescence decreased, indicating statistically significant increase in disorder and a change of average angle to 54 degrees. We argue that these changes were due to mechanochemical interactions between actin and myosin-I.     

Supercontraction in crayfish muscle: correlation with a peculiar actin localization.

Crayfish muscle, like muscles from some other invertebrates, can supercontract. This muscle shortening is characterized by an overlap of thin filaments with crossing of thick filaments through the Z discs. In intact muscle cells, supercontraction does not seem to induce irreversible structural modifications in the tissue. Isolated crayfish myofibrils in the relaxed state cannot be distinguished from vertebrate myofibrils under light microscope, either by phase contrast or by immunofluorescence, with antiactin antibodies, actin being localized in the I bands. However, when isolated crayfish myofibrils are supercontracted, irreversible dammage occurs, most thin filaments being lost. Actin becomes then hardly detectable, being visible, by immunofluorescence, either in the Z discs or evenly distributed in the whole myofibril. During myofibril supercontraction, high amounts of denatured actin, become soluble as shown by SDS-PAGE, by double immunodiffusion, and by DNAse inhibition.     

Covalent and non-covalent chemical engineering of actin for biotechnological applications

The cytoskeletal filaments are self-assembled protein polymers with 8–25 nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways.     

Energy filtered electron tomography of ice-embedded actin and vesicles.

Semiautomatic single-axis tilt electron tomography has been used to visualize the three-dimensional organization of actin filaments in "phantom cells," i.e. lipid vesicles. The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, which was used in the zero-loss imaging mode. Apart from changing the tilt angle, all steps required for automated tomography, such as recentering the image area, refocusing, and centering the energy-selecting slit, were performed by external computer control. This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with improved image contrast compared with that produced by tomography with a conventional electron microscope. In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interaction, single-axis tilt tomography was found to be an appropriate (in fact the only available) method for this kind of investigation. In contrast to random actin networks found in actin gels, actin filaments in and on vesicles with a bending radius of less than approximately 2 microns tend to be arranged in single layers of parallel filaments and often induce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.     

Filament organization revealed in platinum replicas of freeze-dried cytoskeletons

This report presents the appearance of rapidly frozen, freeze-dried cytoskeletons that have been rotary replicated with platinum and viewed in the transmission electron microscope. The resolution of this method is sufficient to visualize individual filaments in the cytoskeleton and to discriminate among actin, microtubules, and intermediate filaments solely by their surface substructure. This identification has been confirmed by specific decoration with antibodies and selective extraction of individual filament types, and correlated with light microscope immunocytochemistry and gel electrophoresis patterns. The freeze-drying preserves a remarkable degree of three-dimensionality in the organization of these cytoskeletons. They look strikingly similar to the meshwork of strands or "microtrabeculae" seen in the cytoplasm of whole cells by high voltage electron microscopy, in that the filaments form a lattice of the same configutation and with the same proportions of open area as the microtrabeculae seen in whole cells. The major differences between these two views of the structural elements of the cytoplasmic matrix can be attributed to the effects of aldehyde fixation and dehydration. Freeze-dried cytoskeletons thus provide an opportunity to study--at high resolution and in the absence of problems caused by chemical fixation--the detailed organization of filaments in different regions of the cytoplasm and at different stages of cell development. In this report the pattern of actin and intermediate filament organization in various regions of fully spread mouse fibroblasts is described.     

Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.     

Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium

A new microscope technique, termed "W" (double view video) microscopy, enables simultaneous observation of two different images of an object through a single video camera or by eye. The image pair may, for example, be transmission and fluorescence, fluorescence at different wavelengths, or mutually perpendicular components of polarized fluorescence. Any video microscope can be converted into a dual imager by simple insertion of a small optical device. The continuous appearance of the dual image assures the best time resolution in existing and future video microscopes. As an application, orientations of actin protomers in individual, moving actin filaments have been imaged at the video rate. Asymmetric calcium influxes into a cell exposed to an intense electric pulse have also been visualized.     

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment–detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.     

Displacement of the mitotic apparatuses by centrifugation reveals cortical actin organization during cytokinesis in cultured tobacco BY-2 cells

In plant cytokinesis, actin is thought to be crucial in cell plate guidance to the cortical division zone (CDZ), but its organization and function are not fully understood. To elucidate actin organization during cytokinesis, we employed an experimental system, in which the mitotic apparatus is displaced and separated from the CDZ by centrifugation and observed using a global–local live imaging microscope that enabled us to record behavior of actin filaments in the CDZ and the whole cell division process in parallel. In this system, returning movement of the cytokinetic apparatus in cultured-tobacco BY-2 cells occurs, and there is an advantage to observe actin organization clearly during the cytokinetic phase because more space was available between the CDZ and the distantly formed phragmoplast. Actin cables were clearly observed between the CDZ and the phragmoplast in BY-2 cells expressing GFP-fimbrin after centrifugation. Both the CDZ and the edge of the expanding phragmoplast had actin bulges. Using live-cell imaging including the global–local live imaging microscopy, we found actin filaments started to accumulate at the actin-depleted zone when cell plate expansion started even in the cell whose cell plate failed to reach the CDZ. These results suggest that specific accumulation of actin filaments at the CDZ and the appearance of actin cables between the CDZ and the phragmoplast during cell plate formation play important roles in the guidance of cell plate edges to the CDZ.     

Motion of actin filaments in the presence of myosin heads and ATP.

We measured, by fluorescence correlation spectroscopy, the motion of actin filaments in solution during hydrolysis of ATP by acto-heavy meromyosin (acto-HMM). The method relies on the fact that the intensity of fluorescence fluctuates as fluorescently labeled actin filaments enter and leave a small sample volume. The rapidity of these number fluctuations is characterized by the autocorrelation function, which decays to 0 in time that is related to the average velocity of translation of filaments. The time of decay of the autocorrelation function of bare actin filaments in solution was 10.59 +/- 0.85 s. Strongly bound (rigor) heads slowed down the diffusion. Direct observation of filaments under an optical microscope showed that addition of HMM did not change the average length or flexibility of actin filaments, suggesting that the decrease in diffusion was not due to a HMM-induced change in the shape of filaments. Rather, slowing down of translational motion was caused by an increase in the volume of the diffusing complex. Surprisingly, the addition of ATP to acto-HMM accelerated the motion of actin filaments. The acceleration was the greatest at the low molar ratios of HMM:actin. Direct observation of filaments under an optical microscope showed that in the presence of ATP the average length of filaments did not change and that the filaments became stiffer, suggesting that acceleration of diffusion was not due to an ATP-induced increase in flexibility of filaments. These results show that some of the energy of splitting of ATP is impaired to actin filaments and suggest that 0.06 +/- 0.02 of HMM interferes with the diffusion of actin filaments during hydrolysis of ATP.     

肌动蛋白体外自组织复合纤维结构的观察

利用原子力显微镜(atomic force microscope,AFM)和透射电子显微镜(transmission electron microscope,WEM)技术,研究了低浓度肌动蛋白在体外简单热力学体系中,形成的自组织复合纤维结构。肌动蛋白在体外通过自组织过程能够聚合形成大尺度的、离散的、复杂的聚集纤维体系,分散的单根微丝较少;在微丝稳定剂鬼笔环肽干预下,肌动蛋白通过受调控的自装配过程,主要形成分散的单根微丝,以及少量由单根微丝组成的微丝束和纤维分支等简单微丝聚集结构。     

Cryoatomic force microscopy of filamentous actin

Cryoatomic force microscopy (cryo-AFM) was used to image phalloidin-stabilized actin filaments adsorbed to mica. The single filaments are clearly shown to be right-handed helical structures with a periodicity of approximately 38 nm. Even at a moderate concentration ( approximately 10 microg/ml), narrow, branched rafts of actin filaments and larger aggregates have been observed. The resolution achieved is sufficient to resolve actin monomers within the filaments. A closer examination of the images shows that the branched rafts are composed of up to three individual filaments with a highly regular lateral registration with a fixed axial shift of approximately 13 nm. The implications of these higher-order structures are discussed in terms of x-ray fiber diffraction and rheology of actin gels. The cryo-AFM images also indicate that the recently proposed model of left-handed F-actin is likely to be an artifact of preparation and/or low-resolution AFM imaging.     

Actin filament organization in the fish keratocyte lamellipodium

From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation- release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid- region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.     

The two actin-binding regions on the myosin heads of cardiac muscle

In the presence of myosin S1 or myosin heads, actin filaments tend to form bundles. The biological meaning of the bundling of actin filaments has been unclear. In this study, we found that the cardiac myosin heads can form the bundles of actin filaments more rapidly than can skeletal S1, as monitored by light scattering and electron microscopy. Moreover, the actin bundles formed by cardiac S1 were found to be more stable against mechanical agitation. The distance between actin filaments in the bundles was approximately 20 nm, which is comparable to the length of a myosin head and two actin molecules. This suggests the direct binding of S1 tails to the adjacent actin filament. The "essential" light chain of cardiac myosin could be cross-linked to the actin molecule in the bundle. When monomeric actin molecules were added to the bundle, the bundles could be dispersed into individual filaments. The three-dimensional structure of the dispersed actin filaments was reconstructed from electron cryo-microscopic images of the single actin filaments dispersed by monomer actin. We were able to demonstrate that cardiac myosin heads bind to two actin molecules: one actin molecule at the conventional actin-binding region and the other at the essential light-chain-binding region. This capability of cardiac myosin heads to bind two actin molecules is discussed in view of lower ATPase activity and slower shortening velocity than those of skeletal ones.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.     

Polymerization and depolymerization of actin with nucleotide states at filament ends

Polymerization induces hydrolysis of ATP bound to actin, followed by γ-phosphate release, which helps advance the disassembly of actin filaments into ADP-G-actin. Mechanical understanding of this correlation between actin assembly and ATP hydrolysis has been an object of intensive studies in biochemistry and structural biology for many decades. Although actin polymerization and depolymerization occur only at either the barbed or pointed ends and the kinetic and equilibrium properties are substantially different from each other, characterizing their properties is difficult to do by bulk assays, as these assays report the average of all actin filaments in solution and are therefore not able to discern the properties of individual actin filaments. Biochemical studies of actin polymerization and hydrolysis were hampered by these inherent properties of actin filaments. Total internal reflection fluorescence (TIRF) microscopy overcame this problem by observing single actin filaments. With TIRF, we now know not only that each end has distinct properties, but also that the rate of γ-phosphate release is much faster from the terminals than from the interior of actin filaments. The rate of γ-phosphate release from actin filament ends is even more accelerated when latrunculin A is bound. These findings highlight the importance of resolving structural differences between actin molecules in the interior of the filament and those at either filament end. This review provides a history of observing actin filaments under light microscopy, an overview of dynamic properties of ATP hydrolysis at the end of actin filament, and structural views of γ-phosphate release.     

Severing of F-actin by yeast cofilin is pH-independent

Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, alpha-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by epsilonATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2-3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green 488 maleimide. The depolymerization of actin by cofilin was faster at high pH.     

Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells

Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells.     

Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding

Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.