Hyper-mobile water is induced around actin filaments

When introduced into water, some molecules and ions (solutes) enforce the hydrogen-bonded network of neighboring water molecules that are thus restrained from thermal motions and are less mobile than those in the bulk phase (structure-making or positive hydration effect), and other solutes cause the opposite effect (structure-breaking or negative hydration effect). Using a method of microwave dielectric spectroscopy recently developed to measure the rotational mobility (dielectric relaxation frequency) of water hydrating proteins and the volume of hydration shells, the hydration of actin filament (F-actin) has been studied. The results indicate that F-actin exhibits both the structure-making and structure-breaking effects. Thus, apart from the water molecules with lowered rotational mobility that make up a typical hydration shell, there are other water molecules around the F-actin which have a much higher mobility than that of bulk water. No such dual hydration has been observed for myoglobin studied as the representative example of globular proteins which all showed qualitatively similar dielectric spectra. The volume fraction of the mobilized (hyper-mobile) water is roughly equal to that of the restrained water, which is two-thirds of the molecular volume of G-actin in size. The dielectric spectra of aqueous solutions of urea and potassium-halide salts have also been studied. The results suggest that urea and I(-) induce the hyper-mobile states of water, which is consistent with their well-known structure-breaking effect. The molecular surface of actin is rich in negative charges, which along with its filamentous structure provides a structural basis for the induction of a hyper-mobile state of water. A possible implication of the findings of the present study is discussed in relation to the chemomechanical energy transduction through interaction with myosin in the presence of ATP.     

Tubular actin filaments in tobacco guard cells

The dynamic remodeling of actin filaments in guard cells functions in stomatal movement regulation. In our previous study, we found that the stochastic dynamics of guard cell actin filaments play a role in chloroplast movement during stomatal movement. In our present study, we further found that tubular actin filaments were present in tobacco guard cells that express GFP-mouse talin; approximately 2.3 tubular structures per cell with a diameter and height in the range of 1–3 µm and 3–5 µm, respectively. Most of the tubular structures were found to be localized in the cytoplasm near the inner walls of the guard cells. Moreover, the tubular actin filaments altered their localization slowly in the guard cells of static stoma, but showed obvious remodeling, such as breakdown and re-formation, in moving guard cells. Tubular actin filaments were further found to be colocalized with the chloroplasts in guard cells, but their roles in stomatal movement regulation requires further investigation.Key words: actin dynamics, tubular actin filaments, chloroplast, guard cell, stomatal movementStomatal movement responses to surrounding environment are mediated by guard cell signaling.^1,2 Actin filaments within guard cells are dynamic cytoarchitectures and function in stomatal development and movement.³ Arrays of actin filaments in guard cells that are dependent on different stomatal apertures have also been reported in references ^4–7. For example, the random or longitudinal orientations of actin filaments in closed stomata change to a radial orientation or ring-like array after stomata opening.^5,6,8 The reorganization of the actin architecture during stomatal movement depends on the depolymerization and repolymerization of actin filaments in guard cells. In contrast to the traditional treadmill model of actin dynamic mechanisms, stochastic dynamics of actin have been revealed in plant cells, such as in the epidermal cells of hypocotyl and root, the pavement cells of Arabidopsis cotyledons, and the guard cells of tobacco (Nicotiana tabacum).^9–11 In this alternative system, the short actin fragments generated from severed long filaments can link with each other to form longer filaments by end-joining activity. The actin regulatory proteins, Arp2/3 complex, capping protein and actin depolymerizing factor (ADF)/cofilin, may also be involved in the stochastic dynamics of actin filaments.^12,13Using tobacco GFP-mouse talin expression lines, we have previously analyzed the stochastic dynamics of guard cell actin filaments and their roles in chloroplast displacement during stomatal movement.^6,11 We found from these analyses that another arrangement of actin filaments, i.e., tubular actin filaments, exists in the guard cells of these tobacco lines. We first found the circle-like actin filaments in 82% of the guard cells (counting 320 cells) in tobacco expressing GFPmouse talin when analyzing a single optical section (Fig. 1A). In a previous study of BY-2 cells expressing GFP-Lifeact labeled actin filaments, Smertenko et al. found similar structures, i.e., quoit-like structures or acquosomes in all of the plant tissues examined except growing root hairs.¹⁰ However, in our present analysis of serial sections, we determined that the circle-like actin filaments in the tobacco guard cells were long tubes (Fig. 1A), as the lengths (about 3–5 µm) of these structures were greater than their diameter (about 1–3 µm). Hence, we denoted these structures as tubular actin filaments to distinguish them from the circular conformations of actin filaments observed previously in other plant cell tissues.^10,14–19 About 2.3 of these tubular actin filaments were found per guard cell, which is less than the number of acquosomes reported in BY-2 cells (about 6.7 per cell).¹⁰ Analysis of serial optical sections at the z-axis revealed that the tubular actin filaments localize in the cytoplasm near the inner walls of the guard cells (Fig. 1B), which is similar to the distribution of chloroplasts in guard cells.¹¹ Longitudinal sections further revealed a colocalization of tubular actin filaments and chloroplasts (Fig. 1B).Open in a separate windowFigure 1Tubular actin filaments in the guard cells of a tobacco (Nicotiana tabacum) line expressing GFP-mouse talin. (A) Optical-sections (interval, 1.5 µm) of guard cells in a moving stoma showing tubular actin filaments (arrow heads). Frames (a1) and (a2) are cross sections of 1.5-µm-picture through the yellow and red lines, respectively, revealing the cross section of the circle structures are parallel lines (arrows). (B) Optical-sections of a stoma from the outer periclinal walls to the inner walls of the guard cells (interval, 1 µm). The tubular actin filaments (arrow heads) are localized in the cytoplasm near to the inner periclinal walls of guard cells. Frame (b1) is the guard cell on the right of the frame “4 µm”; (b2) is the cross section of b1 through the red line; and (b3) is a higher magnification image of the area encompassed by the white square in b2. Arrows indicate the colocalization between the tubular actin filaments and the chloroplast (indicated using a red pseudocolor). (C) Time-series imaging showing the movement of tubular actin filaments in the guard cells of static stomata. Frame (c1) comprises three images colored red (0 S), green (40 S) and blue (80 S), that are merged in a single frame to show the translocation of the tubular actin filaments (arrows). (D) Time-series images of the opening stomata showing the breakdown (arrows) and re-formation (arrowheads) of the tubular actin filaments. All images were captured using a Zeiss LSM 510 META confocal laser scanning microscope, as described by Wang et al.¹¹ Bars, 10 µm.We performed time-lapse imaging and found that the translocation of tubular actin filaments is slow in static stomata in which the distance between two tubular actin filaments typically increased from 2.22 to 2.50 µm after 80 sec (Fig. 1C). In moving stomata, however, the tubular actin filaments showed an obvious dynamic reorganization whereby they could be processed into short fragments and also reemerged after they had disintegrated (Fig. 1D). These results indicate that tubular actin filaments have stochastic dynamics that are similar to the long actin filaments of guard cells.¹¹ In our previous study, we found that the stochastic dynamics of actin filaments correlate with light-induced chloroplast movement in guard cells.¹¹ However, whether the dynamics of the tubular actin filaments are also involved in chloroplast movement during stomatal movement remains to be investigated. In cultured mesophyll cells which had been mechanically isolated from Zinnia elegans, Wilsen et al. previously found a close association between fully closed actin rings and chloroplasts.¹⁸ These authors further found that the average percentage of cells with free actin rings increased at the initial culture stage, and then decreased, which indicates that the formation of actin rings might be a response of the actin cytoskeleton to cellular stress or disturbance.¹⁸ The turgor pressure of guard cells is the fundamental basis of stomatal movement leading to changes in the shape, volume, wall structure, and membrane surface of guard cells.^20–24 We speculate from our current data that there is a relationship between tubular actin filaments and the shape changes of guard cells during stomatal movement.     

Osmotically induced electrical signals from actin filaments.

Actin filaments, F-actin, a major component of the cortical cytoskeleton, play an important role in a variety of cell functions. In this report we have assessed the role of osmotic stress on the electrochemical properties of F-actin. The spontaneous Donnan potential of a polymerized actin solution (5 mg/ml) was -3.93 +/- 1.84 mV, which was linearly reduced by osmotic stress on the order of 1-20 mOsm (0.28 +/- 0.06 mV/mM). Calculated surface charge density was reduced and eventually reversed by increasing the osmotic stress as expected for a phase transition behavior. The electro-osmotic behavior of F-actin disappeared at pH 5.5 and was dependent on its filamentous nature. Furthermore, osmotically stressed F-actin displayed a nonlinear electric response upon application of electric fields on the order of 500-2,000 V/cm. These data indicate that F-actin in solution may display nonideal electro-osmotic properties consistent with ionic "cable" behavior which may be of biological significance in the processing and conduction of electrical signals within the cellular compartment.     

Conformational changes in actin filaments induced by formin binding to the barbed end

Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.     

10 nm filaments in normal and transformed cells.

An antibody was raised against an electrophoretically homogeneous protein from cultured fibroblasts and shown to be directed against 10 nm filaments. The antiserum did not stain microtubules or actin microfilaments. The distribution of 10 nm filaments in normal cells was studied during growth, spreading, locomotion, mitosis, and after treatment with colchicine and cytochalasin B. The 58,000 dalton subunit protein is apparently all polymerized in the filaments which are insoluble in nonionic detergent. The distribution of 10 nm filaments is altered by colchicine treatments which disrupt microtubules. The organization of 10 nm filaments is altered in transformed cells.     

Stomatal opening by fusicoccin is accompanied by depolymerization of actin filaments in guard cells

Actin in guard cells is assembled in a radial pattern when stomata are induced to open under light, but the filaments are disassembled when stomata are closed under darkness or by abscisic acid (S.-O. Eun and Y. Lee, 1997, Plant Physiol. 115: 1491–1498). To test if signals that open stomata commonly generate the polymerized form of actin in guard cells, leaves of Commelina communis L. were treated with a potent stomatal opening agent, fusicoccin, and the actin organization examined by immunolocalization techniques. When stomata were induced to open by fusicoccin, hardly any of the filamentous form of actin was detected; instead, the actin resembled that present in guard cells that had been treated with an antagonist to actin filaments, cytochalasin D, and showed a sharp contrast to the long filaments developed in illuminated guard cells. Furthermore, treatment of illuminated leaves with fusicoccin disintegrated actin filaments that had already been formed in the guard cells. Preincubation of leaves with phalloidin, which interferes with fusicoccin-induced actin depolymerization, delayed fusicoccin-induced opening during the early phase. These observations suggest that the prevention of actin filament formation and/or depolymerization of actin filaments may accelerate the stomatal opening process in response to fusicoccin. Received: 1 October 1999 / Accepted: 29 November 1999     

Disorders of the actin cytoskeleton in transformed epithelial cells

The actin cytoskeleton of 8 transformed epithelial cell lines was studied using electron microscopy of platinum replicas. Seven of these lines belonged to the IAR series of rat liver epithelial cells, being at different stages of neoplastic progression. One cell line (FBT) was derived from the epithelium of bovine fetal trachea. The extent of actin cytoskeleton alteration in cell lines studied has been shown to correlate with other signs of neoplastic transformation. Among various actin-containing cell structures (microfilament bundles, actin meshwork at active edges, cell-cell adherence junctions, and endoplasmic microfilament sheath) the latter was the most sensitive to transformation. The loosening of the sheath and the alteration of its fine structure were observed in all the cell lines. The degree of these changes increased in the following order: FBT; non-tumorigenic IAR lines; IAR lines transformed in vitro; IAR lines obtained from the latter by single or double selection in vivo. The alteration of sheath was the only disturbance of actin cytoskeleton in FBT cells, whereas in other groups of epithelial cell lines some other changes occurred. These involved disruption of actin-containing intercellular junctions, the cell polarization accompanied by progressive shortening of length of the cell active edge containing actin meshwork, and disappearance or reorganization of microfilament bundles.     

Changes in the relationship between actin filaments and the plasma membrane in culturedZinnia cells during tracheary element differentiation investigated by using plasma membrane ghosts

Aggregates of actin filaments appear immediately before secondary wall thickening during tracheary element differentiation in isolatedZinnia cells. An analysis of plasma membrane ghosts revealed that the aggregates were bound to the plasma membrane. The properties of the binding of actin filaments to the plasma membrane were investigated in this system. Present address and for correspondence: Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate, 024 Japan.     

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.     

The presence of ring formed actin filaments in plant cells

Summary Ring formed actin filaments were observed in tobacco BY-2 cells. The change of this structure during culture was followed by fluorescence microscopy.     

Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments

Tropomyosin inhibition of the rate of spontaneous polymerization of actin is associated with binding of tropomyosin to actin filaments. Rate constants determined by using a direct electron microscopic assay of elongation showed that alpha alpha- and alpha beta-tropomyosin have a small or no effect on the rate of elongation at either end of the filaments. The most likely explanation for the inhibition of the rate of polymerization of actin in bulk samples is that tropomyosin reduces the number of filament ends by mechanical stabilization of the filaments.     

Microfilaments and intermediate filaments in epithelial cells transformed by murine sarcoma or leukemia viruses

The murine epithelial cell line MMC-E was used to study changes in the cytoskeletal organization associated with viral transformation of epithelial cells by two different viruses. The cells were transformed with Moloney mouse sarcoma virus (MSV) or murine leukemia virus (MuLV). The expression of actin, myosin and of intermediate filament proteins in the cells was then studied. In MMC-E cells actin and myosin were organized as belt-like structures at the edges of the border cells of the cell islands and also circumferentially in the cells inside the islands. The major change after transformation was the decrease of the actomyosin containing belt extending from cell to cell at the borders of the cell islands. Both MMC-E cells and the MSV-transformed cells contained keratin as a juxtanuclear granular aggregate whereas the MuLV-transformed cells showed bright fibrillar arrays of keratin. Both virus-transformed cell lines showed enhanced vimentin-specific fluorescence and analysis of their cytoskeletal polypeptides confirmed the result. Similar molecular forms of keratin polypeptides were seen in all cells by immunoblotting. Viral transformation of MMC-E epithelial cells thus leads to different changes in their cytoskeletal organization depending on the transforming viral or cellular gene.     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.     

Correlated waves of actin filaments and PIP3 in Dictyostelium cells

Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity.     

Orientation and three-dimensional organization of actin filaments in dividing cultured cells

The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.     

Banding and polarity of actin filaments in interphase and cleaving cells

Heavy meromyosin (HMM) decoration of actin filaments was used to detect the polarity of microfilaments in interphase and cleaving rat kangaroo (PtK2) cells. Ethanol at -20 degrees C was used to make the cells permeable to HMM followed by tannic acid-glutaraldehyde fixation for electron microscopy. Uniform polarity of actin filaments was observed at cell junctions and central attachment plaques with the HMM arrowheads always pointing away from the junction or plaque. Stress fibers were banded in appearance with their component microfilaments exhibiting both parallel and antiparallel orientation with respect to one another. Identical banding of microfilament bundles was also seen in cleavage furrows with the same variation in filament polarity as found in stress fibers. Similarly banded fibers were not seen outside the cleavage furrow in mitotic cells. By the time that a mid-body was present, the actin filaments in the cleavage furrow were no longer in banded fibers. The alternating dark and light bands of both the stress fibers and cleavage furrow fibers are approximately equal in length, each measuring approximately 0.16 micrometer. Actin filaments were present in both bands, and individual decorated filaments could sometimes be traced through four band lengths. Undecorated filaments, 10 nm in diameter, could often be seen within the light bands. A model is proposed to explain the arrangement of filaments in stress fibers and cleavage furrows based on the striations observed with tannic acid and the polarity of the actin filaments.     

Distributionof actin filaments in differentiating cells of Equisetum hyemale root tips

The distribution of F-actin was studied in various cell types in root tips of Equisetum hyemale using rhodamine labelled phalloidin as a probe. In dividing cells no cables could be observed, although a strong diffuse staining, which has the same distribution as the microtubules, occurs in the phragmoplast. During cell development the cables become longer and form meshworks but no relationship with cell expansion and microtubule patterns was observed. The distribution apparently follows the pattern of plasma streaming. In expanded cells in particular the nucleus is surrounded by a web of filaments connected with the larger cables in the cell.     

Dynamic motion of paxillin on actin filaments in living endothelial cells

Our three-dimensional (3-D) images showed that paxillin co-localized on actin filaments as fibrous structures, as well as clusters, in endothelial cells (ECs). In living ECs under flow condition, we monitored concurrently the intracellular dynamics of DsRed2-paxillin and GFP-actin by time-lapse video recording and dual-color fluorescence imaging. The results showed that the dynamic motion of paxillin as fibrous structures was associated with actin filaments, but not with microtubules. Our findings suggest that the actin network plays an important role not only in the assembly/disassembly of paxillin at focal adhesions, but also as a track for the intracellular transport of paxillin, which is involved in signaling pathway.