Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells

The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

The Drosophila javelin gene encodes a novel actin-associated protein required for actin assembly in the bristle

The Drosophila melanogaster bristle is a highly polarized cell that builds specialized cytoskeletal structures. Whereas actin is required for increasing bristle length, microtubules are essential for bristle axial growth. To identify new proteins involved in cytoskeleton organization during bristle development, we focused on identifying and characterizing the javelin (jv) locus. We found that in a jv mutant, the bristle tip is swollen and abnormal organization of bristle grooves is seen over the entire bristle. Using confocal and electron microscopy, we found that in jv mutant bristles, actin bundles do not form properly due to a loss of actin filaments within the bundle. We show that jv is an allele of the predicted CG32397 gene that encodes a protein with no homologs outside insects. Expression of the Jv protein fused to a green fluorescent protein (GFP) shows that the protein is colocalized with actin bundles in the bristle. Moreover, expression of Jv-GFP within the germ line led to the formation of ectopic actin bundles that surround the nucleus of nurse cells. Thus, we report that Jv is a novel actin-associated protein required for actin assembly during Drosophila bristle development.     

Cortical Actin Filaments Potentially Interact with Cortical Microtubules in Regulating Polarity of Cell Expansion in Primary Roots of Maize (Zea mays L.)

Evidence is accumulating implicating cortical microtubules in the directional control of cell expansion. However, the role of actin filaments in this process is still uncertain. To determine the involvement of actin in cell elongation, the organization of actin filaments in primary roots of maize (Zea mays L.) was examined by use of an improved fluorochrome-conjugated phalloidin-labeling method. With this method, a previously undetected state of actin organization was revealed in the elongation and maturation zone of maize roots. Fine transversely oriented cortical actin was observed in all cells of the elongation zone, including the epidermis, cortex, and vascular tissues. The orientation of cortical actin shifted from a predominantly transverse orientation to oblique, longitudinal, and/or random arrangements as the cells matured. The reorientation of cortical actin in maturing root cells mimics the behavior of cortical microtubules reported in other studies. Furthermore, roots treated with the microtubule-stabilizing drug taxol improved the quality of actin preservation as evidenced by the thicker bundles of cortical actin. This suggested that taxol was also capable of stabilizing the cortical actin networks. The elongation of roots exposed to 1 micromole Latrunculin B, an actin-disrupting drug, was inhibited, and after 24 h the roots exhibited moderate swelling particularly along the elongation zone. Latrunculin B also caused microtubules to reorient from transverse to oblique arrays. The results from this study provide evidence that cortical microtubules and actin filaments respond in a coordinated way to environmental signals and may well depend on both elements of the cytoskeleton.     

Distribution of actin-filament bundles in myoid cells,Sertoli cells,and tunica albuginea of rat and mouse testes

Summary Frozen sections of the rat and mouse testes were stained with either FITC-phalloidin or NBD-phallacidin and viewed with conventional fluorescence and confocal laser microscopes in order to demonstrate the arrangment of actin-filament bundles in myoid cells, Sertoli cells and tunica albuginea. Myoid cells are rich in actin-filament bundles crossing at right angles. These bundles running in different directions can also be visualized by means of electron microscopy. Nerve fibers occur in the vicinity of myoid cells, suggesting a neural control of the cell. At Sertoli cell junctions actin filaments occur at the circumference of the cell, where they show a honeycomb pattern. The ratio of the number of Sertoli cells per myoid cell can be calculated by means of confocal microscopy; this technique may provide a new parameter for determining spermatogenic activity. In the tunica albuginea of the juvenile mouse testis, actin filaments are arranged in an alternate fashion.     

Intermediate filaments,microtubules and microfilaments in epidermis of sea urchin tube foot

Summary Tube foot epidermal cells of the sea urchin Strongylocentrotus purpuratus were examined by transmission electron microscopy and fluorescence microscopy to identify the chemical nature of prominent bundles of cytoplasmic filaments. Cross sections revealed filaments of roughly 7–8 nm in diameter closely packed into dense bundles. These bundles, in turn, were each surrounded by a loose sheath of microtubules. The filament size and negative reaction with the fluorescent F-actin binding drug NBD-phallacidin indicated that they were not actin. Indirect immunofluorescence microscopy of whole tissues and frozen sections revealed a strong reaction of the filaments with a monoclonal antibody prepared against porcine stomach desmin. In SDS-polyacrylamide gels of whole tube foot protein, a band of apparent molecular weight around 50 000 daltons reacted with the anti-desmin monoclonal antibody. The combined data provide evidence that the epidermal filament bundles are related to vertebrate intermediate filaments, but further biochemical studies will be necessary to assign them to a particular class of filament proteins.     

Stress fibers in situ in proximal tubules of the rat kidney

Actin bundles in proximal tubules of the rat kidney were examined by immunofluorescence and confocal laser microscopy with special reference to their three-dimensional distribution and identification as stress fibers. Renal tubular segments were prepared from the fresh renal cortex by simple homogenization and centrifugation, and fixed in formaldehyde for staining with fluorescent dye-labeled phalloidin. Segments of the proximal tubules could be identified easily on the bases of their diameter, the height of epithelial cells and prominent brush borders. Confocal laser microscopy clearly demonstrated the overall distribution of actin bundles in the whole-mount proximal tubular segments. Actin bundles in the basal cytoplasm of epithelial cells were observed to run parallel to each other and at a right angle to the tubular axis. In the stereo views reconstructed from serial optical sections, the basal actin bundles appeared as straight rods with both ends tapered. They varied in length and width and extended rather short distances of not more than 10 microns. Often, two or more actin bundles were longitudinally aligned in tandem. Some bundles showed irregular bandings along their length. Each bundle was composed of tightly packed actin filaments which could be decorated with heavy meromyosin subfragment-1 to display a bi-directional arrangement within the bundle. Immunostaining of cryostat sections showed that actin bundles contained myosin and vinculin. Enzymatically isolated proximal tubules contracted upon addition of Mg-ATP. These observations collectively suggest that the actin bundles at the base of renal proximal tubule epithelial cells can be listed among the examples of stress fibers in situ.     

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.     

A morphometric analysis of the redistribution of organelles in columella cells in primary roots of normal seedlings and agravitropic mutants of Hordeum vulgare

The redistribution of organelles in columella cells of horizontally-oriented roots of Hordeum vulgare was quantified in order to determine what structural changes in graviperceptive (i.e., columella) cells are associated with the onset of the root gravicurvature. The sedimentation of amyloplasts is the only major change in cellular structure that correlates positively with the onset of root gravicurvature, which begins within 15 min after re-orientation. There is no consistent contact between sedimented amyloplasts and any other organelles. Nuclei are restricted to the proximal ends of columella cells in vertically-oriented roots, and remain there throughout gravicurvature after roots are oriented horizontally. Root gravicurvature does not involve significant changes in (1) the volume of columella cells, (2) the relative or absolute volumes of organelles in columella cells, or (3) the distribution of endoplasmic reticulum (ER). The size, number and sedimentation rates of amyloplasts in columella cells of non-graviresponsive roots of mutant seedlings are not significantly different from those of graviresponsive roots of normal seedlings. Similarly, there is no significant difference in (1) cellular volume, (2) distribution or surface area of ER, (3) patterns or rates of organelle redistribution in horizontally-oriented roots, (4) relative or absolute volumes of organelles in columella cells of graviresponsive and non-graviresponsive roots. These results suggest that the lack of graviresponsiveness by roots of mutant seedlings is probably not due to either (1) structural differences in columella cells, or (2) differences in patterns or rates of organelle redistribution as compared to that characteristic of graviresponsive roots. Thus, the basis of non-graviresponsiveness in this mutant is probably different from other agravitropic mutants so far studied.     

Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers

In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.     

Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts

The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde. Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 micron in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment 1 and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.     

The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis

In many types of plant cell, bundles of actin filaments (AFs) are generally involved in cytoplasmic streaming and the organization of transvacuolar strands. Actin cross-linking proteins are believed to arrange AFs into the bundles. In root hair cells of Hydrocharis dubia (Blume) Baker, a 135-kDa polypeptide cross-reacted with an antiserum against a 135-kDa actin-bundling protein (135-ABP), a villin homologue, isolated from lily pollen tubes. Immunofluorescence microscopy revealed that the 135-kDa polypeptide co-localized with AF bundles in the transvacuolar strand and in the sub-cortical region of the cells. Microinjection of antiserum against 135-ABP into living root hair cells induced the disappearance of the transvacuolar strand. Concomitantly, thick AF bundles in the transvacuolar strand dispersed into thin bundles. In the root hair cells, AFs showed uniform polarity in the bundles, which is consistent with the in-vitro activity of 135-ABP. These results suggest that villin is a factor responsible for bundling AFs in root hair cells as well as in pollen tubes, and that it plays a key role in determining the direction of cytoplasmic streaming in these cells. Received: 16 September 1999 / Accepted: 3 December 1999     

Influence of cold on organization of actin filaments of different types of root cells in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The effect of the low temperature (+4°C) on the organization of actin filaments (microfilaments) of cells from different growth zones has been studied in the roots of Arabidopsis thaliana (L.). It was found that cold treatment inhibited the growth of the primary root and changed its morphology, causing a formation of large number of deformed (ectopic) root hairs in differentiation zone. The temporal relationship between the disorientation and the organization of actin filaments and the detected changes of growth and morphology of roots after cold treatment was shown. It has been found that actin filaments of root hairs, meristematic cells, cells of elongation zone, and epidermal cells of all root zones of A. thaliana are the most sensitive to the cold.     

Reorganization of actin cytoskeleton of nuclear erythrocytes and leukocytes in fish,frogs, and birds during migration

Using the method of confocal laser scanning microscopy, changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes in fish, frogs, and birds during migration were studied. It has been shown that, during movement, in erythrocytes, like in leukocytes, reorganization of cytoskeleton microfilaments occurs. In the course of migration, in amphibians and birds, red blood cells form pseudopodia filled with bundles of actin filaments arranged in parallel, whereas no pseudopodia are formed in fish erythrocytes. The change in the structure of the actin cytoskeleton of nuclear erythrocytes, like in leukocytes, is responsible for the capability of red blood cells to undergo reactions of migration and phagocytosis.     

Structural similarity between actin bundles from characean algal cells and sea urchin oocytes

The structure of actin bundles from internodal cells of Chara australis, an algal plant, was studied by electron microscopy of negatively stained specimens and optical diffraction. Gently prepared bundles revealed paracrystalline structures resembling the Mg²⁺-induced paracrystals of rabbit skeletal muscle actin (Hanson, 1968). In addition, the algal actin bundles sometimes had transverse striations at intervals of about 130 Å, as has been observed in actin bundles from sea urchin eggs (DeRosier et al., 1977; Spudich & Amos, 1979) and sea urchin coelomocytes (De Rosier & Edds, 1980; Otto & Bryan, 1981). This finding suggests that a common mechanism might be working in a variety of cells to organize actin filaments into functional bundles.     

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.     

Rootcap structure in wild type and in a starchless mutant of Arabidopsis.

Rootcaps of the wild type (WT) and of a starchless, gravitropic mutant (TC7) of Arabidopsis thaliana L. were examined by electron microscopy to identify cellular polarities with respect to gravity. In columella cells, nuclei are located proximally, and the nuclear envelope is continuous with endoplasmic reticulum (ER) that is in turn connected to nearby plasmodesmata. Impregnation of ER with osmium ferricyanide revealed numerous contacts between columella plastids and ER in both genotypes. ER is present mostly in the outer regions of the columella protoplast except in older columella cells that are developing into peripheral cells. In vertical roots, only columella cells that are intermediate in development (story 2 cells) have a higher surface density (S) of ER in the distal compared to proximal regions of the cell. The distal but not the proximal S of the ER is constant throughout columella development. Plastids are less sedimented in TC7 columella cells compared to those of the WT. It is hypothesized that plastid contact with the ER plays a role in gravity perception in both genotypes.     

Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.