Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis

Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern.     

Actin-Binding Proteins in Plant Cells

Abstract: Actinoccurs in all plant cells, as monomers, filaments and filament assemblies. In interphase, actin filaments form a cortical network, co-align with cortical microtubules, and extend throughout the cytoplasm functioning in cytoplasmic streaming. During mitosis, they co-align with microtubules in the preprophase band and phragmoplast and are indispensa ble for cell division. Actin filaments continually polymerise and depolymerise from a pool of monomers, and signal transduction pathways affecting cell morphogenesis modify the actin cytoskeleton. The interactions of actin monomers and filaments with actin-binding proteins (ABP5) control actin dynamics. By binding to actin monomers, ABPs, such as profilin, regulate the pool of monomers available for polymerisation. By breaking filaments or capping filament ends, ABPs, such as actin depoly-merising factor (ADF), prevent actin filament elongation or loss of monomers from filament ends. By bivalent cross-linking to actin filaments, ABPs, such as fimbrin and other members of the spectrin family, produce a variety of higher order assemblies, from bundles to networks. The motor protein ABPs,. which are not covered in this review, move organelles along ac tin filaments. The large variety of ABPs share a number of functional modules. A plant representative of ABPs with particular modules, and therefore particular functions, is treated in this review.     

Reconstitution of an Actin Cortex Inside a Liposome

The composite and versatile structure of the cytoskeleton confers complex mechanical properties on cells. Actin filaments sustain the cell membrane and their dynamics insure cell shape changes. For example, the lamellipodium moves by actin polymerization, a mechanism that has been studied using simplified experimental systems. Much less is known about the actin cortex, a shell-like structure underneath the membrane that contracts for cell movement. We have designed an experimental system that mimicks the cell cortex by allowing actin polymerization to nucleate and assemble at the inner membrane of a liposome. Actin shell growth can be triggered inside the liposome, which offers a useful system for a controlled study. The observed actin shell thickness and estimated mesh size of the actin structure are in good agreement with cellular data. Such a system paves the way for a thorough characterization of cortical dynamics and mechanics.     

Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation.

Several conditional-lethal mutant alleles of the single-copy Saccharomyces cerevisiae beta-tubulin and actin genes were used to evaluate the roles of microtubules and actin filaments in the pheromone-induced extension of mating projections. Mutants defective in tubulin assembly form projections indistinguishable in appearance from those formed by wild-type cells. However, the tubulin mutants are unable to move their nuclei into the projections and to orient the spindle pole body associated with each nucleus toward the projection tip. Actin mutants are defective in spatial orientation of cell-surface growth required for formation of normal mating projections. Migration of nuclei into mating projections and Spa2p segregation to projection tips are also defective in actin mutants. Studies with abp1 null mutants showed that the function of the Abp1p actin-binding protein is either not required for projection formation or there are other proteins in yeast with similar functions. Our findings demonstrate that actin is required to restrict cell-surface growth to a defined region for pheromone-induced morphogenesis and suggest that nuclear position and orientation in mating projections depend on direct or indirect interaction of microtubules with actin filaments.     

An intracellular actin motor in bacteria?

Actin performs structural as well as motor-like functions in eukaryotic cells. Orthologues of actin have also been identified in bacteria, where they perform an essential function during cell growth. Bacterial actins are implicated in the maintenance of rod-shaped cell morphology, and appear to form a cytoskeletal structure, localising as helical filaments underneath the cell membrane. Recently, a plasmid-borne actin orthologue has been shown to perform a mitotic-like function during segregation of a plasmid, and chromosomally encoded actin proteins were found to play an important role in chromosome segregation. Based on the findings that actin filaments are dynamic structures in two bacterial species, we propose that actins perform motor functions rather than a purely structural role in bacteria. We suggest that an intracellular motor exists in bacteria that could be derived from an ancestral actin motor that was present in cells early in evolution.     

Isolation and characterization of actin and myosin from B-lymphocytic guinea pig leukemia cells.

Actin and myosin have been isolated from a guinea pig B cell leukemia line, L2C. The m.w. and amino acid compositions of these proteins are similar to actin and myosin from other nonmuscle cell types. L2C actin polymerizes to form filaments and activates the ATPase activity of skeletal muscle myosin. Actin in crude lymphocyte extracts does not polymerize as well as predicted from the critical concentration of purified lymphocyte actin suggesting that other factors in lymphocyte extracts regulate actin polymerization. Lymphocyte myosin polymerizes to form synthetic filaments at low ionic strength. Lymphocyte myosin binds to actin, but its ATPase activity is not activated by actin. Possible mechanisms for regulation of the lymphocyte contractile apparatus and its importance in a number of lymphocyte functions are discussed.     

Involvement of actin filaments in rhizoid morphogenesis of Spirogyra

The role of actin filaments in rhizoid morphogenesis was studied in Spirogyra . When the algal filaments were severed, new terminal cells started tip growth and finally formed rhizoids. Actin inhibitors, latrunculin B and cytochalasin D, reversibly inhibited the process. A mesh-like structure of actin filaments (AFs) was formed at the tip region. Gd³⁺ inhibited tip growth and decreased AFs in the tip region. Either a decrease in turgor pressure or lowering of the external Ca²⁺ concentration also induced similar results. It was suggested that the mesh-like AF structure is indispensable for the elongation of rhizoids. A possible organization mechanism of the mesh-like AF structure was discussed.     

New twists in actin–microtubule interactions

Actin filaments and microtubules are cytoskeletal polymers that participate in many vital cell functions including division, morphogenesis, phagocytosis, and motility. Despite the persistent dogma that actin filament and microtubule networks are distinct in localization, structure, and function, a growing body of evidence shows that these elements are choreographed through intricate mechanisms sensitive to either polymer. Many proteins and cellular signals that mediate actin–microtubule interactions have already been identified. However, the impact of these regulators is typically assessed with actin filament or microtubule polymers alone, independent of the other system. Further, unconventional modes and regulators coordinating actin–microtubule interactions are still being discovered. Here we examine several methods of actin–microtubule crosstalk with an emphasis on the molecular links between both polymer systems and their higher-order interactions.     

Dynamic polymorphism of actin as activation mechanism for cell motility

Actin filament dynamics are crucial in cell motility. Actin filaments, and their bundles, networks, and gels assemble and disassemble spontaneously according to thermodynamic rules. These dynamically changing structures of actin are harnessed for some of its functions in cells. The actin systems respond to external signals, forces, or environments by biasing the fluctuation of actin assembly structures. In this study, dynamic conformation of actin molecules was studied by monitoring conformational dynamics of actin molecules at the single molecule level in real time. Actin conformation spontaneously fluctuates between multiple conformational states. Regarding myosin motility, the dynamic equilibrium of actin conformation was interpreted as between states that activates and inhibits the motility. The binding of myosin to actin filaments activates myosin motility by shifting the conformational fluctuation of actin towards the state that activates the motility. Thus, the activation mechanism based on thermal fluctuation is suggested at molecular level as well as at cellular level.     

BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development

The actin cytoskeleton is an important regulator of cell expansion and morphogenesis in plants. However, the molecular mechanisms linking the actin cytoskeleton to these processes remain largely unknown. Here, we report the functional analysis of rice (Oryza sativa) FH5/BENT UPPERMOST INTERNODE1 (BUI1), which encodes a formin-type actin nucleation factor and affects cell expansion and plant morphogenesis in rice. The bui1 mutant displayed pleiotropic phenotypes, including bent uppermost internode, dwarfism, wavy panicle rachis, and enhanced gravitropic response. Cytological observation indicated that the growth defects of bui1 were caused mainly by inhibition of cell expansion. Map-based cloning revealed that BUI1 encodes the class II formin FH5. FH5 contains a phosphatase tensin-like domain at its amino terminus and two highly conserved formin-homology domains, FH1 and FH2. In vitro biochemical analyses indicated that FH5 is capable of nucleating actin assembly from free or profilin-bound monomeric actin. FH5 also interacts with the barbed end of actin filaments and prevents the addition and loss of actin subunits from the same end. Interestingly, the FH2 domain of FH5 could bundle actin filaments directly and stabilize actin filaments in vitro. Consistent with these in vitro biochemical activities of FH5/BUI1, the amount of filamentous actin decreased, and the longitudinal actin cables almost disappeared in bui1 cells. The FH2 or FH1FH2 domains of FH5 could also bind to and bundle microtubules in vitro. Thus, our study identified a rice formin protein that regulates de novo actin nucleation and spatial organization of the actin filaments, which are important for proper cell expansion and rice morphogenesis.     

Studies on the organization and localization of actin and myosin in neurons

The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.     

Cell responses regulated by early reorganization of actin cytoskeleton

Microfilaments exist in a dynamic equilibrium between monomeric and polymerized actin and the ratio of monomers to polymeric forms is influenced by a variety of extracellular stimuli. The polymerization, depolymerization and redistribution of actin filaments are modulated by several actin-binding proteins, which are regulated by upstream signalling molecules. Actin cytoskeleton is involved in diverse cellular functions including migration, ion channels activity, secretion, apoptosis and cell survival. In this review we have outlined the role of actin dynamics in representative cell functions induced by the early response to extracellular stimuli.     

Visualization of Actin Filament Patterns in Pollen Tubes of Hosta caerulea Tratt. with a Non-Fixation and TRITC-Phalloidin Method

Actin filament patterns during pollen germination in Hosta caerulea Tratt. were visualized with a simple method in which there was no pre-fixation, with dimethylsulphoxide (DMSO) as a permeabilising agent and staining with TRITC-Phalloidin. The cytoplasm of the vegetative cell of the ungerminated pollen grain contained numerous crystalline fusiform bodies to constitute a storage form of actin. These bodies were transferred to the emerging pollen tube after the germination of the pollen grain. Following the growth of pollen tube, the fusiform bodies were gradually dissociated, branched, slenderized and formed a cross-linked actin network. During the further growth of the pollen tube, the preponderance of longitudinally-oriented thin actin filaments with some anastomoses to form a more complex network present always in the long pollen tube. This was the typical pattern of actin filaments in most cases. In some conditions, actin filaments were assembled to form thick actin cables near the proximate part of the pollen tube tip. The branching and connecting of the cables were probably also seen in some parts. Actin filaments were always entering to the apical region of a tube tip. The significance of the non-fixation and fluorescence-phalloidin (FI-Ph) method and the problems in the future studies are discussed     

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.     

Evidence of direct interaction between actin and membrane lipids

Actin is a protein component of the cystoskeleton and is involved in cell motility. It is believed generally that actin filaments are attached to the cell membrane through an interaction with membranous actin-binding proteins. By using an in vitro system composed of liposomes and actin, we have shown that actin may also interact directly with the phospholipids of the membrane. Actin deposited at the surface of the liposome is organized in two regular patterns: a paracrystalline sheet of parallel filaments in register, or a netlike organization. These interactions of actin with membrane lipids occur only in the presence of millimolar concentrations of Mg2+. These results suggest that the interaction of the cytoskeleton with the membrane involves, at least in part, a direct association of actin with phospholipids.     

Alpha-actinin and actin in the outer retina: a double immunoelectron microscopic study

Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross-linking protein alpha-actinin. Ultrathin sections of chicken retina were double-immunolabelled with monospecific antibodies against actin and alpha-actinin. The highest relative amount of alpha-actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha-actinin to actin labelling are where the actin filaments are most highly cross-linked into bundles and linked to the plasma membrane by alpha-actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell-cell or cell-substrate adherens junctions.     

Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones

Actin filaments and their interactions with cell surface molecules have key roles in tissue cell behaviour. Axonal pathfinding during embryogenesis, an especially complex cell behaviour, is based on the migration of nerve growth cones. We have used fluorescence immunocytochemistry to examine the distribution in growth cones, their filopodia and lamellipodia of several actin-associated proteins and nerve cell adhesion molecules. The leading margins of chick dorsal root ganglion nerve growth cones and their protrusions stain strongly for f-actin, filamin, alpha-actinin, myosin, tropomyosin, talin and vinculin. MAP2 is absent from DRG growth cones, and staining for spectrin fodrin extends into growth cones, but not along filopodia. Thus, organization of the leading margins of growth cones may strongly resemble the leading lamella of migrating fibroblasts. The adhesion-mediating molecules integrin, L1, N-CAM and A-CAM are all found on DRG neurites and growth cones. However, filopodia stain relatively more strongly for integrin and L1 than for A-CAM or N-CAM. In fact, the 180 X 10(3) Mr form of N-CAM may be absent from most of the length of filopodia. DRG neurones cultured in cytochalasin B display differences in immunofluorescence staining which further emphasize that these adhesion molecules interact differentially with the actin filament system of migrating growth cones. Several models for neuronal morphogenesis emphasize the importance of regulation of the expression of adhesion molecules. Our results support hypotheses that cellular distribution and transmembrane interactions are key elements in the functions of these adhesion molecules during axonal pathfinding.     

用非固定的荧光探针方法观察紫萼花粉管肌动蛋白纤丝的形式

用非固定的、二甲基亚砜作为渗透剂的、异硫氰四甲基罗丹明标记的鬼笔环肽染色方法,观察了紫萼(Hosta caerulea Tratt.)未萌发的花粉粒及不同生长时期的花粉管中的肌动蛋白纤丝的形式。显示未萌发的花粉粒中具有结晶状的梭状体,为肌动蛋白的一种贮藏形式。花粉萌发时,这种梭状体转移到短的花粉管中,逐渐松解、分支和形成肌动蛋白纤丝交错的网络。在花粉管迅速生长和达到一定长度时,肌动蛋白纤丝形成以与花粉管长轴平行的细丝占优势的网络系统,这是在大多数情况中紫萼花粉管典型的肌动蛋白纤丝的形式。在某些条件下,在花粉管接近顶端的前部,肌动蛋白纤丝可集合成长的粗束,这种粗束也常有分支和并合。肌动强白纤丝一直分布到花粉管的末端。讨论了研究肌动蛋白纤丝的非固定方法的重要性和进一步研究花粉管肌动蛋白纤丝值得注意的问题。     

VASP is a processive actin polymerase that requires monomeric actin for barbed end association

Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (K(d) = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (K(off) = 0.69 s(-1)) polymerases that deliver multiple actin monomers per barbed end-binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.     

Actin dynamics is essential for myosin-based transport of membrane organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.