Actin cytoskeleton in plants: from transport networks to signaling networks.

The plant actin cytoskeleton is characterized by a high diversity in regard to gene families, isoforms, and degree of polymerization. In addition to the most abundant F-actin assemblies like filaments and their bundles, G-actin obviously assembles in the form of actin oligomers composed of a few actin molecules which can be extensively cross-linked into complex dynamic meshworks. The role of the actomyosin complex as a force generating system - based on principles operating as in muscle cells - is clearly established for long-range mass transport in large algal cells and specialized cell types of higher plants. Extended F-actin networks, mainly composed of F-actin bundles, are the structural basis for this cytoplasmic streaming of high velocities On the other hand, evidence is accumulating that delicate meshworks built of short F-actin oligomers are critical for events occurring at the plasma membrane, e.g., actin interventions into activities of ion channels and hormone carriers, signaling pathways based on phospholipids, and exo- and endocytotic processes. These unique F-actin arrays, constructed by polymerization-depolymerization processes propelled via synergistic actions of actin-binding proteins such as profilin and actin depolymerizing factor (ADF)/cofilin are supposed to be engaged in diverse aspects of plant morphogenesis. Finally, rapid rearrangements of F-actin meshworks interconnecting endocellular membranes turn out to be especially important for perception-signaling purposes of plant cells, e.g., in association with guard cell movements, mechano- and gravity-sensing, plant host-pathogen interactions, and wound-healing.     

The roles of focal adhesion and cytoskeleton systems in fluid shear stress-induced endothelial cell response

Focal adhesions are polyproteins linked to extracellular matrix and cytoskeleton, which play an important rolein the process of transforming force signals into intracellular chemical signals and subsequently triggering relatedphysiological or pathological reactions. The cytoskeleton is a network of protein fibers in the cytoplasm, which iscomposed of microfilaments, microtubules, intermediate filaments, and cross-linked proteins. It is a very importantstructure for cells to maintain their basic morphology. This review summarizes the process of fluid shear stresstransduction mediated by focal adhesion and the key role of the cytoskeleton in this process, which focuses on thefocal adhesion and cytoskeleton systems. The important proteins involved in signal transduction in focal adhesion areintroduced emphatically. The relationship between focal adhesion and mechanical transduction pathways arediscussed. In this review, we discuss the relationship between fluid shear stress and associated diseases such asatherosclerosis, as well as its role in clinical research and drug development.     

New anti-actin drugs in the study of the organization and function of the actin cytoskeleton.

The high degree of structural and molecular complexity of the actin-based cytoskeleton, combined with its ability to reorganize rapidly and locally in response to stimuli, and its force-generating properties, have made it difficult to assess how the different actin structures are assembled in cells, and how they regulate cell behavior. An obvious approach to study the relationships between actin organization, dynamics, and functions is the specific perturbation of actin structures using pharmacological means. Until recently there were only a few agents available that interfered with cellular activities by binding to actin and most of our knowledge concerning the involvement of actin in basic cellular processes was based on the extensive use of the cytochalasins. In recent years we have identified an increasing number of actin-targeted marine natural products, including the latrunculins, jasplakinolides (jaspamides), swinholide A, misakinolide A, halichondramides, and pectenotoxin II, which are discussed in this article. All these marine-sponge-derived compounds are unusual macrolides and can be classified into several major families, each with its own distinct chemical structures. We describe the current state of knowledge concerning the actin-binding properties of these compounds and show that each class of drugs alters the distribution patterns of actin in a unique way, and that even within a chemical class, structurally similar compounds can have different biochemical properties and cellular effects. We also discuss the effects of these new drugs on fenestrae formation in liver endothelial cells as an example of their usefulness as powerful tools to selectively unmask actin-mediated dynamic processes.     

Structural organization of the cytoskeleton in SV40 human corneal epithelial cells cultured on nano- and microscale grooves

The basement membrane of human corneal epithelial cells (HCECs) has a three-dimensional nanoscale architecture, which includes pores, bumps and fibers that may influence cell-substrate adhesion and spreading in the overlying cells. We previously demonstrated that nano- and microscale groove and ridge patterns influence the morphological response and the adhesive response of HCECs to a nominal wall shear stress. Cell-substrate adhesion is mediated by adhesion receptors that bind to extracellular matrix components and anchor the cytoskeleton (CSK) of cells to extracellular elements. Here we investigate the CSK organization in SV40-transformed HCECs grown on nano- and microscale groove and ridge patterns. X-ray lithography was used to fabricate uniform groove and ridge patterns with features ranging in size from 200 nm to 2 microm grooves. Scanning electron microscopy and transmission electron microscopy were used to investigate CSK structure and the distribution of -beta1 integrin adhesion receptors. CSK elements aligned with the patterns; however, the spatial organization of these elements was influenced by feature size. Larger CSK bundles lay on top of the ridges and ran parallel to the patterns, whereas smaller CSK bundles, whose width was proportional to the groove size, spanned the grooves. -Beta1 integrins co-localized with the CSK and had a higher density at the poles of aligned spindle-shaped cells. Differences in organization seen on the different topographical feature sizes may be indicative of differences in extracellular matrix organization. This may explain, in part, previous observations regarding the dependence of cell adhesive responses on the size of topographic features in the substrate.     

Role of the actin cytoskeleton in insulin action.

Insulin has diverse effects on cells, including stimulation of glucose transport, gene expression, and alterations of cell morphology. The hormone mediates these effects by activation of signaling pathways which utilize, 1) adaptor molecules such as the insulin receptor substrates (IRS), the Src and collagen homologs (Shc), and the growth factor receptor binding protein 2 (Grb2); 2) lipid kinases such as phosphatidylinositol 3-kinase (PI 3-Kinase); 3) small G proteins; and 4) serine, threonine, and tyrosine kinases. The activation of such signaling molecules by insulin is now well established, but we do not yet fully understand the mechanisms integrating these seemingly diverse pathways. Here, we discuss the involvement of the actin cytoskeleton in the propagation and regulation of insulin signals. In muscle cells in culture, insulin induces a rapid actin filament reorganization that coincides with plasma membrane ruffling and intense accumulation of pinocytotic vesicles. Initiation of these effects of insulin requires an intact actin cytoskeleton and activation of PI 3-kinase. We observed recruitment PI 3-kinase subunits and glucose transporter proteins to regions of reorganized actin. In both muscle and adipose cells, actin disassembly inhibited early insulin-induced events such as recruitment of glucose transporters to the cell surface and enhanced glucose transport. Additionally, actin disassembly inhibited more prolonged effects of insulin, including DNA synthesis and expression of immediate early genes such as c-fos. Intact actin filaments appear to be essential for mediation of early events such as association of Shc with Grb2 in response to insulin, which leads to stimulation of gene expression. Preliminary observations support a role for focal adhesion signaling complexes in insulin action. These observations suggest that the actin cytoskeleton facilitates propagation of the morphological, metabolic, and nuclear effects of insulin by regulating proper subcellular distribution of signaling molecules that participate in the insulin signaling pathway.     

激光共聚焦显微成像技术在植物细胞微丝骨架三维动态观察中的应用

激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)是目前生物学领域应用最广泛、分辨率高的仪器。可用于细胞内形态结构观察及三维重建、组分空间定位、实时动态变化监测等研究,图像分析软件还能提供荧光强度、空间距离定量测定的丰富信息。本文以携带GFP融合拟南芥丝束蛋白1(AtFIM1)的肌动蛋白结合结构域2(fABD2)基因的B Y-2转基因细胞系为材料,运用LSCM技术观察到间期细胞的网络状微丝结构并重构出胞内微丝的三维网络结构;实时动态监测细胞有丝分裂过程中微丝骨架的动态变化;通过细胞内荧光强度的分布直观地看出BY-2细胞胞质分裂过程中微丝骨架的动态变化。这些结果显示出LSCM在研究植物细胞微丝骨架的三维网络动态结构及图像荧光强度分析与统计方面的优越性。     

Role of the cytoskeleton during early development.

Oocytes, eggs, and embryos from a diverse array of species have evolved cytoskeletal specializations which allow them to meet the needs of early embryogenesis. While each species studied possesses one or more specializations which are unique, several cytoskeletal features are widely conserved across different animal phyla. These features include highly-developed cortical cytoskeletal domains associated with developmental information, microtubule-mediated pronuclear transport, and rapid intracellular signal-regulated control of cytoskeletal organization.     

Relationships between the actin cytoskeleton and cell volume regulation.

The actin cytoskeleton mediates a variety of essential biological functions in cells, including division, shape changes, and movement. A number of studies have suggested that the abundant submembranous actin cytoskeleton present in the cortex of many cell types is involved in the regulation of cell volume. This relationship is supported by numerous works which document the changes in the structural organization of the actin cytoskeleton which accompany cell volume changes and the F-actin-dependence of the regulatory volume responses. In addition, other studies demonstrate structural and functional relationships between the actin cytoskeleton and the membrane transporters known to be involved in cell volume homeostasis. This review provides a summary of the current level of knowledge in this area and discusses the mechanisms which may underlie the linkage between the actin cytoskeleton and cell volume regulation.     

Optimization of adhesion mode atomic force microscopy resolves individual molecules in topography and adhesion.

The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force-distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force-distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60 x 60 nm2.     

The cytoskeleton in fish melanophore melanosome positioning

Melanophore melanosomes organelles can be regulated to move and locate correspondingly to many other different organelle types. Comparing lessons from analysis of a specific melanosome distribution can, therefore, contribute to the understanding of distribution of other organelles, and vice versa. From such data, it is now generally accepted that microtubules provide directed long-distance movement, while cell peripheral movements include microfilaments. In fish melanophores, both actin and dynein exhibit counter-forces to the kinesin-like protein in maintaining the evenly dispersed state, while actin and kinesin exhibit counter-forces to dynein in many other systems. Lessons from elevating cAMP levels indicate the presence of a peripheral feedback regulatory system involved in maintaining the evenly dispersed state. Studies from dynein inhibition suggest that the kinesin-like protein involved in fish melanosome dispersal is regulated in contrast to many other systems. One would further expect melanosome transport to be regulated also on actin/myosin, in order to prevent actin-dependent capture of melanosomes during the microtubule-dependent aggregation and dispersion. General findings will be discussed in comparison with positioning and movement of other organelle types in cells. Finally, recent data on melanosome-dependent organising of microtubules show that dynein is involved in nucleating microtubules extending from melanosome aggregates in melanophore fragments.     

实验室建设与管理的新思路

由于我国医疗器械的发展,对相关的医疗器械质量监督检验所提出更高的要求,为此,结合医疗器械质量监督检验所的发展,从实验室组建、机构设置和实验室管理提出新的思路,供同行参考。     

The cytoskeleton in plant and fungal cell tip growth

Tip-growing cells have a particular lifestyle that is characterized by the following features: (1) the cells grow in one direction, forming a cylindrical tube; (2) tip-growing cells are able to penetrate their growth environment, thus having to withstand considerable external forces; (3) the growth velocity of tip-growing cells is among the fastest in biological systems. Tip-growing cells therefore appear to be a system well suited to investigating growth processes. The cytoskeleton plays an important role in cell growth in general, which is why tip-growing cells provide an excellent model system for studying this aspect. The cytoskeletal system comprises structural elements, such as actin filaments and microtubules, as well as proteins that link these elements, control their configuration or are responsible for transport processes using the structural elements as tracks. Common aspects as well as differences in configuration and function of the cytoskeleton in various types of tip-growing cells reveal the general principles that govern the relationship between the cytoskeleton and cell growth.     

刀具化学镀结合力研究

通过对刀具化学镀中影响镀层与基体问结合力的因素研究和讨论，提出了相应的改进方法，并总结出了一些具体的试验工艺。     

Organization and functional roles of the cytoskeleton in oligodendrocytes

Mature oligodendrocytes are characterized by their numerous cytoplasmic extensions and flat membranous sheets. These sheets contain an extensive cytoskeletal network of microtubules (MTs) that maintain the cellular morphology, are specifically important for cellular sorting, and provide the rails for organelle trafficking. Mitochondria are localized in the primary and secondary processes and follow the tracks of the MTs in the cytoplasmic extensions. Oligodendrocytes express microtubule associated proteins (MAPs), specifically MAP2 and tau, which might be involved in the regulation and stabilization of the dynamic MT network in the myelin-containing cellular processes. Tau and MAP2 heterogeneity increases during oligodendroglia maturation, and in mature oligodendrocytes tau mRNA with four MT binding domains are more prominent than in progenitor cells. Filamentous cell inclusions are a unifying mechanism underlying a variety of late-onset neurodegenerative disorders and have mainly been viewed as neuron-specific. Recent evidence indicated that glial changes occur in CNS degenerative diseases and seem to be a more common feature than previously thought. Glial fibrillary tangles (GFTs) in oligodendrocytes were observed in familial multiple system tauopathy, and glial cytoplasmic inclusions (GCIs) and oligodendroglia degeneration are the histological hallmark of multiple system atrophy (MSA). GCIs are associated with MTs and contain stress proteins and MAPs. Thus, neurons and glial cells share common cytoskeletal pathologies. During health and disease, MAPs might be important regulators of the structural stability and plasticity of the oligodendroglia cytoskeleton.     

Dictyostelium as model system for studies of the actin cytoskeleton by molecular genetics.

The actin cytoskeleton is an essential structure for most movements at the cellular and intracellular level. Whereas for contraction a muscle cell requires a rather static organisation of cytoskeletal proteins, cell motility of amoeboid cells relies on a tremendously dynamic turnover of filamentous networks in a matter of seconds and at distinct regions inside the cell. The best model system for studying cell motility is Dictyostelium discoideum. The cells live as single amoebae but can also start a developmental program that leads to multicellular stages and differentiation into simple types of tissues. Thus, cell motility can be studied on single cells and on cells in a tissue-like aggregate. The ability to combine protein purification and biochemistry with fairly easy molecular genetics is a unique feature for investigation of the cytoskeleton and cell motility. The actin cytoskeleton in Dictyostelium harbours essentially all classes of actin-binding proteins that have been found throughout eukaryotes. By conventional mutagenesis, gene disruption, antisense approaches, or gene replacements many genes that code for cytoskeletal proteins have been disrupted, and altered phenotypes in transformants that lacked one or more of those cytoskeletal proteins allowed solid conclusions about their in vivo function. In addition, tagging the proteins or selected domains with green fluorescent protein allows the monitoring of protein redistribution during cell movement. Gene tagging by restriction enzyme mediated integration of vectors and the ongoing international genome and cDNA sequencing projects offer the chance to understand the dynamics of the cytoskeleton by identification and functional characterisation of all proteins involved.