The distribution of cofilin and DNase I in vivo

Actin is the principal component of the cytoskeleton, a structure that can be disassembled and reassembled in a matter of seconds in vivo. The state of assembly of actin in vivo is primarily regulated by one or more actin binding proteins (ABPs). Typically, the actions of ABPs have been studied one by one, however, we propose that multiple ABPs, acting cooperatively, may be involved in the control of actin filament length. Cofilin and DNase I are two ABPs that have previously been demonstrated to form a ternary complex with actin in vitro. This is the first report to demonstrate their co-localisation in vivo, and differences in their distributions. Our observations strongly suggest a physiological role for higher order complexes of actin in regulation of cytoskeletal assembly during processes such as cell division.     

The distribution of cofilin and DNase I in vivo

Actin is the principal component of the cytoskeleton,a structure that can be disassembled and reassem-bled in a matter of seconds in vivo.The state of assembly of actin vivo is primarily regulated by one or more actin binding proteins(ABPs).Typically,the actions of ABPs have been studied one by one,however,we propose that multiple ABPs ,acting cooperatively,may be involved in the control of actin filament length.Cofilin and Dnase Iare two ABPs that have previously been demonstrated to form a ternary complex with actin in vitro.This is the first report to demonstrate their co-localisation in vivo,and differences in their distributions.Our observations strongly suggest a physiological role for higher order complexes of actin in regulation of cytoskeletal assembly during processes such as cell division.     

Characterization of MR-1, a novel myofibrillogenesis regulator in human muscle

Myofibrils are components of both cardiac and skeletalmuscle cells. Myofibrillogenesis is a highly complexprocess that involves the expression and assembly ofmuscle proteins into sarcomeres [1]. The key componentsof sarcomeres are two filamentary proteins, actin andmyosin. Sarcomeric myosin is the molecular motor thattransduces energy from the hydrolysis of ATP into directedmovement and drives sarcomere shortening and musclecontraction. Each myosin hexamer is composed of twoheavy chains, two…     

Apoptosis commitment--translating survival signals into decisions on mitochondria

Most defective and unwanted cells die by apoptosis, cells without damaging the surrounding tissue. Once a an exquisitely controlled genetic programme for removing such cell has committed to apoptosis, the process is remarkably efficient, and is completed within a few minutes of initiation. This point of no retum for an apoptotic cell is commonly held to be the point at which the outer mitochondrial membrane is permeabilised, a process regulated by the Bcl-2 family of proteins. How these proteins regulate this decision point is central to diseases such as cancer where apoptotic control is lost. In this review, we will discuss apoptotic signalling and how a cell makes the irreversible decision to die. We will focus on one set of survival signals, those derived by cell adhesion to the extracellular matrix （ECM）, and use these to highlight the complexities of apoptotic signalling. In particular, we will illustrate how multiple signalling pathways converge to determine critical cell fate decisions.     

Plant villins:Versatile actin regulatory proteins

Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology.Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Villins contain six gelsolin homology domains(G1–G6) and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a de fined role in modifying actin dynamics in the pollen Invitube; most of their in vivo activities remain to be ascertained.Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review,we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understanding of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.     

Regulation of Actin Dynamics in Pollen Tubes： Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

Dynamic Actin Controls Polarity Induction de novo in Protoplasts

Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.     

Entry of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus(SFTSV) is a globe-shaped virus covered by a dense icosahedral array of glycoproteins Gn/Gc that mediate the attachment of the virus to host cells and the fusion of viral and cellular membranes. Several membrane factors are involved in virus entry, including C-type lectins and nonmuscle myosin heavy chain ⅡA. The post-fusion crystal structure of the Gc protein suggests that it is a class Ⅱ membrane fusion protein, similar to the E/E1 protein of flaviviruses and alphaviruses. The virus particles are internalized into host cell endosomes through the clathrin-dependent pathway, where the low pH activates the fusion of the virus with the cell membrane. With information from studies on other bunyaviruses, herein we will review our knowledge of the entry process of SFTSV.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca^2＋ （[Ca^2＋]cyt） is an ubiquitous second messenger in plant cell signaling, and [Ca^2＋]cyt elevation is associated with Ca^2＋-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca^2＋ channels and their regulation remains limited in planta. A type of voltage- dependent Ca^2＋-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba^2＋ and Ca^2＋, and their activities can be inhibited by micromolar Gd^3＋. The unitary conductance and the reversal potential of the channels depend on the Ca^2＋ or Ba^2＋ gradients across the plasma membrane. The inward whole-cell Ca^2＋ （Ba^2＋） current, as well as the unitary current amplitude and NPo of the single Ca^2＋ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

Isolation of microvillar microfilaments and associated transmembrane complex from ascites tumor cell microvilli

The association of microvillar microfilaments with the microvillar membrane actin-containing transmembrane complex of MAT-C1 13762 ascites tumor cell microvilli has been investigated by differential centrifugation, gel electrophoresis and electron microscopy of detergent extracts of the isolated microvilli. Several methods have been used to reduce breakdown and solubilization of the microfilament core actin during the detergent extractions for preparation of microvillar core microfilaments. Gel electrophoresis of differential centrifugation fractions demonstrated that over 70% of the total microvillus actin could be pelleted with microfilament cores at 10 000 g under extraction conditions which reduce filament breakdown. Transmission electron microscopy (TEM) of all of the core preparations showed arrays of microfilaments and small microfilament bundles. The major protein components of the microfilament cores, observed by sodium dodecyl sulfate (SDS) electrophoresis, were actin and alpha-actinin. Among the less prominent polypeptide components was a 58 000 Dalton polypeptide (58 K), previously identified as a member of the MAT-Cl transmembrane complex. This three-component complex contains, in addition to 58 K, actin associated directly and stably with a cell surface glycoprotein (Carraway, CAC, Jung, G & Carraway, K L, Proc. natl acad. sci. US 80 (1983) 430). Evidence that the apparent association of complex with the microfilament core was not due simply to co-sedimentation was provided by myosin affinity precipitation. These results provide further evidence that the transmembrane complex is a site for the interaction of microfilaments with the microvillar plasma membrane.     

Actin microfilament dynamics and actin side-binding proteins in plants

Actin microfilaments are highly organized and essential intracellular components of organelle movement and cell morphogenesis in plants. The organization of these microfilaments undergoes dynamic changes during cell division, elongation, and differentiation. Recent live-cell imaging of plant actin microfilaments has revealed their native organization and remarkable dynamics. In addition, characterization of plant actin side-binding proteins has progressed rapidly by genetic, biochemical, and bioinformatic approaches. The gathering and integration of microscopy-based information from actin microfilament dynamics and the molecular identification of actin side-binding proteins have provided considerable insights into actin microfilament-dependent events and actin microfilament organization in plants.     

Specific Localization of Serine 19 Phosphorylated Myosin II during Cell Locomotion and Mitosis of Cultured Cells

Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells. To examine how such phosphorylation is regulated in space and time within cells during coordinated cell movements, including cell locomotion and cell division, we generated a phosphorylation-specific antibody.

Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated myosin along the direction of cell movement. The level of myosin phosphorylation is high in an anterior region near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involved in cell locomotion. Phosphorylated myosin is also concentrated in cortical microfilament bundles, indicating that cortical filaments are under tension. The enrichment of phosphorylated myosin in the moving edge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated myosin. On the other hand, the phosphorylation level of circumferential microfilament bundles in cell–cell contacts is low. These observations suggest that peripheral microfilaments at the edge are involved in force production to drive the cell margin forward while microfilaments in cell–cell contacts play a structural role. During cell division, both fibroblastic and epithelial cells exhibit an increased level of myosin phosphorylation upon cytokinesis, which is consistent with our previous biochemical study (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129–137). In the case of the NRK epithelial cells, phosphorylated myosin first appears in the midzones of the separating chromosomes during late anaphase, but apparently before the formation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis.

     

考虑生化刺激的细胞分裂的力学模型

为了解释动物细胞胞质分裂的力学机理 ,基于大量的细胞卵裂实验数据 ,在Zinemanas和Nir的流体动力学模型基础上 ,对微丝的局部集中函数改为随同质膜移动 ,增加了由于生化刺激引起主动微丝的影响系数。数值计算表明 :此模型能较好的预测细胞在胞质分裂过程中 ,细胞的总体和局部变形 ,以及卵裂沟处的张力和细胞内压。     

Electron microscopic localization of myosin II and ABP-120 in the cortical actin matrix of Dictyostelium amoebae using IgG-gold conjugates

To narrow the field of possible functions of an actin-binding protein (ABP-120) and myosin II, we have used high resolution immunocytochemistry with IgG-colloidal gold conjugates to identify the types of actin containing structures with which these proteins are associated in the isolated cell cortex. Staining for myosin II and ABP-120 is associated with distinct regions of the actin cytoskeleton in isolated cortices. Myosin II is localized to lateral arrays of filaments, where it is clustered and has a density that is unrelated to distance from the plasma membrane. Staining for myosin II is associated also with unidentified cytoplasmic vesicles. However, staining for ABP-120 is concentrated in dense networks of branched microfilaments that are adjacent to the plasma membrane or in surface projections (residual pseudopods and lamellopods). These results are consistent with a role for ABP-120 in the formation of filament networks in vivo and further suggest that networks of branched microfilaments are unlikely to participate in motility that is mediated by myosin II.     

Changes in the organization of actin and myosin in non-muscle cells induced by N-ethylmaleimide

There is evidence from in vitro studies that the SH reagent N-ethylmaleimide (NEM) causes the formation of ATP-resistant rigor-complexes between actin and myosin, and NEM-modified heavy meromyosin has been used by Cande et al. to study the contractile process during cytokinesis. It is reported here that treatment of tissue-cultured cells with NEM causes an immediate cessation of all motile activities and a simultaneous stabilization of the ultrastructure of the cell visualized on lysis with detergent-containing buffers. After NEM treatment a 5- to 10-fold increase in the amount of myosin was found associated with the detergent-resistant cell residues. As suggested by immunoelectron microscopy, using antibodies to non-muscle myosin together with gold-labelled protein A, increasing amounts of myosin filaments became associated with the microfilament assemblies of the cell with time of NEM treatment. In addition to this there was a slow, progressive reorganization of the cortical wave of microfilaments. The structures interpreted as myosin filaments were visualized at relatively high resolution. The immunoelectron microscopy finally also indicated the presence of a non-filamentous form of myosin in agreement with the results of others.     

Cortical actin turnover during cytokinesis requires myosin II

The involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling.     

Microfilament-associated proteins in tissue culture cells viewed by stereo immunofluorescence microscopy.

Stereo immunofluorescence microscopy avoids the problem of juxtaposition of structures often encountered in normal fluorescence microscopy. The procedure has been used in conjunction with antibodies against microfilament associated proteins to reveal the arrangement of microfilaments in a rat mammary cell line both in the fully spread state and in cells during the process of spreading on the substratum. use of antibodies to myosin, tropomyosin, alpha-actinin and filamin emphasizes that at early times during the spreading process these proteins are abundantly present underneath the upper plasma membrane, suggesting that the cortical layer present underneath this membrane may be contractile. In addition the results emphasize that even in well spread cells microfilament bundles are expressed both above and below the nucleus, in agreement with the assumption that microfilaments may form a supporting layer underneath the plasma membrane.     

The mechanism of matrix vesicle formation. Studies on the composition of chondrocyte microvilli and on the effects of microfilament-perturbing agents on cellular vesiculation

The mechanism of matrix vesicle (MV) formation by growth plate chondrocytes in primary cell culture was assessed both by using drugs which interfere with assembly or disassembly of microfilaments and microtubules, as well as by comparison of the composition of chondrocyte microvilli with MV. Cytochalasin D, which is known to inhibit assembly of actin microfilaments, was found to stimulate the release of alkaline phosphatase-rich MV. This stimulatory effect was confirmed by studies with [3H]palmitate- and 32P-prelabeled cells which showed that cytochalasin D enhanced the release of labeled MV. In contrast, phalloidin, which blocks disassembly of microfilaments, suppressed release of cellular alkaline phosphatase into MV. The phospholipid composition of vesicles released by cells treated with cytochalasin D and phalloidin was virtually identical with that of the controls. In contrast, colchicine, which interferes with the assembly of microtubules, was found to cause fragmentation of the cells, producing large vesicles significantly different in lipid composition from MV. Microscopic studies revealed that cytochalasin D caused marked rounding and retraction of the cells, with evidence of actin withdrawal from the cell periphery. This led to cell surface blebbing and formation of small zeiotic bodies at the tips of cell processes. In contrast, phalloidin enhanced and stabilized the actin network within the cells. Chemical analysis of microvilli prepared from isolated chondrocytes revealed high levels of alkaline phosphatase and a phospholipid composition almost identical to MV. Electrophoretic profiles of microvillar proteins were again like that of MV, except for the presence of high levels of actin. This cytoskeletal protein was nondetectable in MV. Taken together with the effects of the drugs, the data indicate that cell surface microvilli are the precursors of MV and that retraction of the supporting microfilament network is essential for the release of these structures.     

Sequential rearrangement and nuclear polymerization of actin in baculovirus-infected Spodoptera frugiperda cells.

Proper assembly of nucleocapsids of the baculovirus Autographa californica nuclear polyhedrosis virus is prevented by cytochalasin D, a drug that interferes with actin microfilament function. To investigate the involvement of microfilaments in A. californica nuclear polyhedrosis virus replication, a fluorescence microscopy study was conducted that correlated changes in distribution of microfilaments with events in the life cycle of the virus. Tetramethylrhodamine isothiocyanate-labeled phalloidin was used to label microfilaments, and monoclonal antibody was used to label p39, the major viral capsid protein. Three microfilament arrangements were found in infected cells. During uptake of virus, thick cables were formed. These were insensitive to cycloheximide, indicating that this configuration was a rearrangement of preexisting cellular actin mediated by a component of the viral inoculum. At the time of cell rounding and before viral DNA replication, ventral aggregates of actin were observed. These were sensitive to cycloheximide but not to aphidicolin, indicating that an early viral gene mediated this actin rearrangement. Ventral aggregates did not result from the rounding process itself. Uninfected cells prerounded with colchicine did not form ventral aggregates. Cells prerounded with colchicine and then infected did form aggregates. At the time of exponential production of progency virus, microfilaments were found in the nucleus surrounding the virogenic stroma. In this area (where nucleocapsid assembly is known to take place) microfilaments colocalized with p39. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis identified p39 among proteins retained on an f-actin affinity column. We postulate that microfilaments in the nucleus provide a scaffold to position capsids for proper assembly and filling with DNA.