Homocysteine Induces Hypophosphorylation of Intermediate Filaments and Reorganization of Actin Cytoskeleton in C6 Glioma Cells

In this study, we investigated the actions of high homocysteine (Hcy) levels (100 and 500 μM) on the cytoskeleton of C6 glioma cells. Results showed that the predominant cytoskeletal response was massive formation of actin-containing filopodia at the cell surface that could be related with Cdc42 activation and increased vinculin immunocontent. In cells treated with 100 μM Hcy, folic acid, trolox, and ascorbic acid, totally prevented filopodia formation, while filopodia induced by 500 μM Hcy were prevented by ascorbic acid and attenuated by folic acid and trolox. Moreover, competitive NMDA ionotropic antagonist DL-AP5 totally prevented the formation of filopodia in both 100 and 500 μM Hcy treated cells, while the metabotropic non-selective group I/II antagonist MCPG prevented the effect of 100 μM Hcy but only slightly attenuated the effect induced by of 500 μM Hcy on actin cytoskeleton. The competitive non-NMDA ionotropic antagonist CNQX was not able to prevent the effects of Hcy on the reorganization of actin cytoskeleton in the two concentrations used. Also, Hcy-induced hypophosphorylation of vimentin and glial fibrillary acidic protein (GFAP) and this effect was prevented by DL-AP5, MCPG, and CNQX. In conclusion, our results show that Hcy target the cytoskeleton of C6 cells probably by excitoxicity and/or oxidative stress mechanisms. Therefore, we could propose that the dynamic restructuring of the actin cytoskeleton of glial cells might contribute to the response to the injury provoked by elevated Hcy levels in brain.     

Actin in Growing Nerve Cells

AXONAL growth from developing and regenerating nerve cells is a very rapid process. The growth rate is often as high as 50 µm/h¹ and the axon that emerges from a cell less than 0.1 mm in diameter can have a final length of more than a metre. Such a vigorous process, occurring in a non-dividing cell, will probably dominate the biosynthetic activity of the neurone and it is likely that proteins made in large amounts will be major components of the growing fibres. For this reason we have examined the proteins synthesized by growing neurones in the hope that there would be components we could identify and, perhaps, to which we could assign a role in neurite assembly. We have used explanted neurones growing in tissue culture because they can be obtained largely free of other cell types; and we have chosen embryonic sympathetic neurones because of the extremely rapid outgrowth of processes which can be induced by addition of the nerve growth factor (NGF)².     

CfGST转基因拟南芥抗寒性和叶肉细胞超微结构研究

CfGST基因克隆于加拿大森林害虫云杉蚜虫的幼虫基因组.研究CfGST转基因拟南芥表型特征,以及低温条件下叶肉细胞超微结构与存活率.结果表明,与野生型拟南芥相比,CfGST转基因拟南芥的茎粗、叶宽,植株高度、分枝数、荚果数皆降低,生长速度减慢.低温(5±1)℃处理后,转基因拟南芥叶片的叶绿体和线粒体膜结构完整清晰,存活率提高25.14%.     

The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning

In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1–PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.Plants constantly monitor the properties of light in their natural environment to optimize light capture for photosynthesis and growth (e.g. shade avoidance and phototropism) and to time important developmental transitions (e.g. germination and flowering; Neff et al., 2000; Briggs and Christie, 2002; Franklin and Whitelam, 2005). To do so, plants have a multitude of photoreceptors that allow them to sense changes in light period, direction, wavelength composition, and intensity. The main types of photoreceptors are the red/far-red light-absorbing phytochromes and the UV-A/blue light-sensing phototropins, cryptochromes, and Zeitlupe protein families (Chen et al., 2004; Jiao et al., 2007; Demarsy and Fankhauser, 2009). The signaling pathways triggered by these photoreceptors are integrated to fine-tune responses to ever-changing light environments (Casal, 2000; Franklin and Whitelam, 2004; Iino, 2006).In Arabidopsis (Arabidopsis thaliana), phototropin1 (phot1) and its paralog phot2 were discovered as primary photoreceptors for blue light-induced hypocotyl phototropism and for high light-induced chloroplast avoidance movements, respectively (Liscum and Briggs, 1995; Huala et al., 1997; Jarillo et al., 2001; Kagawa et al., 2001). Subsequent studies have shown that phototropins regulate a wide set of physiological and developmental responses, including chloroplast accumulation under low light, stomatal opening, leaf flattening, and phototropism of the root, inflorescence stem, and petiole (Sakai et al., 2001). Thus, phototropins are proposed to optimize the photosynthetic potential of plants, particularly under unfavorable environments such as extremely high light, weak illumination, and drought (Kasahara et al., 2002; Takemiya et al., 2005; Galen et al., 2007).Phot1 and phot2 regulate these processes selectively and in a fluence-dependent manner. Phot1 mediates the chloroplast accumulation, leaf positioning, and phototropic responses under very low light (Demarsy and Fankhauser, 2009). Under higher light intensities, the phot2 pathway becomes activated and acts redundantly with phot1 in these processes (Sakai et al., 2001). Phot2 also specifically controls the chloroplast avoidance response induced by high light (Jarillo et al., 2001; Kagawa et al., 2001). For stomatal opening, phot1 and phot2 act redundantly over a broad range of light intensity (Kinoshita et al., 2001; Doi et al., 2004).Phototropins are Ser/Thr kinases belonging to the AGC family (for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and phospholipids-dependent protein kinase C; Bogre et al., 2003). Two LOV (for light, oxygen, or voltage) photosensory domains that bind to the blue light-absorbing chromophore FMN regulate the kinase activity (Christie, 2007). Phototropin activation and early signaling events at the level of the photoreceptor itself have been extensively studied (Tokutomi et al., 2008; Demarsy and Fankhauser, 2009). However, downstream signaling is less well understood. Light-induced phot1 autophosphorylation has recently been shown to be an essential signaling event, but apart from the photoreceptor itself, no direct substrate for the kinase activity has been identified in planta (Inoue et al., 2008b; Sullivan et al., 2008). Nonetheless, several proteins are known to interact with phot1. These include Broad-Complex, Tramtrack, Bric-à-Brac (BTB) proteins belonging to the 33-member NONPHOTOTROPIC HYPOCOTYL3 (NPH3)/ROOT PHOTOTROPISM2-LIKE (NRL) subfamily, 14-3-3 proteins, and ADP-ribosylation factors (members of the Ras superfamily of GTP-binding proteins that play important roles in the assembly and disassembly of coat proteins associated with driving vesicle budding and fusion; Motchoulski and Liscum, 1999; Sullivan et al., 2009).Genetic experiments showed that NPH3 is required for phot1- and phot2-mediated phototropism and for phot1-controlled leaf positioning but is not involved in stomatal opening or chloroplast movements (Inada et al., 2004; Inoue et al., 2008a). In addition, RPT2 acts in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. RPT2 can associate with phot1 in vitro and in vivo, but there is no evidence for a direct interaction with phot2 (Inada et al., 2004). NPH3 is also known to interact with phot1 in vivo, but an interaction with phot2 has not been reported (Motchoulski and Liscum, 1999; Lariguet et al., 2006). Thus, phototropin signaling is believed to branch quickly, and phot1 and phot2 appear to recruit different signaling components to trigger distinct physiological processes. NPH3 and RPT2 are proposed to mediate protein scaffolding using their protein-protein interaction domains (BTB/Pox virus and Zinc finger as well as coiled coil) and by these means may provide signaling specificity via interaction with specific targets in different tissues and subcellular compartments (Celaya and Liscum, 2005). The phototropins may regulate such interactions by modifying the phosphorylation status of the signaling protein (e.g. NPH3 and 14-3-3 proteins; Pedmale and Liscum, 2007; Sullivan et al., 2009).The nature of phototropin-controlled responses is diverse. On the one hand, chloroplast movements and stomatal opening are rapid, cell-autonomous, and reversible processes. On the other hand, phototropic responses and leaf flattening are slower symmetric growth processes coordinated by cell expansion and division. Such growth coordination is under tight hormonal regulation, and the hormone auxin is a central regulator of phototropism (Holland et al., 2009), leaf flattening (Keller and Van Volkenburgh, 1997; Li et al., 2007; Bainbridge et al., 2008; Braun et al., 2008), and leaf positioning (Tao et al., 2008; Millenaar et al., 2009). An important task is to identify points of convergence between phototropin signaling and auxin signaling. Hypocotyl phototropism is triggered by blue light-induced auxin redistribution and signaling across the organ (Esmon et al., 2006; Holland et al., 2009). Recent reports suggest that the phototropins achieve this by directly regulating the activity of auxin transporters. First, the three main classes of auxin transporters (AUXIN RESISTANT1 [AUX1]/LIKE AUX1, PIN-FORMED [PIN], and P-glycoproteins [PGP]) are involved in the regulation of phototropism (Friml et al., 2002; Noh et al., 2003; Blakeslee et al., 2004; Nagashima et al., 2008; Stone et al., 2008). Second, phot1 is required for the relocalization of PIN1 upon blue light exposure (Blakeslee et al., 2004). Third, the phototropin-related AGC kinase PINOID (PID) is a crucial regulator of PIN1 intracellular cycling, which suggests an important role for AGC kinases in the regulation of auxin transport polarity (Michniewicz et al., 2007; Robert and Offringa, 2008). The link between the phototropins and auxin has not been firmly established in the cases of leaf flattening and leaf positioning.NPH3 is a strong candidate to provide a link between phototropins and auxin transport. First, NPH3 acts specifically in phototropin-controlled processes that involve growth regulation. Second, the rice (Oryza sativa) homolog of NPH3 called COLEOPTILE PHOTOTROPISM1 (CPT1) is an essential mediator of auxin redistribution in coleoptiles during the phototropin response (Haga et al., 2005). Third, an Arabidopsis homolog of NPH3 named MACCHIBOU4/ENHANCER OF PINOID/NAKED PINS IN YUC MUTANTS1 (MAB4/ENP/NPY1) is involved in organogenesis synergistically with PID by controlling PIN1 localization in embryo and inflorescence stems (Cheng et al., 2007; Furutani et al., 2007). However, beyond these correlative observations, the mechanisms of auxin transport regulation by phototropin signaling remain poorly understood (Holland et al., 2009).PHYTOCHROME KINASE SUBSTRATE (PKS) proteins were initially identified as phytochrome signaling components that regulate developmental processes such as deetiolation and growth orientation of roots and hypocotyls (Fankhauser et al., 1999; Lariguet et al., 2003; Khanna et al., 2006; Boccalandro et al., 2008; Molas and Kiss, 2008; Schepens et al., 2008). PKS1, PKS2, and PKS4 interact with phytochrome A and PKS1 is phosphorylated by phytochrome A in vitro (Fankhauser et al., 1999; Lariguet et al., 2003; Schepens et al., 2008). Recently, we have shown that PKS1 also interacts with phot1 and NPH3 in vivo and is required for phot1-mediated root and hypocotyl phototropism (Lariguet et al., 2006; Boccalandro et al., 2008). The importance of PKS proteins for phototropism prompted us to test their involvement in phototropin-mediated responses more globally. Here, we show that PKS2 acts in phot1 and phot2 signaling pathways controlling leaf positioning and leaf flattening but not chloroplast movements and stomatal opening. Interestingly, PKS2 and NPH3 selectively control phototropin-mediated growth responses and interact genetically. Several lines of evidence, including auxin transport assays in mesophyll protoplasts, suggest that PKS2 may regulate these developmental light responses by modulating auxin homeostasis.     

The Actin Cytoskeleton in Myelinating Cells

Neurochemical Research - Myelinating cells of both the peripheral and central nervous systems (CNSs) undergo dramatic cytoskeletal reorganization in order to differentiate and produce myelin....     

Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses.     

pH Change in Culture Medium Induces Reorganization of Intranuclear Actin in Two-Cell Mouse Embryos

Actin is a permanent component of the cell nucleus involved in many nuclear processes. However, some nuclear functions of actin remain insufficiently explored. The role played by various extracellular stimuli in regulation of nuclear actin still remains enigmatic. Deviation of basic parameters of culture medium from optimal values is a member of the group of extracellular stimuli that are very important for mammalian embryos cultured in vitro. Change in culture medium pH from the level optimal for embryo homeostasis is one such signals. The purpose of this study was to investigate the intranuclear actin distribution in nuclei of two-cell mouse embryos under stress conditions induced by changes in extracellular pH. The pattern of actin localization has been tracked after short-term culturing of the embryos at optimal (pH 7.2), increased (pH 7.8), or decreased (pH 6.5) pH conditions. Analysis was carried out with confocal microscopy using methods of direct fluorescent and indirect immunofluorescent identification of actin. It has been shown that the change of culture medium pH from the optimum value is the signal that alters intranuclear actin distribution in nuclei of the embryonic cells. Culture of two-cell mouse embryos in suboptimal pH conditions (pH 6.5 and pH 7.8) induced alterations in the intranuclear actin localization, which, in particular, were expressed in accumulation of monomeric actin and the appearance of phalloidin-stainable actin in the nuclei. These changes, in our opinion, show some signs of similarity with stress-induced changes in nuclear-actin distribution, which, as has been reported earlier by a number of researchers, have been observed in the nuclei of somatic cells.     

Cytochemical Localization of Actin and Myosin Aggregates in Interphase Nuclei in Situ

Biochemical and ultrastructural studies on isolated nuclear compartments have previously shown actin and myosin to be constituents of interphase nuclei. In the present work, immunocytochemistry, in conjunction with confocal microscopy and ultrastructural immunogold techniques, shows that interphase nuclei of intact dorsal root ganglion neurons and of PC12 cells contain actin and myosin. Nuclear actin was observed to be distributed throughout the nucleoplasm occurring as distinct aggregates. Frequently, prominent actin aggregates were associated with the nucleolar periphery, often near nucleolar satellites. Ultrastructurally, actin was observed to be associated with linear, electrondense structures, putatively identified as chromatin fibers, extending from nucleoli. Use of three antibodies against subclasses of α-actin isoforms revealed that nuclear actin is more closely related to α-sarcomeric actin than to α-smooth muscle actin. Those aggregates associated with the nucleolus were found to be in the polymerized F-actin form, in a small fraction of neurons, as assessed by FITC-phalloidin. A myosin-like antigen was also observed to occur as intranuclear aggregates. Quantitative assays of the distribution of actin and myosin aggregates by nearest neighbour analysis indicated a distribution characterized as uniform and failed to reveal statistically significant associations between any set of aggregates, The evidence presented herein indicates that actin and myosin are constituent proteins of interphase nuclei in situ of both normal mammalian and transformed mammalian cells.     

Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism''s life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca²⁺-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.     

LINC Complexes Mediate the Positioning of Cone Photoreceptor Nuclei in Mouse Retina

It has long been observed that many neuronal types position their nuclei within restricted cytoplasmic boundaries. A striking example is the apical localization of cone photoreceptors nuclei at the outer edge of the outer nuclear layer of mammalian retinas. Yet, little is known about how such nuclear spatial confinement is achieved and further maintained. Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes) consist of evolutionary-conserved macromolecular assemblies that span the nuclear envelope to connect the nucleus with the peripheral cytoskeleton. Here, we applied a new transgenic strategy to disrupt LINC complexes either in cones or rods. In adult cones, we observed a drastic nuclear mislocalization on the basal side of the ONL that affected cone terminals overall architecture. We further provide evidence that this phenotype may stem from the inability of cone precursor nuclei to migrate towards the apical side of the outer nuclear layer during early postnatal retinal development. By contrast, disruption of LINC complexes within rod photoreceptors, whose nuclei are scattered across the outer nuclear layer, had no effect on the positioning of their nuclei thereby emphasizing differential requirements for LINC complexes by different neuronal types. We further show that Sun1, a component of LINC complexes, but not A-type lamins, which interact with LINC complexes at the nuclear envelope, participate in cone nuclei positioning. This study provides key mechanistic aspects underlying the well-known spatial confinement of cone nuclei as well as a new mouse model to evaluate the pathological relevance of nuclear mispositioning.     

Improved Visualization of Cell Nuclei in Unstained Cultured Cells

Nuclear morphology is useful in tissue culture studies in determining the presence and grade of transformed cells as well as in determining the heterogeneity of the cell population (Grogan el al. 1981, Hustin 1976, Siracky et al. 1978, Siracky 1979). The ratio of long and short nuclear axes provides a useful numerical expression of nuclear shape (Hustin 1976). Clear visualization of nuclei is critical for making the necessary measurements.     

Ultrastructural Localization of Filamentous Actin within Neuronal Interphase Nuclei in Situ

Previous biochemical studies utilizing isolated nuclei and nuclear matrices have shown actin to be a constituent of the interphase nucleus. In addition, recent ultrastructural work has shown the presence of actin and myosin within nuclei of interphase cells in situ. It was unclear, however, whether this intranuclear actin is present in the unpolymerized globular actin or the filamentous (F)-actin form. The present work, using confocal microscopy and ultrastructural cytochemical techniques, demonstrates the presence of F-actin within interphase nuclei of intact, uncompromised, dorsal root ganglion neurons in vitro and in vivo. Labeling by FITC-phalloidin detected the presence of intranuclear F-actin adjacent to the nucleolar periphery in a small fraction of cells in vitro, an observation confirmed by three-dimensional reconstruction. Ultrastructural analyses of cells exposed to heavy meromyosin (HMM), showed the presence of typical "arrowhead" complexes. The observation that these complexes were associated with nucleoli confirms that the intranuclear ligand detected by FITC-phalloidin indeed represents F-actin. Postembedding labeling with HMM conjugated to 20-nm colloidal gold (HMM-Au₂₀) resulted in labeling similar to that obtained with HMM. However, HMM-Au₂₀ was found to label a much larger fraction of cells, both in vitro and in vivo, than did FITC-phalloidin or HMM. This finding indicates that labeling with HMM-Au₂₀ more accurately reflects the extent of actin polymerization in nuclei. Results from double labeling with HMM-Au₂₀ and an antibody to α-sarcomeric actin confirmed that only a small amount of nuclear actin is in the F-form. Together, these results represent a first ultrastructural demonstration of the presence of F-actin in nuclei of neurons. While the role of nuclear F-actin has yet to be defined, the results suggest that F-actin may represent a component of the molecular motor responsible for the dynamic positioning of specific chromatin domains into the tissue-specific, nonrandom patterns observed in many cell types.     

Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization

Most animal cells use a combination of actin-myosin–based contraction and actin polymerization– based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389–399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53–62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401–410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.