Effects induced by an actin-polymerizing peptide in goldfish mauthner neurons

In goldfishes, we studied (i) manifestations of functional activity of Mauthner neurons (MNs) reflected in motor behavior and (ii) changes in 3-D morphometry (ratio of volumes) and ultrastructure of MNs after applications of an actin-polymerizing peptide obtained from scorpion venom and after vestibular rotational stimulations (trainings) inducing natural modification of functions of the MNs (adaptation). In MNs subjected to application of the peptide, the increase in the functional resistance and morphological stability caused by long-lasting stimulation directly depended on the dose of the applied peptide or on the effectiveness of trainings, whereas in intact and control MNs such stimulation resulted in significant decreases in the activity and volumes of these cells. At the ultrastructure level, both applications of the peptide and trainings caused the formation of extensive bundles of actin filaments (“stress-fibers”) in the cytoplasm of MNs and led to an increase in the dimension of desmosome-like contacts (DLCs) in afferent synapses. At chemical synapses, the effect looked like a reciprocal decrease in the length of the active zones (a structural sign of long-term depression, LTD), while at mixed synapses this was manifested in an increase in the number of fibrillar bridges in the gaps of DLCs (a structural sign of long-term potentiation, LTP). The data obtained allow us to hypothesize that LTD of the efficacy of transmission through chemical synapses is involved in the formation of the adaptation state of the MNs and that polymerization of actin in the cytoplasm and DLCs underlies the mechanism of LTD and adaptation. The development of ultrastructural manifestations of LTP at mixed synapses after polymerization of actin by the peptide, which is related to a reciprocal increase in the efficacy of “mixed” afferent inputs, explains the maintenance of a high integral level of activity of the MNs, despite a drop in the functional activity of “ chemical” afferent inputs. Therefore, the actin cytoskeleton plays a clearly significant role in the maintenance of the balance of excitation at the neuronal level. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 389–401, September–December, 2006.     

Bacterial actin MreB assembles in complex with cell shape protein RodZ

Bacterial actin homologue MreB is required for cell shape maintenance in most non‐spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix‐turn‐helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB–RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm.     

Diverse roles of actin in <Emphasis Type="Italic">C. elegans</Emphasis>early embryogenesis

Background  

The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics.     

Oxidation and reduction of actin: Origin,impact in vitro and functional consequences in vivo

Actin is among the most abundant proteins in eukaryotic cells and assembles into dynamic filamentous networks regulated by many actin binding proteins. The actin cytoskeleton must be finely tuned, both in space and time, to fulfill key cellular functions such as cell division, cell shape changes, phagocytosis and cell migration. While actin oxidation by reactive oxygen species (ROS) at non-physiological levels are known for long to impact on actin polymerization and on the cellular actin cytoskeleton, growing evidence shows that direct and reversible oxidation/reduction of specific actin amino acids plays an important and physiological role in regulating the actin cytoskeleton. In this review, we describe which actin amino acid residues can be selectively oxidized and reduced in many different ways (e.g. disulfide bond formation, glutathionylation, carbonylation, nitration, nitrosylation and other oxidations), the cellular enzymes at the origin of these post-translational modifications, and the impact of actin redox modifications both in vitro and in vivo. We show that the regulated balance of oxidation and reduction of key actin amino acid residues contributes to the control of actin filament polymerization and disassembly at the subcellular scale and highlight how improper redox modifications of actin can lead to pathological conditions.     

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology,mycelial growth and plant infection

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4′,6‐diamidino‐2‐phenylindole (DAPI) staining and PcSDA1‐green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non‐filamentous yeasts and human cells.     

Actin‐mediated plasma membrane plasticity of the intracellular parasite Theileria annulata

Pathogen–host interactions are modulated at multiple levels by both the pathogen and the host cell. Modulation of host cell functions is particularly intriguing in the case of the intracellular Theileria parasite, which resides as a multinucleated schizont free in the cytosol of the host cell. Direct contact between the schizont plasma membrane and the cytoplasm enables the parasite to affect the function of host cell proteins through direct interaction or through the secretion of regulators. Structure and dynamics of the schizont plasma membrane are poorly understood and whether schizont membrane dynamics contribute to parasite propagation is not known. Here we show that the intracellular Theileria schizont can dynamically change its shape by actively extending filamentous membrane protrusions. We found that isolated schizonts bound monomeric tubulin and in vitro polymerized microtubules, and monomeric tubulin polymerized into dense assemblies at the parasite surface. However, we established that isolated Theileria schizonts free of host cell microtubules maintained a lobular morphology and extended filamentous protrusions, demonstrating that host microtubules are dispensable both forthe maintenance of lobular schizont morphology and for the generation of membrane protrusions. These protrusions resemble nanotubes and extend in an actin polymerization‐dependent manner; using cryo‐electron tomography, we detected thin actin filaments beneath these protrusions, indicating that their extension is driven by schizont actin polymerization. Thus the membrane of the schizont and its underlying actin cytoskeleton possess intrinsic activity for shape control and likely function as a peri‐organelle to interact with and manipulate host cell components.     

Protein Kinase Activities Associated with Actin Cytoskeleton in Xenopus laevis Oocytes and Eggs

Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [-³²P]ATP has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [-³²P]ATP in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca²⁺-dependent kinases, as well as MAP, p34^cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed.     

Actin study of the synapses of surviving hippocampal slices during long-term potentiation

A study was made of the synaptic actin ultrastructural localization in the hippocampal slices at long-lasting potentiation of area CA, using myosin subfragment-1 labeling. A specific qualitative ultrastructural sign of the potentiated hippocampal synapses was revealed for the first time - the formation in spines of rodlike bundles of actin filaments resembling the cilia. They penetrate the spine stalks to pass through the spine core towards the postsynaptic densities of active zones. The thinner bridges link the filament bundles with the actin cytoskeleton meshwork, with spine apparatus cisterns and with postsynaptic membranes of the active zones. Besides the increasing density of the presynaptic actin meshwork was shown. The changes in the actin cytoskeleton being taken into consideration, its contractile properties account for some morphofunctional features of the potentiated synapses known before and predict previously unknown ones.     

Cotton LIM domain‐containing protein GhPLIM1 is specifically expressed in anthers and participates in modulating F‐actin

As one form of actin binding protein (ABP), LIM domain protein can trigger the formation of actin bundles during plant growth and development. In this study, a cDNA (designated GhPLIM1) encoding a LIM domain protein with 216 amino acid residues was identified from a cotton flower cDNA library. Quantitative RT‐PCR indicated that GhPLIM1 is specifically expressed in cotton anthers, and its expression levels are regulated during anther development of cotton. GhPLIM1:eGFP transformed cotton cells display a distributed network of eGFP fluorescence, suggesting that GhPLIM1 protein is mainly localised to the cell cytoskeleton. In vitro high‐speed co‐sedimentation and low co‐sedimentation assays indicate that GhPLIM1 protein not only directly binds actin filaments but also bundles F‐actin. Further biochemical experiments verified that GhPLIM1 protein can protect F‐actin against depolymerisation by Lat B. Thus, our data demonstrate that GhPLIM1 functions as an actin binding protein (ABP) in modulating actin filaments in vitro, suggesting that GhPLIM1 may be involved in regulating the actin cytoskeleton required for pollen development in cotton.     

HopW1 from Pseudomonas syringae Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.     

Apple S‐RNase interacts with an actin‐binding protein,MdMVG, to reduce pollen tube growth by inhibiting its actin‐severing activity at the early stage of self‐pollination induction

In S‐RNase‐mediated self‐incompatibility, S‐RNase secreted from the style destroys the actin cytoskeleton of the self‐pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S‐RNase does not bind or sever filamentous actin (F‐actin). In apple (Malus domestica), we identified an actin‐binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S‐RNase and directly binds and severs F‐actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S‐RNase inhibits the F‐actin‐severing activity of MdMVG in vitro. In vivo, the addition of S‐RNase to self‐pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self‐pollination induction in the presence of MdMVG overexpression. By generating 25 single‐, double‐ and triple‐point mutations in the amino acid motif E‐E‐K‐E‐K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple‐point mutant, MdMVG^{(E167A/E171A/K185A)}, that no longer has F‐actin‐severing activity or interacts with any of the four S‐haplotype S‐RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S‐RNases. We conclude that apple S‐RNase interacts with MdMVG to reduce self‐pollen tube growth by inhibiting its F‐actin‐severing activity.     

Sodium fluoride induces podosome formation in endothelial cells

Background information. Fluoride is a well‐known G‐protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin‐based structures endowed with adhesion and matrix‐degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells. Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell‐division cycle 42), Rac1 and RhoA activities are stimulated in NaF‐treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF‐induced podosomes are fully competent for matrix protein degradation. Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.     

Effects of Intracellular Calcium and Actin Cytoskeleton on TCR Mobility Measured by Fluorescence Recovery

Background

The activation of T lymphocytes by specific antigen is accompanied by the formation of a specialized signaling region termed the immunological synapse, characterized by the clustering and segregation of surface molecules and, in particular, by T cell receptor (TCR) clustering.

Methodology/Principal Findings

To better understand TCR motion during cellular activation, we used confocal microscopy and photo-bleaching recovery techniques to investigate the lateral mobility of TCR on the surface of human T lymphocytes under various pharmacological treatments. Using drugs that cause an increase in intracellular calcium, we observed a decrease in TCR mobility that was dependent on a functional actin cytoskeleton. In parallel experiments measurement of filamentous actin by FACS analysis showed that raising intracellular calcium also causes increased polymerization of the actin cytoskeleton. These in vitro results were analyzed using a mathematical model that revealed effective binding parameters between TCR and the actin cytoskeleton.

Conclusion/Significance

We propose, based on our results, that increase in intracellular calcium levels leads to actin polymerization and increases TCR/cytoskeleton interactions that reduce the overall mobility of the TCR. In a physiological setting, this may contribute to TCR re-positioning at the immunological synapse.     

Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function,the Actin Cytoskeleton,and Cell Adhesion

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.     

Mannose,but not glucose or sucrose,disturbs actin cytoskeleton in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

A dynamic cytoskeleton able to recognize and respond to both abiotic and biotic stimuli is necessary for the proper functioning of a living cell. The cytoskeleton is involved in cell growth and division, maintenance of cell shape, cytoplasmic streaming, and organelle movements. Numerous studies have focused on the relationships between sugar metabolism, sugar signaling, and the cytoskeleton in yeast and animal cells. Data on such connections in plants are scarce. In the present study we investigated the effects of exogenously delivered sugars on the plant actin cytoskeleton. Detached Arabidopsis thaliana leaves were incubated with sugars for 2 days and the cytoskeleton was visualized using fluorescent-labeled phalloidin. Glucose and sucrose did not influence the pattern of the actin cytoskeleton. In contrast, mannose caused the disappearance of filamentous structures and generated actin foci. The symptoms started to be visible after 24 h of the exposure to mannose. The effect did not occur in Nicotiana tabacum mesophyll cells. This insensitivity was probably due to the presence of phosphomannose isomerase in tobacco cells. Mannose is commonly used as a selection marker for the transformation of plants lacking the enzymes responsible for the metabolism of mannose-6-phosphate. Exposure to this hexose has been linked with DNA fragmentation and a release of cytochrome c from mitochondria. Both responses are treated as features of programmed cell death. However, in our experiments no DNA laddering was observed in mannose-treated Arabidopsis leaves.     

Dopaminergic Modulation of the Voltage-Gated Sodium Current in the Cochlear Afferent Neurons of the Rat

The cochlear inner hair cells synapse onto type I afferent terminal dendrites, constituting the main afferent pathway for auditory information flow. This pathway receives central control input from the lateral olivocochlear efferent neurons that release various neurotransmitters, among which dopamine (DA) plays a salient role. DA receptors activation exert a protective role in the over activation of the afferent glutamatergic synapses, which occurs when an animal is exposed to intense sound stimuli or during hypoxic events. However, the mechanism of action of DA at the cellular level is still not completely understood. In this work, we studied the actions of DA and its receptor agonists and antagonists on the voltage-gated sodium current (I_Na) in isolated cochlear afferent neurons of the rat to define the mechanisms of dopaminergic control of the afferent input in the cochlear pathway. Experiments were performed using the voltage and current clamp techniques in the whole-cell configuration in primary cultures of cochlear spiral ganglion neurons (SGNs). Recordings of the I_Na showed that DA receptor activation induced a significant inhibition of the peak current amplitude, leading to a significant decrease in cell excitability. Inhibition of the I_Na was produced by a phosphorylation of the sodium channels as shown by the use of phosphatase inhibitor that produced an inhibition analogous to that caused by DA receptor activation. Use of specific agonists and antagonists showed that inhibitory action of DA was mediated both by activation of D1- and D2-like DA receptors. The action of the D1- and D2-like receptors was shown to be mediated by a G_αs/AC/cAMP/PKA and G_αq/PLC/PKC pathways respectively. These results showed that DA receptor activation constitutes a significant modulatory input to SGNs, effectively modulating their excitability and information flow in the auditory pathway.     

Distinct developmental roles for direct and indirect talin-mediated linkage to actin

Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is essential to connect extracellular matrix-bound integrins to the cytoskeleton. Talin can connect to the cytoskeleton either directly, through its actin-binding motifs, or indirectly, by recruiting other actin-binding proteins. Talin's carboxy-terminal end contains a well-characterized actin-binding domain (ABD). We tested the role of the C-terminal ABD of talin in integrin function in Drosophila. We found that introduction of mutations that reduced actin binding in vitro into the isolated C-terminal Talin-ABD impaired actin binding in vivo. Moreover, when engineered into full-length talin, these mutations disrupted a subset of integrin-mediated adhesion-dependent developmental events. Specifically, morphogenetic processes that involve dynamic, short-term integrin-mediated adhesion were particularly sensitive to impaired function of the C-terminal Talin-ABD. We propose that during development talin connects integrins to the cytoskeleton in distinct ways in different types of integrin-mediated adhesion: directly in transient adhesions and indirectly in stable long-lasting adhesions. Our results provide insight into how a similar array of molecular components can contribute to diverse adhesive processes throughout development.     

On the Origin of Extracellular Glutamate Levels Monitored in the Basal Ganglia of the Rat by In Vivo Microdialysis

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ-aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K⁺ depolarization, (b) Na⁺ channel blockade, (c) removal of extracellular Ca²⁺, and (d) depletion of presynaptic vesicles by local administration of α-latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K⁺ depolarization and were increased by perfusing with tetrodotoxin or with Ca²⁺-free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K⁺-depolarizing conditions by α-latrotoxin. Because the effect of K⁺ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l -trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K⁺ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.     

Soluble Axoplasm Enriched from Injured CNS Axons Reveals the Early Modulation of the Actin Cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology.     

Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.