Cytoskeleton Network Research Articles

A schizophrenia associated CMYA5 allele displays differential binding with desmin

CMYA5 is a candidate gene for schizophrenia because of the genetic association of variant rs10043986 (C > T) to this severe mental disorder. Studies of CMYA5 and its gene product, myospryn, in the brain and neuronal cells have not been previously reported. The SNP rs10043986 changes the 4,063rd amino acid from Pro to Leu, which is likely to alter protein function. To understand its potential role in the brain, we examined the neuronal expression of myospryn and its binding partner, desmin, an intermediate filament (IF) protein, and investigated how the two alleles of myospryn affect its binding to desmin. Myospryn and desmin are shown to be expressed in the brain and myospryn is shown to localize to the cytoplasm and nucleus of myoblast, neuroblastoma, and glioblastoma cell lines. Peripherin and vimentin, known brain IF proteins, have high protein similarity to desmin but were found not to interact with myospryn using yeast two-hybrid (Y2H). Using a quantitative Y2H assay and surface plasmon resonance, the T allele (Leu) of rs10043986 was found to have stronger binding to desmin than the C allele (Pro). Based on findings described in this report, we hypothesize that the interaction between myospryn to IF provides structural support and efficient rearrangement of the cytoskeleton network during early neuritogenesis.

Isotype Specific Assembly of B Cell Antigen Receptors and Synergism With Chemokine Receptor CXCR4

Expression of the membrane-bound form of the immunoglobulin (Ig) as part of the antigen receptor is indispensable for both the development and the effector function of B cells. Among five known isotypes, IgM and IgD are the common B cell antigen receptors (BCRs) that are co-expressed in naïve B cells. Despite having identical antigen specificity and being associated with the same signaling heterodimer Igα/Igβ (CD79a/CD79b), IgM and IgD-BCR isotypes functionally differ from each other in the manner of antigen binding, the formation of isolated nanoclusters and in their interaction with co-receptors such as CD19 and CXCR4 on the plasma membrane. With recent developments in experimental techniques, it is now possible to investigate the nanoscale organization of the BCR and better understand early events of BCR engagement. Interestingly, the cytoskeleton network beneath the membrane controls the BCR isotype-specific organization and its interaction with co-receptors. BCR triggering results in reorganization of the cytoskeleton network, which is further modulated by isotype-specific signals from co-receptors. For instance, IgD-BCR is closely associated with CXCR4 on mature B cells and this close proximity allows CXCR4 to employ the BCR machinery as signaling hub. In this review, we discuss the functional specificity and nanocluster assembly of BCR isotypes and the consequences of cross-talk between CXCR4 and IgD-BCR. Furthermore, given the role of BCR and CXCR4 signaling in the development and survival of leukemic B cells, we discuss the consequences of the cross-talk between CXCR4 and the BCR for controlling the growth of transformed B cells.

Cellular protein profiles altered by PRRSV infection of porcine monocytes-derived dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play an important role in inducing primary antigen-specific immune responses. Some viruses have evolved to specifically target DCs to circumvent the host immune responses for their persistence in the host. One example is porcine reproductive and respiratory syndrome virus (PRRSV) that causes a persistent infection in pigs through modulating DC-mediated antiviral response. To study the cellular protein responses in PRRSV-infected monocyte-derived dendritic cells (MoDCs), two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins in PRRSV-infected MoDCs and the control cells. A total of 252 cellular proteins in MoDCs that were significantly altered at different time periods post-infection were identified. Differentially expressed proteins that are involved in the endocytosis pathway, actin cytoskeleton network, antigen processing and presentation, JAK-STAT signaling pathway and PRRSV receptors were identified and further analyzed. Among them, the expression changes of STAT1, Mx1, PICALM and SLA-DR were further verified by Western blotting. The protein profiles associated with PRRSV infection of MoDCs should offer novel insights to further investigation of PRRSV-mediated antiviral evasion mechanism and its pathobiology in swine.

Age-dependent migratory behavior of human endothelial cells revealed by substrate microtopography

Cell migration is part of many important in vivo biological processes and is influenced by chemical and physical factors such as substrate topography. Although the migratory behavior of different cell types on structured substrates has already been investigated, up to date it is largely unknown if specimen's age affects cell migration on structures. In this work, we investigated age-dependent migratory behavior of human endothelial cells from young (≤ 31 years old) and old (≥ 60 years old) donors on poly(dimethylsiloxane) microstructured substrates consisting of well-defined parallel grooves. We observed a decrease in cell migration velocity in all substrate conditions and in persistence length perpendicular to the grooves in cells from old donors. Nevertheless, in comparison to young cells, old cells exhibited a higher cell directionality along grooves of certain depths and a higher persistence time. We also found a systematic decrease of donor age-dependent responses of cell protrusions in orientation, velocity and length, all of them decreased in old cells. These observations lead us to hypothesize a possible impairment of actin cytoskeleton network and affected actin polymerization and steering systems, caused by aging.

Weak power frequency magnetic fields induce microtubule cytoskeleton reorganization depending on the epidermal growth factor receptor and the calcium related signaling

We have shown previously that a weak 50 Hz magnetic field (MF) invoked the actin-cytoskeleton, and provoked cell migration at the cell level, probably through activating the epidermal growth factor receptor (EGFR) related motility pathways. However, whether the MF also affects the microtubule (MT)-cytoskeleton is still unknown. In this article, we continuously investigate the effects of 0.4 mT, 50 Hz MF on the MT, and try to understand if the MT effects are also associated with the EGFR pathway as the actin-cytoskeleton effects were. Our results strongly suggest that the MF effects are similar to that of EGF stimulation on the MT cytoskeleton, showing that 1) the MF suppressed MT in multiple cell types including PC12 and FL; 2) the MF promoted the clustering of the EGFR at the protein and the cell levels, in a similar way of that EGF did but with higher sensitivity to PD153035 inhibition, and triggered EGFR phosphorylation on sites of Y1173 and S1046/1047; 3) these effects were strongly depending on the Ca2+ signaling through the L-type calcium channel (LTCC) phosphorylation and elevation of the intracellular Ca2+ level. Strong associations were observed between EGFR and the Ca2+ signaling to regulate the MF-induced-reorganization of the cytoskeleton network, via phosphorylating the signaling proteins in the two pathways, including a significant MT protein, tau. These results strongly suggest that the MF activates the overall cytoskeleton in the absence of EGF, through a mechanism related to both the EGFR and the LTCC/Ca2+ signaling pathways.

Mutually inhibitory Ras-PI(3,4)P2 feedback loops mediate cell migration

Signal transduction and cytoskeleton networks in a wide variety of cells display excitability, but the mechanisms are poorly understood. Here, we show that during random migration and in response to chemoattractants, cells maintain complementary spatial and temporal distributions of Ras activity and phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P2]. In addition, depletion of PI(3,4)P2 by disruption of the 5-phosphatase, Dd5P4, or by recruitment of 4-phosphatase INPP4B to the plasma membrane, leads to elevated Ras activity, cell spreading, and altered migratory behavior. Furthermore, RasGAP2 and RapGAP3 bind to PI(3,4)P2, and the phenotypes of cells lacking these genes mimic those with low PI(3,4)P2 levels, providing a molecular mechanism. These findings suggest that Ras activity drives PI(3,4)P2 down, causing the PI(3,4)P2-binding GAPs to dissociate from the membrane, further activating Ras, completing a positive-feedback loop essential for excitability. Consistently, a computational model incorporating such a feedback loop in an excitable network model accurately simulates the dynamic distributions of active Ras and PI(3,4)P2 as well as cell migratory behavior. The mutually inhibitory Ras-PI(3,4)P2 mechanisms we uncovered here provide a framework for Ras regulation that may play a key role in many physiological processes.

Dynamic stability of the actin ecosystem.

In cells, actin filaments continuously assemble and disassemble while maintaining an apparently constant network structure. This suggests a perfect balance between dynamic processes. Such behavior, operating far out of equilibrium by the hydrolysis of ATP, is called a dynamic steady state. This dynamic steady state confers a high degree of plasticity to cytoskeleton networks that allows them to adapt and optimize their architecture in response to external changes on short time-scales, thus permitting cells to adjust to their environment. In this Review, we summarize what is known about the cellular actin steady state, and what gaps remain in our understanding of this fundamental dynamic process that balances the different forms of actin organization in a cell. We focus on the minimal steps to achieve a steady state, discuss the potential feedback mechanisms at play to balance this steady state and conclude with an outlook on what is needed to fully understand its molecular nature.

Effects of Mechanical Stimuli on Profilin- and Formin-Mediated Actin Polymerization.

Self-assembling actin filaments not only form the basis of the cytoskeleton network in cells but also are utilized as nanosized building blocks to make novel active matter in which the dynamic polymerization and depolymerization of actin filaments play a key role. Formins belong to a main family of actin nucleation factors that bind to the barbed end of actin filaments and regulate actin polymerization through an interaction with profilin. Due to actomyosin contractility and relative rotation between formin and actin filaments, formin-dependent actin polymerization is subject to force and rotation constraints. However, it remains unclear how force and rotation constraints affect formin-dependent actin polymerization in the presence of profilin. Here, we show that for rotation-unconstrained actin filaments, elongation is accelerated by both force and profilin. The combined effect leads to surprisingly fast actin elongation that can approach the diffusion-limited rate at forces of a few piconewtons. The elongation of rotation-constrained filaments is also accelerated by profilin but is insensitive to applied force. We show that FH2, the main actin binding domain, plays the primary mechanosensing role. Together, the findings not only significantly advance our understanding of the mechanochemical regulation of formin-mediated actin polymerization in cells but also can potentially be utilized to make novel actin-based active matter.

Effect of extraction Alkaloids from Zephyranthes candida induces cell death through destruction microtubules in (China hamster cell line)

Background: In eukaryotic cells the cytoskeleton network consists of three major structural elements, microtubules, microfilament,and intermediate filaments. Tubulin is the basic protein of the MTs, molecules of tubulin arranged in dimmers consisting of two forms, α- tubulin and β-tubulin. They are continuously changeable structures.Taxanes and vinca alkaloids are inhibitors MTC that destabilize microtubules, there by suppressing their dynamics which required for proper mitotic function and effectively blocking cell regulation progression resulting in cell death. Objective: To evaluate the capability of alkaloids taken away from leaves of Zephyranthes candidato divert the MTC network of (CHO), which is an aggressive metastasis cell line. Patients and Methods: In our experiments we used the mouse CHO cell line. This cell line was obtained from the Department of Biology,Faculty of Medicine, Wuhan University, China. The cells were grown in Dulbecco’s Minimal Essential Medium (DMEM) (PAA Laboratories GmbH, Linz, Austria), supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 Uml–1 penicillin, and 100 μg ml–1 streptomycin (PAA Laboratories, Austria) in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C.Zephyranthes candida crude alkaloid extract treated cells were cultivated over microscopic cover slips, washed three Times for 4 min with PBS (pH 6.9) and fixed by 3% paraformaldehydein PBS. The ability of alkaloid extract to inhibit CHO tumor cells growth in vivo was assessed; forty mice were injected subcutaneouslyin the right dorsal with this tumor cells, after the tumor developed to 8-1 mm2, and eliminating the animalsthat did not develop tumors, the animals were divided to three groups each one with ten individuals. Results: The extractor was capable of divert the MTC of the cells below realization after 1hr of in sinuation in a focus as little as possible 20 μg/ml. while DAPI staining used, the cells death was not check in this focus and time. The cell death have been spotted when the focus of the alkaloid reproducer raised up to 80 and 100 μg/ml through the aforesaid exposure time. The cells were able to improving there native MTC contraction after 12 hr of the alkaloid deletion. The excerpt focus of 1mg/Kg/Bw competently prevent CHO cell line cancer development in vivo to 97.14% after 3 weeks therapy contrast to natural control. Conclusion: The invasive species H22 cells offer changesin the preparation of their MTC at a focus of Zephyranthes candida leaf alkaloids excerpt as low as 20 μg/ml focus after 1hr of exposuretime.

Numerical method for vesicle movement analysis in a complex cytoskeleton network

The detection of the precise movement of a vesicle during transport in a live cell provides key information for the intracellular delivery process. Here we report a novel numerical method for analyzing three-dimensional vesicle movement. Since the vesicle moves along a linear cytoskeleton during the active transport, our method first detects the orientation and position of the cytoskeleton as a linear section based on angle correlation and linear regression, after noise reduction. Then, the precise vesicle movement is calculated using vector analysis, in terms of rotation angle and translational displacement. Using this method, various vesicle trajectories obtained via high spatiotemporal resolution microscopy can be understood..

Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light

BackgroundArtificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. ObjectiveThe purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. MethodsThe spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. ResultsWe developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450 nm, 525 nm and 625 nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99 J/cm2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the “mitochondria” and “integrin signaling” categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. ConclusionArtificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede normal cellular functions and contribute to premature skin aging. The present results extend our knowledge of the effects of the low-energy wavelengths that are increasingly used to treat skin disorders.

Modeling the neuron as a nanocommunication system to identify spatiotemporal molecular events in neurodegenerative disease.

AimIn tauopathies such as Alzheimer’s disease (AD), molecular changes spanning multiple subcellular compartments of the neuron contribute to neurodegeneration and altered axonal signaling. Computational modeling of end-to-end linked events benefit mechanistic analysis and can be informative to understand disease progression and accelerate development of effective therapies. In the calcium-amyloid beta model of AD, calcium ions that are an important regulator of neuronal function undergo dysregulated homeostasis that disrupts cargo loading for neurotrophic signaling along axonal microtubules (MTs). The aim of the present study was to develop a computational model of the neuron using a layered architecture simulation that enables us to evaluate the functionalities of several interlinked components in the calcium-amyloid beta model.MethodsThe elevation of intracellular calcium levels is modeled upon binding of amyloid beta oligomers (AβOs) to calcium channels or as a result of membrane insertion of oligomeric Aβ1-42 to form pores/channels. The resulting subsequent Ca2+ disruption of dense core vesicle (DCV)-kinesin cargo loading and transport of brain-derived neurotrophic factor (BDNF) on axonal MTs are then evaluated. Our model applies published experimental data on calcium channel manipulation of DCV-BDNF and incorporates organizational complexity of the axon as bundled polar and discontinuous MTs. The interoperability simulation is based on the Institute of Electrical and Electronics Engineers standard association P1906.1 framework for nanoscale and molecular communication.ResultsOur analysis provides new spatiotemporal insights into the end-to-end signaling events linking calcium dysregulation and BDNF transport and by simulation compares the relative impact of dysregulation of calcium levels by AβO-channel interactions, oligomeric Aβ1-42 pores/channel formation, and release of calcium by internal stores. The flexible platform of our model allows continued expansion of molecular details including mechanistic and morphological parameters of axonal cytoskeleton networks as they become available to test disease and treatment predictions.ConclusionThe present model will benefit future drug studies on calcium homeostasis and dysregulation linked to measurable neural functional outcomes. The algorithms used can also link to other multiscale multi-cellular modeling platforms to fill in molecular gaps that we believe will assist in broadening and refining multiscale computational maps of neurodegeneration.

P-163 - Smokeless tobacco chewing mediates plethora of physiological hazard and neuronal health

Smokeless tobacco (SLT) or chewing tobacco is very common addiction practice in India across the ages making a somber threat and huge disease burden. Oral use of SLT is often assorted with many ingredients and is capable to induce extreme toxicological challenges and carcinogenic developments. The current study investigated the systemic stress in human subjects using blood parameters of SLT chewers. Perturbations in parameters of metabolism, inflammation, RBC morphology and enhanced apoptosis of PBMC in SLT-chewers compared to non-chewer human subjects were found. The study further delved whether SLT mediated any neurodegeration. In vitro studies using SLT showed the neuronal cell death in a dose dependent manner compromising the mitochondrial health. Apoptosis was induced with the consequence of generation of reactive species, induction of DNA damage, alteration of membrane potential, mitochondrial morphology, activation of caspase-cascade, apoptotic proteins and disruption of the neuronal cytoskeleton network. To correlate the neurobiological insult of SLT, mouse behavioral studies with open-field test and plus maze and molecular analyses were investigated. This unique mechanistic study with molecular mechanism validated fatal effects of SLT and SLT-mediated neuronal cell death.

Honokiol, a Polyphenol Natural Compound, Attenuates Cisplatin-Induced Acute Cytotoxicity in Renal Epithelial Cells Through Cellular Oxidative Stress and Cytoskeleton Modulations.

Cisplatin is a potent anti-cancer drug that has been widely used in the treatment of various cancers; however, cisplatin administration results in severe nephrotoxicity and impedes its clinical applications. In this study, we showed that honokiol, a polyphenol constituent extracted from Magnolia officinalis exhibited a short-term protective effect against cisplatin-induced damages in renal epithelial cells in vitro. The protective effects of honokiol were resulted from the combination of (1) reduced cellular oxidative stress ranging from 53 to 32% reduction during a 24-h incubation, (2) the maintenance of cellular antioxidant capacity and (3) the stabilization of cytoskeletal structure of the kidney epithelial cells. By promoting the polymerization of actin (1.6-fold increase) and tubulin (1.8-fold increase) cytoskeleton, honokiol not only maintained epithelial cell morphology, but also stabilized cellular localizations of tight junction protein Occludin and adhesion junction protein E-Cadherin. With stabilized junction protein complexes and structural polymerized cytoskeleton network, honokiol preserved epithelial cell polarity and morphology and thus reduced cisplatin-induced cell disruption and damages. Our data demonstrated for the first time that honokiol could counteract with cisplatin-induced damages in renal epithelial cells in vitro, future in vivo studies would further validate the potential clinical application of honokiol in cisplatin-based cancer treatments with reduced nephrotoxicity.

TGF\u03b21 reinforces arterial aging in the vascular smooth muscle cell through a long-range regulation of the cytoskeletal stiffness

Here we report exquisitely distinct material properties of primary vascular smooth muscle (VSM) cells isolated from the thoracic aorta of adult (8 months) vs. aged (30 months) F344XBN rats. Individual VSM cells derived from the aged animals showed a tense internal network of the actin cytoskeleton (CSK), exhibiting increased stiffness (elastic) and frictional (loss) moduli than those derived from the adult animals over a wide frequency range of the imposed oscillatory deformation. This discrete mechanical response was long-lived in culture and persistent across a physiological range of matrix rigidity. Strikingly, the pro-fibrotic transforming growth factor β1 (TGFβ1) emerged as a specific modifier of age-associated VSM stiffening in vitro. TGFβ1 reinforced the mechanical phenotype of arterial aging in VSM cells on multiple time and length scales through clustering of mechanosensitive α5β1 and αvβ3 integrins. Taken together, these studies identify a novel nodal point for the long-range regulation of VSM stiffness and serve as a proof-of-concept that the broad-based inhibition of TGFβ1 expression, or TGFβ1 signal transduction in VSM, may be a useful therapeutic approach to mitigate the pathologic progression of central arterial wall stiffening associated with aging.

SP600125 blocks the proteolysis of cytoskeletal proteins in apoptosis induced by gas signaling molecule (NO) via decreasing the activation of caspase-3 in rabbit chondrocytes

NO plays a key role in the pathological mechanisms of articular diseases. As cytoskeletal proteins are responsible for the polymerization, stabilization, and dynamics of the cytoskeleton network, we investigated whether cytoskeletal proteins are the intracellular pathological targets of NO. We aimed at clarifying whether the cytoskeleton perturbations involved in apoptosis are induced in rabbit articular chondrocytes by NO, which can be liberated by sodium nitroprusside (SNP) treatment. The first passage rabbit articular chondrocytes were cultured as monolayer for the experiments, and the effects of NO were tested in the presence of JNK-specific inhibitor, SP600125. SNP treatment of cultured chondrocytes caused significant apoptosis in a concentration-dependent manner (time and dose), as evaluated by TUNEL assay and Annexin V flow cytometry, while the apoptosis was reduced by the SP600125 addition 30 min before SNP treatment. Besides, SP600125 decreased significantly the protein expression of total caspase-3 and the intracellular gene expression of caspase-3, measured by Western blot analysis and PCR. SP600125 also increased the cytoskeletal protein expressions. These results suggested that JNK pathway plays a critical role in the NO-induced chondrocyte apoptosis, and SP600125 treatment blocks the dissolution of the cytoskeletal proteins via activation of caspase-3 pathways.

Mechanotransmission and Mechanosensing of Human Alpha-actinin 1

α-actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework of the cytoskeleton through crosslinking the actin filaments, as well as in focal adhesion maturation. However, the molecular mechanisms underlying its functions are unclear. In this work, by mechanical manipulation of single human α-actinin 1 using magnetic tweezers, we determined the mechanical stability and kinetics of the functional domains in α-actinin 1 as well as the mechanical strength of the α-actinin 1 dimerization interaction. Moreover, we identified the force-dependence of vinculin binding to α-actinin 1, with the demonstration that force is required to expose the high-affinity binding site for vinculin binding. Based on the mechanical stability and kinetics of α-actinin 1, a novel role of the α-actinin 1 as molecular suspensions for the cytoskeleton network is revealed. Our results provide the first comprehensive analysis of the force dependent stability and interactions of α-actinin 1, which sheds new light on the molecular mechanisms underlying its mechanotransmission and mechanosensing functions.

Mechanotransmission and Mechanosensing of Human alpha-Actinin 1

α-Actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework of thecytoskeleton through crosslinking the actin filaments, as well as in focal adhesion maturation. However, the molecular mechanisms underlying its functions are unclear. Here, by mechanical manipulation of single human α-actinin 1 using magnetic tweezers, we determined the mechanical stability and kinetics of the functional domains in α-actinin 1. Moreover, we identified the force-dependence of vinculin binding to α-actinin 1, with the demonstration that force isrequired to expose the high-affinity binding site for vinculin binding. Further, a role of the α-actinin 1 as molecular shock absorber for the cytoskeleton network is revealed. Our results provide a comprehensive analysis of the force-dependent stability and interactions of α-actinin 1, which sheds important light on the molecular mechanisms underlyingitsmechanotransmission and mechanosensing functions.

Going with the Flow: Water Flux and Cell Shape during Cytokinesis

Cell shape changes during cytokinesis in eukaryotic cells have been attributed to contractile forces from the actomyosin ring and the actomyosin cortex. Here we propose an additional mechanism where active pumping of ions and water at the cell poles and the division furrow can also achieve the same type of shape change during cytokinesis without myosin contraction. We develop a general mathematical model to examine shape changes in a permeable object subject to boundary fluxes. We find that hydrodynamic flows in the cytoplasm and the relative drag between the cytoskeleton network phase and the water phase also play a role in determining the cell shape during cytokinesis. Forces from the actomyosin contractile ring and cortex do contribute to the cell shape, and can work together with water permeation to facilitate cytokinesis. To influence water flow, we osmotically shock the cell during cell division, and find that the cell can actively adapt to osmotic changes and complete division. Depolymerizing the actin cytoskeleton during cytokinesis also does not affect the contraction speed. We also explore the role of membrane ion channels and pumps in setting up the spatially varying water flux.

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR3 Is Required for Controlling Aphid Feeding from the Phloem.

The actin cytoskeleton network has an important role in plant cell growth, division, and stress response. Actin-depolymerizing factors (ADFs) are a group of actin-binding proteins that contribute to reorganization of the actin network. Here, we show that the Arabidopsis (Arabidopsis thaliana) ADF3 is required in the phloem for controlling infestation by Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is an important phloem sap-consuming pest of more than fifty plant families. In agreement with a role for the actin-depolymerizing function of ADF3 in defense against the GPA, we show that resistance in adf3 was restored by overexpression of the related ADF4 and the actin cytoskeleton destabilizers, cytochalasin D and latrunculin B. Electrical monitoring of the GPA feeding behavior indicates that the GPA stylets found sieve elements faster when feeding on the adf3 mutant compared to the wild-type plant. In addition, once they found the sieve elements, the GPA fed for a more prolonged period from sieve elements of adf3 compared to the wild-type plant. The longer feeding period correlated with an increase in fecundity and population size of the GPA and a parallel reduction in callose deposition in the adf3 mutant. The adf3-conferred susceptibility to GPA was overcome by expression of the ADF3 coding sequence from the phloem-specific SUC2 promoter, thus confirming the importance of ADF3 function in the phloem. We further demonstrate that the ADF3-dependent defense mechanism is linked to the transcriptional up-regulation of PHYTOALEXIN-DEFICIENT4, which is an important regulator of defenses against the GPA.