Dominant-negative alleles of 14-3-3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae

14-3-3 Proteins are thought to function as adapters in signaling complexes [1,2], thereby participating in cellular processes including vesicle trafficking and exocytosis [3,4]. To delineate further the function of 14-3-3 proteins during vesicle trafficking, we generated dominant-negative alleles of the two 14-3-3 homologues, Bmh1p and Bmh2p, in budding yeast and analyzed their phenotype in respect to exocytosis. Cells overexpressing the carboxy-terminal region of Bmh2p failed to polarize vesicular transport although bulk exocytosis remained unaffected and showed a disrupted actin cytoskeleton. Our data suggest that 14-3-3 proteins may act primarily on the actin cytoskeleton to regulate vesicle targeting.     

A role for protein kinase casein kinase2 α-subunits in the Arabidopsis circadian clock

Circadian rhythms are autoregulatory, endogenous rhythms with a period of approximately 24 h. A wide variety of physiological and molecular processes are regulated by the circadian clock in organisms ranging from bacteria to humans. Phosphorylation of clock proteins plays a critical role in generating proper circadian rhythms. Casein Kinase2 (CK2) is an evolutionarily conserved serine/threonine protein kinase composed of two catalytic α-subunits and two regulatory β-subunits. Although most of the molecular components responsible for circadian function are not conserved between kingdoms, CK2 is a well-conserved clock component modulating the stability and subcellular localization of essential clock proteins. Here, we examined the effects of a cka1a2a3 triple mutant on the Arabidopsis (Arabidopsis thaliana) circadian clock. Loss-of-function mutations in three nuclear-localized CK2α subunits result in period lengthening of various circadian output rhythms and central clock gene expression, demonstrating that the cka1a2a3 triple mutant affects the pace of the circadian clock. Additionally, the cka1a2a3 triple mutant has reduced levels of CK2 kinase activity and CIRCADIAN CLOCK ASSOCIATED1 phosphorylation in vitro. Finally, we found that the photoperiodic flowering response, which is regulated by circadian rhythms, was reduced in the cka1a2a3 triple mutant and that the plants flowered later under long-day conditions. These data demonstrate that CK2α subunits are important components of the Arabidopsis circadian system and their effects on rhythms are in part due to their phosphorylation of CIRCADIAN CLOCK ASSOCIATED1.     

Proteomics on brefeldin A-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton

The growing importance of vesicular trafficking and cytoskeleton dynamic reorganization during plant development requires the exploitation of novel experimental approaches. Several genetic and cell biological studies have used diverse pharmaceutical drugs that inhibit vesicular trafficking and secretion to study these phenomena. Here, proteomic and cell biology approaches were applied to study effects of brefeldin A (BFA), an inhibitor of vesicle recycling and secretion, in Arabidopsis roots. The main aim of this study was to obtain an overview of proteins affected by BFA, but especially to identify new proteins involved in the vesicular trafficking and its cross-talk to the actin cytoskeleton. The results showed that BFA altered vesicular trafficking and caused the formation of BFA-compartments which was accompanied by differential expression of several proteins in root cells. Some of the BFA-up-regulated proteins belong to the class of the vesicular trafficking proteins, such as V-ATPase and reversibly glycosylated polypeptide, while others, such as profilin 2 and elongation factor 1 alpha, are rather involved in the remodeling of the actin cytoskeleton. Upregulation of profilin 2 by BFA was verified by immunoblot and live imaging at subcellular level. The latter approach also revealed that profilin 2 accumulated in BFA-compartments which was accompanied by remodeling of the actin cytoskeleton in BFA-treated root cells. Thus, profilin 2 seems to be involved in the cross-talk between vesicular trafficking and the actin cytoskeleton, in a BFA-dependent manner.     

The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Insulin-induced endothelial cell cortical actin filament remodeling: a requirement for trans-endothelial insulin transport

Insulin's trans-endothelial transport (TET) is critical for its metabolic action on muscle and involves trafficking of insulin bound to its receptor (or at high insulin concentrations, the IGF-I receptor) via caveolae. However, whether caveolae-mediated insulin TET involves actin cytoskeleton organization is unknown. Here we address whether insulin regulates actin filament organization in bovine aortic endothelial cells (bAEC) and whether this affects insulin uptake and TET. We found that insulin induced extensive cortical actin filament remodeling within 5 min. This remodeling was inhibited not only by disruption of actin microfilament organization but also by inhibition of phosphatidylinositol 3-kinase (PI3K) or by disruption of lipid rafts using respective specific inhibitors. Knockdown of either caveolin-1 or Akt using specific small interfering RNA also eliminated the insulin-induced cortical actin filament remodeling. Blocking either actin microfilament organization or PI3K pathway signaling inhibited both insulin uptake and TET. Disruption of actin microfilament organization also reduced the caveolin-1, insulin receptor, and IGF-I receptor located at the plasma membrane. Exposing bAEC for 6 h to either TNFα or IL-6 blocked insulin-induced cortical actin remodeling. Extended exposure (24 h) also inhibited actin expression at both mRNA and protein levels. We conclude that insulin-induced cortical actin filament remodeling in bAEC is required for insulin's TET in a PI3K/Akt and plasma membrane lipid rafts/caveolae-dependent fashion, and proinflammatory cytokines TNFα and IL-6 block this process.     

Arabidopsis response regulators ARR3 and ARR4 play cytokinin-independent roles in the control of circadian period

Light and temperature are potent environmental signals used to synchronize the circadian oscillator with external time and photoperiod. Phytochrome and cryptochrome photoreceptors integrate light quantity and quality to modulate the pace and phase of the clock. PHYTOCHROME B (phyB) controls period length in red light as well as the phase of the clock in white light. phyB interacts with ARABIDOPSIS RESPONSE REGULATOR4 (ARR4) in a light-dependent manner. Accordingly, we tested ARR4 and other members of the type-A ARR family for roles in clock function and show that ARR4 and its closest relative, ARR3, act redundantly in the Arabidopsis thaliana circadian system. Loss of ARR3 and ARR4 lengthens the period of the clock even in the absence of light, demonstrating that they do so independently of active phyB. In addition, in white light, arr3,4 mutants show a leading phase similar to phyB mutants, suggesting that circadian light input is modulated by the interaction of phyB with ARR4. Although type-A ARRs are involved in cytokinin signaling, the circadian defects appear to be independent of cytokinin, as exogenous cytokinin affects the phase but not the period of the clock. Therefore, ARR3 and ARR4 are critical for proper circadian period and define an additional level of regulation of the circadian clock in Arabidopsis.     

Uncoated endocytic vesicles require the unconventional myosin, Myo6, for rapid transport through actin barriers

After clathrin-mediated endocytosis, clathrin removal yields an uncoated vesicle population primed for fusion with the early endosome. Here we present the first characterization of uncoated vesicles and show that myo6, an unconventional myosin, functions to move these vesicles out of actin-rich regions found in epithelial cells. Time-lapse microscopy revealed that myo6-associated uncoated vesicles were motile and exhibited fusion and stretching events before endosome delivery, processes that were dependent on myo6 motor activity. In the absence of myo6 motor activity, uncoated vesicles remained trapped in the actin mesh, where they exhibited Brownian-like motion. Exit from the actin mesh occurred by a slow diffusion-based mechanism, delaying transferrin trafficking to the early endosome. Expression of a myo6 mutant that bound tightly to F-actin produced immobilized vesicles and blocked trafficking. Depolymerization of the actin cytoskeleton rescued this block and specifically accelerated transferrin delivery to the early endosome without affecting earlier steps in endocytosis. Therefore actin is a physical barrier impeding uncoated vesicle trafficking, and myo6 is recruited to move the vesicles through this barrier for fusion with the early endosome.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 form a positive feedback regulatory loop in the Arabidopsis circadian clock

In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.     

Functional nonequivalency of actin isovariants in Arabidopsis.

Plants encode at least two ancient and divergent classes of actin, reproductive and vegetative, and each class produces several subclasses of actin isovariants. To gain insight into the functional significance of the actin isovariants, we generated transgenic Arabidopsis lines that expressed a reproductive actin, ACT1, under the control of the regulatory sequences of a vegetative actin gene, ACT2. In the wild-type plants, ACT1 is predominantly expressed in the mature pollen, growing pollen tubes, and ovules, whereas ACT2 is constitutively and strongly expressed in all vegetative tissues and organs, but not in pollen. Misexpression of ACT1 in vegetative tissues causes dwarfing of plants and altered morphology of most organs, and the effects are in direct proportion to protein expression levels. Similar overexpression of ACT2 has little effect. Immunolocalization of actin in leaf cells from transgenic plants with highest levels of ACT1 protein revealed massive polymerization, bundling, and reorganization of actin filaments. This phenomenon suggests that misexpression of ACT1 isovariant in vegetative tissues affects the dynamics of actin and actin-associated proteins, in turn disrupting the organization of actin cytoskeleton and normal development of plants.     

Multi-level control of actin dynamics by protein kinase D

Dynamic actin remodeling is fundamental to processes such as cell motility, vesicle trafficking, and cytokinesis. Protein kinase D (PKD) is a serine–threonine kinase known to be involved in diverse biological functions ranging from vesicle fission at the Golgi complex to regulation of cell motility and invasion. This review addresses the role of PKD in the organization of the actin cytoskeleton with a particular emphasis on the substrates associated with this function. We further highlight the multi-level control of actin dynamics by PKD and suggest that the tight spatio-temporal control of PKD activity is critical for the coordination of directed cell migration.     

Isolation and characterization of two forms of a cytoskeleton

Isolated petaloid coelomocytes from the sea urchin Strongylocentrotus droebachiensis transform to a filopodial morphology in hypotonic media. Electron micrographs of negatively stained Triton-insoluble cytoskeletons show that the petaloid form consists of a loose net of microfilaments while the filopodial form consists of paracrystalline bundles of microfilaments. Actin is the major protein of both forms of the cytoskeleton. Additional polypeptides have molecular weights of approximately 220,000, 64,000, 57,000, and 27,000 daltons. Relative to actin the filopodial cytoskeletons have an average of 2.5 times as much 57k polypeptide as the petaloid cytoskeletons. Treatment with 0.25 M NaCl dissociates the filament bundles into individual actin filaments free of the actin-associated polypeptides. Thus, one or more of these actin-associated polypeptides may be responsible for crosslinking the actin filaments into bundles and maintaining the three-dimensional nature of the cytoskeletons.     

Miconazole induces changes in actin cytoskeleton prior to reactive oxygen species induction in yeast

The antifungal compound miconazole inhibits ergosterol biosynthesis and induces reactive oxygen species (ROS) in susceptible yeast species. To further uncover the mechanism of miconazole antifungal action and tolerance mechanisms, we screened the complete set of haploid Saccharomyces cerevisiae gene deletion mutants for mutants with an altered miconazole sensitivity phenotype. We identified 29 S. cerevisiae genes, which when deleted conferred at least 4-fold hypersensitivity to miconazole. Major functional groups encode proteins involved in tryptophan biosynthesis, membrane trafficking including endocytosis, regulation of actin cytoskeleton, and gene expression. With respect to the antifungal activity of miconazole, we demonstrate an antagonism with tryptophan and a synergy with a yeast endocytosis inhibitor. Because actin dynamics and induction of ROS are linked in yeast, we further focused on miconazole-mediated changes in actin cytoskeleton organization. In this respect, we demonstrate that miconazole induces changes in the actin cytoskeleton, indicative of increased filament stability, prior to ROS induction. These data provide novel mechanistic insights in the mode of action of a ROS-inducing azole.