Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

MEK1/2 and p38-like MAP kinase successively mediate H2O2 signaling in Vicia guard cell

As a second messenger, H₂O₂ generation and signal transduction is subtly controlled and involves various signal elements, among which are the members of MAP kinase family. The increasing evidences indicate that both MEK1/2 and p38-like MAP protein kinase mediate ABA-induced H₂O₂ signaling in plant cells. Here we analyze the mechanisms of similarity and difference between MEK1/2 and p38-like MAP protein kinase in mediating ABA-induced H₂O₂ generation, inhibition of inward K⁺ currents, and stomatal closure. These data suggest that activation of MEK1/2 is prior to p38-like protein kinase in Vicia guard cells.Key words: H₂O₂ signaling, ABA, p38-like MAP kinase, MEK1/2, guard cellAn increasing number of literatures elucidate that reactive oxygen species (ROS), especially H₂O₂, is essential to plant growth and development in response to stresses,^1–4 and involves activation of various signaling events, among which are the MAP kinase cascades.^1–3,5 Typically, activation of MEK1/2 mediates NADPH oxidase-dependent ROS generation in response to stresses,^4,6–8 and the facts that MEK1/2 inhibits the expression and activation of antioxidant enzymes reveal how PD98059, the specific inhibitor of MEK1/2, abolishes abscisic acid (ABA)-induced H₂O₂ generation.^6,8,9 It has been indicated that PD98059 does not to intervene on salicylic acid (SA)-stimulated H₂O₂ signaling regardless of SA mimicking ABA in regulating stomatal closure.^2,6,8,10 Generally, activation of MEK1/2 promotes ABA-induced stomatal closure by elevating H₂O₂ generation in conjunction with inactivating anti-oxidases.Moreover, activation of plant p38-like protein kinase, the putative counterpart of yeast or mammalian p38 MAP kinase, has been reported to participate in various stress responses and ROS signaling. It has been well documented that p38 MAP kinase is involved in stress-triggered ROS signaling in yeast or mammalian cells.^11–13 Similar to those of yeast and mammals, many studies showed the activation of p38-like protein kinase in response to stresses in various plants, including Arabidopsis thaliana,^14–16 Pisum sativum,¹⁷ Medicago sativa¹⁸ and tobacco.¹⁹ The specific p38 kinase inhibitor SB203580 was found to modulate physiological processes in plant tissues or cells, such as wheat root cells,²⁰ tobacco tissue²¹ and suspension-cultured Oryza sativa cells.²² Recently, we investigate how activation of p38-like MAP kinase is involved in ABA-induced H₂O₂ signaling in guard cells. Our results show that SB203580 blocks ABA-induced stomatal closure by inhibiting ABA-induced H₂O₂ generation and decreasing K⁺ influx across the plasma membrane of Vicia guard cells, contrasting greatly with its analog SB202474, which has no effect on these events.^23,24 This suggests that ABA integrate activation of p38-like MAP kinase and H₂O₂ signaling to regulate stomatal behavior. In conjunction with SB203580 mimicking PD98059 not to mediate SA-induced H₂O₂ signaling,^23,24 these results generally reveal that the activation of p38-like MAP kinase and MEK1/2 is similar in guard cells.On the other hand, activation of p38-like MAP kinase^23,24 is not always identical to that of MEK1/2^8,25 in ABA-induced H₂O₂ signaling of Vicia guard cells. For example, H₂O₂^- and ABA-induced stomatal closure was partially reversed by SB203580. The maximum inhibition of both regent-induced stomatal closure were observed at 2 h after treatment with SB203580, under which conditions the stomatal apertures were 89% and 70% of the control values, respectively. By contrast, when PD98059 was applied together with ABA or H₂O₂, the effects of both ABA- and H₂O₂-induced stomatal closure were completely abolished (Fig. 1). These data imply that the two members of MAP kinase family are efficient in H₂O₂-stimulated stomatal closure, but p38-like MAP kinase is less susceptive than MEK1/2 to ABA stimuli.Open in a separate windowFigure 1Effects of SB203580 and PD98059 on ABA- and H₂O₂-induced stomatal closure. The experimental procedure and data analysis are according to the previous publication.^8,23,24It has been reported that ABA or NaCl activate p38 MAP kinase in the chloronema cells of the moss Funaria hygrometrica in 2∼10 min.²⁶ Similar to this, SB203580 improves H₂O₂-inhibited inward K⁺ currents after 4 min and leads it to the control level (100%) during the following 8 min (Fig. 2). However, the activation of p38-like MAP kinase in response to ABA need more time, and only recovered to 75% of the control at 8 min of treatment (Fig. 2). These results suggest that control of H₂O₂ signaling is required for the various protein kinases including p38-like MAP kinase and MEK1/2 in guard cells,^1,2,8,23,24 and the ABA and H₂O₂ pathways diverge further downstream in their actions on the K⁺ channels and, thus, on stomatal control. Other differences in action between ABA and H₂O₂ are known. For example, Köhler et al. (2001) reported that H₂O₂ inhibited the K⁺ outward rectifier in guard cells shows that H₂O₂ does not mimic ABA action on guard cell ion channels as it acts on the K⁺ outward rectifier in a manner entirely contrary to that of ABA.²⁷Open in a separate windowFigure 2Effect of SB203580 on ABA- and H₂O₂-inhibited inward K⁺ currents. The experimental procedure and data analysis are according to the previous publication.²⁴ SB203580 directs ABA- and H₂O₂-inactivated inward K⁺ currents across plasma membrane of Vicia guard cells. Here the inward K⁺ currents value is stimulated by −190 mV voltage.Based on the similarity and difference between PD98059 and SB203580 in interceding ABA and H₂O₂ signaling, we speculate the possible mechanism is that the member of MAP kinase family specially regulate signal event in ABA-triggered ROS signaling network,^1–4 and the signaling model as follows (Fig. 3).Open in a separate windowFigure 3Schematic illustration of MAP kinase-mediated H₂O₂ signaling of guard cells. The arrows indicate activation. The line indicates enhancement and the bar denotes inhibition.     

The Twisted Dwarf's ABC: How Immunophilins Regulate Auxin Transport

There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.Key Words: Twisted Dwarf1, plant development, auxin, immunophilin, P-glycoprotein, ABC transporterFK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.^1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.¹ All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.² The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca²⁺ release channels^6,7 and associates with the cell cycle regulator TGF-β.⁸ Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,⁹ leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.^3–5Open in a separate windowFigure 2Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,¹⁸ the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates¹⁷ because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers¹⁵ and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel).     

ARF-GTPase as a Molecular Switch for Polar Auxin Transport Mediated by Vesicle Trafficking in Root Development

Polar auxin transport (PAT), which is controlled precisely by both auxin efflux and influx facilitators and mediated by the cell trafficking system, modulates organogenesis, development and root gravitropism. ADP-ribosylation factor (ARF)-GTPase protein is catalyzed to switch to the GTP-bound type by a guanine nucleotide exchange factor (GEF) and promoted for hybridization to the GDP-bound type by a GTPase-activating protein (GAP). Previous studies showed that auxin efflux facilitators such as PIN1 are regulated by GNOM, an ARF-GEF, in Arabidopsis. In the November issue of The Plant Journal, we reported that the auxin influx facilitator AUX1 was regulated by ARF-GAP via the vesicle trafficking system.¹ In this addendum, we report that overexpression of OsAGAP leads to enhanced root gravitropism and propose a new model of PAT regulation: a loop mechanism between ARF-GAP and GEF mediated by vesicle trafficking to regulate PAT at influx and efflux facilitators, thus controlling root development in plants.Key Words: ADP-ribosylation factor (ARF), ARF-GAP, ARF-GEF, auxin, GNOM, polar transport of auxinPolar auxin transport (PAT) is a unique process in plants. It results in alteration of auxin level, which controls organogenesis and development and a series of physiological processes, such as vascular differentiation, apical dominance, and tropic growth.² Genetic and physiological studies identified that PAT depends on efflux facilitators such as PIN family proteins and influx facilitators such as AUX1 in Arabidopsis.Eight PIN family proteins, AtPIN1 to AtPIN8, exist in Arabidopsis. AtPIN1 is located at the basal side of the plasma membrane in vascular tissues but is weak in cortical tissues, which supports the hypothesis of chemical pervasion.³ AtPIN2 is localized at the apical side of epidermal cells and basally in cortical cells.^1,4 GNOM, an ARF GEF, modulates the localization of PIN1 and vesicle trafficking and affects root development.^5,6 The PIN auxin-efflux facilitator network controls root growth and patterning in Arabidopsis.⁴ As well, asymmetric localization of AUX1 occurs in the root cells of Arabidopsis plants,⁷ and overexpression of OsAGAP interferes with localization of AUX1.¹ Our data support that ARF-GAP mediates auxin influx and auxin-dependent root growth and patterning, which involves vesicle trafficking.¹ Here we show that OsAGAP overexpression leads to enhanced gravitropic response in transgenic rice plants. We propose a model whereby ARF GTPase is a molecular switch to control PAT and root growth and development.Overexpression of OsAGAP led to reduced growth in primary or adventitious roots of rice as compared with wild-type rice.¹ Gravitropism assay revealed transgenic rice overxpressing OsAGAP with a faster response to gravity than the wild type during 24-h treatment. However, 1-naphthyl acetic acid (NAA) treatment promoted the gravitropic response of the wild type, with no difference in response between the OsAGAP transgenic plants and the wild type plants (Fig. 1). The phenotype of enhanced gravitropic response in the transgenic plants was similar to that in the mutants atmdr1-100 and atmdr1-100/atpgp1-100 related to Arabidopsis ABC (ATP-binding cassette) transporter and defective in PAT.⁸ The physiological data, as well as data on localization of auxin transport facilitators, support ARF-GAP modulating PAT via regulating the location of the auxin influx facilitator AUX1.¹ So the alteration in gravitropic response in the OsAGAP transgenic plants was explained by a defect in PAT.Open in a separate windowFigure 1Gravitropism of OsAGAP overexpressing transgenic rice roots and response to 1-naphthyl acetic acid (NAA). (A) Gravitropism phenotype of wild type (WT) and OsAGAP overexpressing roots at 6 hr gravi-stimulation (top panel) and 0 hr as a treatment control (bottom panel). (B) Time course of gravitropic response in transgenic roots. (C and D) results correspond to those in (A and B), except for treatment with NAA (5 × 10⁻⁷ M).The polarity of auxin transport is controlled by the asymmetric distribution of auxin transport proteins, efflux facilitators and influx carriers. ARF GTPase is a key member in vesicle trafficking system and modulates cell polarity and PAT in plants. Thus, ARF-GDP or GTP bound with GEF or GAP determines the ARF function on auxin efflux facilitators (such as PIN1) or influx ones (such as AUX1).ARF1, targeting ROP2 and PIN2, affects epidermal cell polarity.⁹ GNOM is involved in the regulation of PIN1 asymmetric localization in cells and its related function in organogenesis and development.⁶ Although VAN3, an ARF-GAP in Arabidopsis, is located in a subpopulation of the trans-Golgi transport network (TGN), which is involved in leaf vascular network formation, it does not affect PAT.¹⁰ OsAGAP possesses an ARF GTPase-activating function in rice.¹¹ Specifically, our evidence supports that ARF-GAP bound with ARF-GTP modulates PAT and gravitropism via AUX1, mediated by vesicle trafficking, including the Golgi stack.¹Therefore, we propose a loop mechanism between ARF-GAP and GEF mediated by the vascular trafficking system in regulating PAT at influx and efflux facilitators, which controls root development and gravitropism in plants (Fig. 2). Here we emphasize that ARF-GEF catalyzes a conversion of ARF-bound GDP to GTP, which is necessary for the efficient delivery of the vesicle to the target membrane.¹² An opposite process of ARF-bound GDP to GTP is promoted by ARF-GTPase-activating protein via binding. A loop status of ARF-GTP and ARF-GDP bound with their appurtenances controls different auxin facilitators and regulates root development and gravitropism.Open in a separate windowFigure 2Model for ARF GTPase as a molecular switch for the polar auxin transport mediated by the vesicle traffic system.     

Leaf carbohydrate metabolism during defense: Intracellular sucrose-cleaving enzymes do not compensate repression of cell wall invertase

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild type (wt) tobacco Nicotiana tabacum L. Samsun NN (SNN) with plants with RNA interference-mediated repression of cwINV (SNN::cwINV) during the interaction with the oomycetic phytopathogen Phytophthora nicotianae. We have previously shown that the transgenic plants developed normally under standard growth conditions, but exhibited weaker defense reactions in infected source leaves and were less tolerant to the pathogen. Here, we show that repression of cwINV was not accompanied by any compensatory activities of intracellular sucrose-cleaving enzymes such as vacuolar and alkaline/neutral invertases or sucrose synthase (SUSY), neither in uninfected controls nor during infection. In wt source leaves vacuolar invertase did not respond to infection, and the activity of alkaline/neutral invertases increased only slightly. SUSY however, was distinctly stimulated, in parallel to enhanced cwINV. In SNN::cwINV SUSY-activation was largely repressed upon infection. SUSY may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions. Its activity, however, seems to be directly affected by cwINV and the related reflux of carbohydrates from the apoplast into the mesophyll cells.Key words: cell wall invertase, apoplastic invertase, alkaline invertase, neutral invertase, sucrose synthase, plant defense, Nicotiana tabacum, Phytophthora nicotianaePlant defense against pathogens is costly in terms of energy and carbohydrates.^1,2 Sucrose (Suc) and its cleavage products glucose and fructose are central molecules for metabolism and sensing in higher plants (reviewed in refs. ³ and ⁴). Rapid mobilization of these carbohydrates seems to be an important factor determining the outcome of plant-pathogen interactions. In particular in source cells reprogramming of the carbon flow from Suc to hexoses may be a crucial process during defense.^1,2There are two alternative routes of sucrolytic carbohydrate mobilization. One route is reversible and involves an uridine 5′-diphosphate (UDP)-dependent cleavage catalyzed by sucrose synthase (SUSY). Its activity is limited by the concentrations of Suc and UDP in the cytosol, as the affinity of the enzyme to its substrate is relatively low (K_m for Suc 40–200 mM). The other route is the irreversible, hydrolytic cleavage by invertases (INVs), which exhibit high affinity to Suc (K_m 7–15 mM).⁵Plants possess three different types of INV isoenzymes, which can be distinguished by their solubility, subcellular localization, pH-optima and isoelectric point. Usually, they are subdivided into cell wall (cwINV), vacuolar (vacINV), and alkaline/neutral (a/nINVs) INVs.cwINV, also referred to as extracellular or apoplastic INV, is characterized by a low pH-optimum (pH 3.5–5.0) and usually ionically bound to the cell wall. It is the key enzyme of the apoplastic phloem unloading pathway and plays a crucial role in the regulation of source/sink relations (reviewed in refs. ^{3, 6–8}). A specific role during plant defense has been suggested, based on observations that cwINV is often induced during various plant-pathogen interactions, and the finding that overexpression of a yeast INV in the apoplast increases plant resistance.^6,8–10 It was shown, that a rapid induction of cwINV is, indeed, one of the early defense-related reactions in resistant tobacco source leaves after infection with Phytophthora nicotianae (P. nicotianae).¹¹ Finally, the whole infection area in wt leaves was covered with hypersensitive lesions, indicating that all cells had undergone hypersensitive cell death (Fig. 1A).^1,11 When the activity of cwINV was repressed by an RNAi construct, defense-related processes were impaired, and the infection site exhibited only small spots of hypersensitive lesions. Finally, the pathogen was able to sporulate, indicating a reduced resistance of these transgenic plants (Fig. 1A).¹Open in a separate windowFigure 1Defense-induced changes in the activity of intracellular sucrose-cleaving enzymes and their contribution to defense. (A) The repression of cwINV in source leaves of tobacco leads to impaired pathogen resistance and can not be compensated by other sucrose-cleaving enzymes. The intensity of defense reactions is amongst others indicated by the extent of hypersensitive lesions. (B and C) Absolute activity of vacuolar (B) and alkaline/neutral (C) INVs at the infection site (white symbols, control; black symbols, infection site). (D) Increase in SUSY activity at the infection site. All data points taken from noninfected control parts of the plants in each individual experiment and each point along the time scale of an experiment are set as 0%. At least three independent infections are averaged and their means are presented as percentage changes ± SE (circles, SNN; triangles, SNN::cwINV). Insets show the means of the absolute amount of activities (white symbols, control; black symbols, infection site). Material and methods according to Essmann, et al.¹vacINV, also labeled as soluble acidic INV, is characterized by a pH optimum between pH 5.0–5.5. Among others it determines the level of Suc stored in the vacuole and generates hexose-based sugar signals (reviewed in refs. ³ and ¹²). Yet, no specific role of vacINV during pathogen response has been reported. Although vacINV and cwINV are glycoproteins with similar enzymatic and biochemical properties and share a high degree of overall sequence homology and two conserved amino acid motifs,⁴ the activity of vacINV in tobacco source leaves was not changed due to the repression of the cwINV (Fig. 1B).¹ After infection with P. nicotianae the activity of vacINV in wt SNN did not respond under conditions where cwINV was stimulated.¹ There was also no significant change in the transgenic SNN::cwINV (Fig. 1B). This suggests that during biotic stress, there is no crosstalk between the regulation of cwINV and vacINV.a/nINVs exhibit activity maxima between pH 6.5 and 8.0, are not glycosylated and thought to be exclusively localized in the cytosol. But recent reports also point to a subcellular location in mitochondria and chloroplasts.^13,14 Only a few a/nINVs have been cloned and characterized, and not much is known about their physiological functions (reviewed in refs. ^{4, 14} and ¹⁵). Among other things they seem to be involved in osmotic or low-temperature stress response.^14,15 During the interaction between tobacco and P. nicotianae the activity of a/nINVs rose on average 17% in the resistant wt SNN between 1 to 9 hours post infection (Fig. 1C). By contrast, in SNN::cwINV the a/nINVs activities remained unchanged in control leaves and even after infection (Fig. 1C). This suggests that the defense related stimulation in a/nINVs activities is rather a secondary phenomenon, possibly in response to the enhanced cwINV activity and the related carbohydrate availability in the cytosol.SUSY can be found as a soluble enzyme in the cytosol, bound to the inner side of the plasma membrane or the outer membrane of mitochondria, depending on the phosphorylation status. It channels hexoses into polysaccharide biosynthesis (i.e., starch, cellulose and callose) and respiration.^12,16 There is also evidence that SUSY improves the metabolic performance at low internal oxygen levels¹⁷ but little is known about its role during plant defense. Callose formation is presumably one of the strongest sink reactions in plant cells.^1,18 Defense-related SUSY activity may serve to allocate Suc into callose deposition and other carbohydrate-consuming defense reactions. In fact, in the resistant wt the activity of SUSY increased upon interaction with P. nicotianae in a biphasic manner (Fig. 1D). The time course is comparable to that of cwINV activity and correlates with callose deposition and enhanced respiration.^1,11 However, repression of cwINV leads in general to a reduction of SUSY activity in source leaves of tobacco.¹ After infection the activation of SUSY was also significantly impaired (Fig. 1D). At the same time, the early defense-related callose deposition in infected mesophyll cells of SNN::cwINV plants is substantially delayed.¹ It is known that expression of SUSY isoforms is differentially controlled by sugars,¹² and there is evidence that hexoses generated by the defense-induced cwINV activity deliver sugar signals to the infected cells.¹ In this sense, the reduction of defense-related, cwINV-generated sugar signals could be responsible for the repression of SUSY activity in SNN::cwINV plants after infection with P. nicotianae.Only limited hexoses or hexose-based sugar signals could be generated by cytoplasmic Suc cleavage.¹² The reduction of soluble carbohydrates for sugar signaling and also as fuel for metabolic pathways that support defense reactions could be responsible for the impaired resistance in SNN::cwINV plants (Fig. 1A).Obviously, neither intracellular INV isoforms, nor SUSY can compensate for the reduced carbohydrate availability due to cwINV repression during plant defense. The data also suggest that the activity of SUSY is affected by cwINV and related reflux of carbohydrates. It is known that SUSY activity can be controlled, e.g., by sugar-mediated phosphorylation¹² and one may speculate that posttranslational modulation of the protein is affected by the defense-related carbohydrate status of the cell.     

Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and thioflavin T binding

Characterization of aggregation profiles of monoclonal antibodies (mAb) is gaining importance because an increasing number of mAb-based therapeutics are entering clinical studies and gaining marketing approval. To develop a successful formulation, it is imperative to identify the critical biochemical properties of each potential mAb drug candidate. We investigated the conformational change and aggregation of a human IgG1 using external dye-binding experiments with fluorescence spectroscopy and compared the aggregation profiles obtained to the results of size-exclusion chromatography. We show that using an appropriate dye at selected mAb concentration, unfolding or aggregation can be studied. In addition, dye-binding experiments may be used as conventional assays to study therapeutic mAb stability.Key words: therapeutic monoclonal antibody, protein aggregation, conformational change, stability and shelf-life prediction, accelerated studiesMonoclonal antibodies (mAbs) have emerged as a novel class of protein drugs and are utilized for a variety of mostly incurable and debilitating diseases such as cancer and rheumatoid arthritis.^1–4 For treatment of chronic diseases, it is desirable for these drugs to be administered subcutaneously, in which case high protein concentrations (>100 mg/mL) are generally needed.^5,6 Protein-based drugs containing mAbs must contain minimum amounts of aggregation and fragmentation and conserve their structural integrity during storage because degraded or aggregated protein may induce immunogenicity or reduce efficacy. Currently, size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is the most commonly used method to characterize mAb aggregation profiles;⁷ however it is time consuming, expensive and requires expertise. SEC-HPLC cannot be used to obtain accurate biophysical profiles of mAbs at high concentrations because dilution during the experiment might lead to reversible aggregation. Furthermore, the potential interaction of aggregates with surfaces, e.g., needle, tubing, column, will lead to the loss of sample and thus an inaccurate analysis.^8,9 Additional drawbacks of the technique are that different conformations such as partially unfolded monomers also cannot be distinguished by SEC-HPLC and large aggregates may be totally excluded during the injection into the column.External dye binding assays have been used to characterize protein stability and aggregation,^10–12 and studies involving biopharmaceuticals have been reported recently, e.g., for thermostability screening¹⁰ and detection of aggregation.^11–14 These methods are not limited by protein quantity and are more sensitive because they are fluorescence-based. We studied the accelerated unfolding of an IgG1 mAb with the hydrophobic dye 1-anilino-8-naphthale-nesulfonate (ANS), and its accelerated aggregation with aggregate specific Thioflavin T (ThT). We have also conducted accelerated aggregation studies with SEC-HPLC7 and compared the findings to the ThT binding results. We hypothesize that key structures formed during mAb aggregation can be probed selectively by the appropriate dyes (Fig. 1) with specific mAb concentrations.Open in a separate windowFigure 1Key structures of the mAb probed by fluorescent dyes. N and U are native and unfolded monomers, respectively. “n” reactive monomers form aggregates.     

Significance of microtubule catastrophes at focal adhesion sites

Directional cell migration requires cell polarization and asymmetric distribution of cell signaling. Focal adhesions and microtubules are two systems which are essential for these. It was shown that these two systems closely interact with each other. It is known that microtubule targeting stimulates focal adhesion dissociation. Our recent study shows that focal adhesions, in turn, specifically induce microtubule catastrophe via a biochemical mechanism. We were able to track down one of the focal adhesion proteins paxillin which is involved in this process. Paxillin phosphorylation was previously shown to be the key component in the regulation of focal adhesion assembly or disassembly. Since microtubule catastrophe dynamic differs at the leading edge and cell rear, similar to paxillin phosphorylation levels, we suggest a model connecting asymmetric distribution of focal adhesions and asymmetric distribution of microtubule catastrophes at adhesion sites as a feedback loop.Key words: microtubule catastrophe, focal adhesion, microtubules, paxillin, cell motilityCell migration is important for many biological processes. It requires organized asymmetric dynamics of focal adhesions (FAs), sites where cells interact with extra cellular matrix. FAs appear at the leading edge as small transient dot-like structures termed focal complexes (FXs).^1,2 FX assembly and disassembly is regulated by phosphorilation status of paxillin a major FX protein.^3,4 Most of FXs form and disassemble rapidly. However, some adhesions mature in a force-dependent manner, into larger late adhesions. This process, involves both an increase in size and change in molecular composition^3,5 and is accompanied by a reduction in local paxillin phosphorylation.⁴ Late adhesions are more stable, immobile and undergo forced disassembly by multiple microtubule targeting events⁶ only underneath the approaching cell body or transform into fibrillar adhesions by a Src-dependent mechanism.⁷Similarly to the leading edge, proper adhesion patterns at the cell rear are also essential. Most trailing adhesions are initiated in protrusions at the rear and flanks of the cell as FX rapidly mature in response to tension and transform into sliding trailing adhesions.⁸ The process of sliding is complex. While adhesion proteins coupled with the actin cytoskeleton can be translocated relative to substratum, those that are associated with the membrane are thought to undergo treadmilling within the adhesion site.^9,10 Treadmilling, which includes disassembly of adhesion proteins at the distal end and reassembly at the proximal end,¹⁰ is accompanied by fusion with new adhesions formed in front of the sliding one.⁶ Thus, despite a protein composition similar to late adhesions, sliding adhesions are more dynamic. Not surprisingly, sliding adhesions have high paxillin phosphorylation at the distal end of the adhesion site, indicating very dynamic assembly/disassembly rates.⁴Several mechanisms have been proposed for the regulation of adhesion turnover (reviewed in ref. ¹¹). However, these have not accounted for the observed asymmetry of adhesion turnover. Understanding this requires examining the connection with another asymmetric intracellular system, the microtubule network. This dynamic network closely interacts with FAs. Microtubules play an essential role in cell migration and polarized distribution of signals within the cell. Multiple microtubule targeting to FA leads to their disassembly both at the leading edge and at the cell rear.⁶Unlike microtubule growth in other cell regions, growth at its leading edge is persistent, characterized by short periods of shrinkage.⁸ Simultaneous observation of microtubules and FAs show that microtubules specifically target adhesion sites.¹² More detailed analysis of microtubule dynamics reveals that FAs are preferable sites for microtubule catastrophes.¹³ Although FAs cover only about 5% of cell area more than 40% of catastrophes occur at these sites. The likelihood of microtubule catastrophe is seven times higher when a microtubule grows through a FA rather than through an adhesion-free area¹³ and about 90% of microtubules approaching adhesion sites undergo catastrophe. Although most of the catastrophes occur at late adhesions, due to their increased stability and lifespan, there is no difference in efficiency of catastrophe induction between small focal complexes and large rigid late adhesions.¹³ As FX do not have dense adhesion or actin plaque, it is likely that microtubule catastrophe is triggered by a biochemical mechanism rather than mechanical rigidity. This is also supported by the fact that mechanical obstacles in a cell do not necessarily cause microtubule catastrophe.¹³At the cell rear, microtubule dynamics differ from those at the leading edge. Microtubules spend less time in a growing phase and more time in pauses and shrinkage.⁸ Polymerization and depolymerization occur within a very limited area close to the cell edge.⁸ Live-cell imaging of cells expressing both microtubule and focal adhesion markers show that this complex dynamic sequence often happens within a single sliding adhesion. Microtubules that are captured at the proximal end of adhesion undergo multiple repetitive catastrophes at the distal end (Fig. 1) accompanied by rescue at the capture site. Thus, the capture mechanism significantly increases the lifetime of a microtubule and ensures that repetitive catastrophes occur at the single adhesion. This scenario leads to high catastrophe frequency at the cell rear, resulting in intensive catastrophe-dependent regulation in this cell region.Open in a separate windowFigure 1Multiple microtubule catastrophes at the sliding adhesion. (A) Frame from TIRF video sequence of a fish fibroblast cell (CAR) co-transfected with GFP-tubulin (green) to visualize microtubules and Cherry-Zyxin (red) to mark focal adhesions. The boxed region is presented in the kymograph in (B). Bar, 10 µm. (B) Kymograph of microtubule dynamics at a trailing end focal adhesion. Top panel shows microtubule (MT) only. Bottom panel shows life history plot of MT (green line shows movement of MT end) in relation to focal adhesions (red). Arrows show catastrophes at the distal end of adhesion, arrowheads show capture at the proximal end of adhesion.Detailed analysis of microtubule catastrophe localization shows that they occur at the areas of FAs where paxillin is enriched and highly phosphorylated.^4,13 Paxillin was shown to interact with microtubules through its Lim2/Lim3 domain.¹⁴ Purified GST-Lim2/Lim3 fragment injected into the cell localizes to FAs, displacing endogenous paxillin.¹³ This leads to a 40% decrease in the number of microtubule catastrophe events at adhesion sites,¹³ indicating that paxillin is needed for catastrophe initiation.In summary, we conclude that microtubule catastrophes at focal adhesions are specific events that are triggered by a biochemical mechanism. This process involves the focal adhesion protein paxillin, which may serve as a docking site for microtubules and/or microtubule catastrophe factors. The nature of catastrophe factors remains to be clarified. Possible mechanisms include molecules which induce microtubule catastrophe directly, such as stathmin,¹⁵ or molecules which regulate catastrophe-inducing factors activity. Alternatively, catastrophe factors at adhesion sites could act by removing stabilizing factors from microtubule tips. Thus, allowing already active catastrophe-inducing molecules such as kinesin-13 family member MCAK^16,17 to complete their function. Furthermore, microtubule catastrophe at paxillin-enriched areas, followed by release of microtubule-associated factors, may be involved in paxillin phosphorylation. This local regulation of adhesion disassembly would close the feed-back loop to microtubule regulation of FA turnover.In this model, asymmetric distribution of microtubule catastrophes is tightly linked to asymmetric regulation of FA. Since asymmetric FA dynamics in a cell are critical for organization of the actin cytoskeleton, tensile force distribution and directional cell migration, we conclude that microtubule catastrophes serve as important regulatory events for asymmetric signaling and dynamics of the whole cell (Fig. 2).Open in a separate windowFigure 2Model for asymmetric focal adhesion and microtubule dynamics. Focal complexes at the leading edge either disassemble or mature in response to tension. Microtubules undergo catastrophe both at focal complexes and late adhesions. Late adhesions disassemble in response to multiple microtubule targeting. At the cell rear a microtubule is captured at the proximal end of sliding adhesion and undergoes multiple catastrophes at its distal end, supporting disassembly of this region.     

Reactive oxygen species as transducers of sphinganine-mediated cell death pathway

Long chain bases or sphingoid bases are building blocks of complex sphingolipids that display a signaling role in programmed cell death in plants. So far, the type of programmed cell death in which these signaling lipids have been demonstrated to participate is the cell death that occurs in plant immunity, known as the hypersensitive response. The few links that have been described in this pathway are: MPK6 activation, increased calcium concentrations and reactive oxygen species (ROS) generation. The latter constitute one of the more elusive loops because of the chemical nature of ROS, the multiple possible cell sites where they can be formed and the ways in which they influence cell structure and function.Key words: hydrogen peroxide, long chain bases, programmed cell death, reactive oxygen species, sphinganine, sphingoid bases, superoxideA new transduction pathway that leads to programmed cell death (PCD) in plants has started to be unveiled.^1,2 Sphingoid bases or long chain bases (LCBs) are the distinctive elements in this PCD route that naturally operates in the entrance site of a pathogen as a way to contend its spread in the plant tissues.^2,3 This defense strategy has been known as the hypersensitive response (HR).^4,5As a lately discovered PCD signaling circuit, three connected transducers have been clearly identified in Arabidopsis: the LCB sphinganine (also named dihydrosphingosine or d18:0); MPK6, a mitogen activated kinase and superoxide and hydrogen peroxide as reactive oxygen species (ROS).^1,2 In addition, calcium transients have been recently allocated downstream of exogenously added sphinganine in tobacco cells.⁶Contrary to the signaling lipids derived from complex glycerolipid degradation, sphinganine, a metabolic precursor of complex sphingolipids, is raised by de novo synthesis in the endoplasmic reticulum to mediate PCD.^1,2 Our recent work demonstrated that only MPK6 and not MPK3 (commonly functionally redundant kinases) acts in this pathway and is positioned downstream of sphinganine elevation.² Although ROS have been identified downstream of LCBs in the route towards PCD,¹ the molecular system responsible for this ROS generation, their cellular site of formation and their precise role in the pathway have not been unequivocally identified. ROS are produced in practically all cell compartments as a result of energy transfer reactions, leaks from the electron transport chains, and oxidase and peroxidase catalysis.⁷Similar to what is observed in pathogen defense,³ increases in endogenous LCBs may be elicited by addition of fumonisin B1 (FB1) as well; FB1 is a mycotoxin that inhibits ceramide synthase. This inhibition results in an accumulation of its substrate, sphinganine and its modified forms, leading to the activation of PCD.^1,2,8 The application of FB1 is a commonly used approach for the study of PCD elicitation in Arabidopsis.^1,2,9–11An early production of ROS has been linked to an increase of LCBs. For example, an H₂O₂ burst is found in tobacco cells after 2–20 min of sphinganine supplementation,¹² and superoxide radical augmented in the medium 60 min after FB1 or sphinganine addition to Arabidopsis protoplasts (Fig. 1A). In consonance with this timing, both superoxide and H₂O₂ were detected in Arabidopsis leaves after 3–6 h exposure to FB1 or LCBs.¹ However, the source of ROS generation associated with sphinganine elevation seems to not be the same in both species: in tobacco cells, ROS formation is apparently dependent on a NADPH oxidase activity, a ROS source consistently implicated in the HR,^13,14 while in Arabidopsis, superoxide formation was unaffected by diphenyliodonium (DPI), a NADPH oxidase inhibitor (Fig. 1A). It is possible that the latter oxidative burst is due to an apoplastic peroxidase,¹⁵ or to intracellular ROS that diffuse outwards.^16,17 These results also suggest that both tobacco and Arabidopsis cells could produce ROS from different sources.Open in a separate windowFigure 1ROS are produced at early and long times in the FB1-induced PCD in Arabidopsis thaliana (Col-0). (A) Superoxide formation by Arabidopsis protoplasts is NADPH oxidase-independent and occurs 60 min after FB1 or sphinganine (d18:0) exposure. Protoplasts were obtained from a cell culture treated with cell wall lytic enzymes. Protoplasts were incubated with 10 µM FB1 or 10 µM sphinganine for 1 h. Then, cells were vacuum-filtered and the filtrate was used to determine XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt] reduction as described in references ²⁸ and ²⁹. DPI was used at 50 µM. (B) H₂O₂ formation in Arabidopsis wt and lcb2a-1 mutant in the presence and absence of FB1. Arabidopsis seedlings were exposed to 10 µM FB1 and after 48 h seedlings were treated with DA B (3,3-diaminobencidine) to detect H₂O₂ according to Thordal-Christensen et al.³⁰It has been suggested that the H₂O₂ burst associated with the sphinganine signaling pathway leads to the expression of defense-related genes but not to the PCD itself in tobacco cells.¹² It is possible that ROS are involved in the same way in Arabidopsis, since defense gene expression is also induced by FB1 in Arabidopsis.⁹ In this case, it will be important to define how the early ROS that are DPI-insensitive could contribute to the PCD manifestation mediated by sphinganine.The generation of ROS (4–60 min) found in Arabidopsis was associated to three conditions: the addition of sphinganine (Fig. 1A), FB1 (Fig. 1A) or pathogen elicitors.¹⁵ This is consistent with the MPK6 activation time, which is downstream of sphinganine elevation and occurs as early as 15 min of FB1 or sphinganine exposure.² All of them are events that appear as initial steps in the relay pathway that produces PCD.In order to explore a possible participation of ROS at more advanced times of PCD progression, we detected in situ H₂O₂ formation in Arabidopsis seedlings previously exposed to FB1 for 48 h. As shown in Figure 1B, formation of the brown-reddish precipitate corresponding to the reaction of H₂O₂ with 3,3′-diaminobenzidine (DAB) was only visible in the FB1-exposed wild type plants, as compared to the non-treated plants. However, when lcb2a-1 mutant seedlings were used, FB1 exposure had a subtle effect in ROS formation. This mutant has a T-DNA insertion in the gene encoding subunit LCB2a from serine palmitoyltransferase (SPT), which catalyzes the first step in sphingolipid synthesis¹⁸ and the mutant has a FB1-resistant phenotype.² These results indicate that mutations in the LCB1¹ and LCB2a² genes (coding for the subunits of the heterodimeric SPT) that lead to a non-PCD phenotype upon the FB1 treatment, are unable to produce H₂O₂. In addition, they suggest that high levels of hydrogen peroxide are produced at advanced times in the PCD mediated by LCBs in Arabidopsis.Exposure of Arabidopsis to an avirulent strain of Pseudomonas syringae produces an endogenous elevation of LCBs as a way to implement defense responses that include HR-PCD.³ In this condition, we clearly detected H₂O₂ formation inside chloroplasts (Fig. 2A). When ultrastructure of the seedlings tissues exposed to FB1 for 72 h was analyzed, integrity of the chloroplast membrane system was severely affected in Arabidopsis wild-type seedlings exposed to FB1.² Therefore, we suggest that ROS generation-LCB induced in the chloroplast could be responsible of the observed membrane alteration, as noted by Liu et al. who found impairment in chloroplast function as a result of H₂O₂ formation in this organelle from tobacco plants. Interestingly, these plants overexpressed a MAP kinase kinase that activated the kinase SIPK, which is the ortholog of the MPK6 from Arabidopsis, a transducer in the PCD instrumented by LCBs.²Open in a separate windowFigure 2Conditions of LCBs elevation produce H₂O₂ formation in the chloroplast and perturbation in the membrane morphology of mitochondria. (A) Exposure of Arabidopsis leaves to the avirulent strain Pseudomonas syringae pv. tomato DC3000 (avrRPM1) (or Pst avrRPM1) induces H₂O₂ formation in the chloroplast. Arabidopsis leaves were infiltrated with 1 × 10⁸ UFC/ml Pst avrRPM1 and after 18 h, samples were treated to visualize H₂O₂ formation with the DAB reaction. Controls were infiltrated with 10 mM MgCl₂ and then processed for DAB staining. Then, samples were analyzed in an optical photomicroscope Olympus Provis Model AX70. (B) Effect of FB1 on mitochondria ultrastructure. Wild type Arabidopsis seedlings were treated with FB1 for 72 h and tissues were processed and analyzed according to Saucedo et al.² Ch, chloroplast; M, mitochondria; PM, plasma membrane. Arrows show mitochondrial cisternae. Bars show the correspondent magnification.In addition, we have detected alterations in mitochondria ultrastructure as a result of 72 h of FB1 exposure (Fig. 2B). These alterations mainly consist in the reduced number of cristae, the membrane site of residence of the electron transport complexes. In this sense, it has been shown that factors that induce PCD such as the victorin toxin, methyl jasmonate and H₂O₂ produce alterations in mitochondrial morphology.^20–22 In fact, some of these studies propose that ROS are formed in the mitochondria and then diffuse to the chloroplasts.^22–24It is reasonable to envisage that damage of the membrane integrity of these two organelles reflects the effects of vast amounts of ROS produced by the electron transport chains.^25,26 Recent evidence supports the destruction of the photosynthetic apparatus associated to the generation of ROS in the HR.²⁶ At this time of PCD progression, ROS could be contributing to shut down the energy machinery in the cell, which ultimately would become the point of no-return of PCD²⁷ as part of the execution program of the cell death mediated by LCBs.In conclusion, we propose that ROS can display two different functional roles in the PCD process driven by LCBs. These roles depend on the time of ROS expression, the cellular site where they are generated, the enzymes that produce them, and the magnitude in which they are formed.     

Induction as well as suppression: How aphid saliva may exert opposite effects on plant defense

Aphids ingest from the sieve tubes and by doing so they are confronted with sieve-tube occlusion mechanisms, which are part of the plant defense system. Because aphids are able to feed over longer periods, they must be able to prevent occlusion of the sieve plates induced by stylet penetration. Occlusion probably depends upon Ca²⁺-influx into the sieve element (SE) lumen. Aphid behavior, biochemical tests and in vitro experiments demonstrated that aphid''s watery saliva, injected during initial phase of a stylet penetration into the SE lumen, contains proteins that are able to bind calcium and prevent calcium-induced SE occlusion. In this addendum, we speculate on the consequences of saliva secretion for plant resistance. (a) The release of elicitors (e.g., oligogalacturonides) due to cell wall digestion by gel saliva enzymes may increase the resistance of cortex, phloem parenchyma cells and companion cells (CC) around the puncture site. (b) Ca²⁺-binding by aphid watery saliva may suppress the local defense responses in the SEs. (c) Signaling cascades triggered in CCs may lead to systemic resistance.Key words: aphid saliva, calcium binding, elicitor, oligogalacturonides, local plant defense, systemic plant defense, phloem translocation, aphid/plant-interactionAfter having penetrated the sieve-element (SE) plasma membrane, aphids encounter unspecific wound-induced occlusion reactions to prevent sap leakage.^1–4 Occlusion mechanisms by callose, structural P-proteins and forisomes are likely induced by a sudden calcium influx into the sieve-tube lumen.⁵ Calcium possibly enters the sieve-tube lumen through the stylet wounding-site in the plasma membrane and/or stretch-activated calcium-channels.^6–8 After SE penetration, aphids secrete watery saliva that contains calcium-binding proteins presumed to sabotage sieve-plate occlusion.^9,10We demonstrated that Megoura viciae (Buckton) is most likely able to prevent or reduce sieve-tube occlusion in Vicia faba by secretion of watery saliva. By in vitro confrontation of isolated forisomes, protein bodies responsible for sieve-tube occlusion in Fabaceaen,⁵ and watery saliva concentrate, we were able to show that salivary proteins convey forisomes from a dispersed (+Ca²⁺) into a condensed (−Ca²⁺) state.¹⁰ The dispersed forisome functions in vivo as a plug, leading to stoppage of mass flow.⁵This in vitro evidence was corroborated by aphid behavior in response to leaf tip burning, which triggers an electrical potential wave (EPW) along the sieve tubes. Such an EPW induces Ca²⁺-influx and corresponding SE occlusion along the pathway.¹¹ The passage of the EPW is associated with a prolonged secretion of watery saliva of aphids. This is interpreted as an attempt to unplug the SEs by calcium binding.¹⁰ Similar behavioral changes in response to leaf-tip burning were observed in an extended set of aphid/plant species combinations, indicating that attempted sabotage of sieve-tube occlusion by aphid saliva is a widespread phenomenon (unpublished).Aphid feeding was reported to induce local (on the same leaf) and systemic (in distant leaves) reactions of the host plant. The local response led to enhanced feeding,^12–14 while the systemic response showed reduced ingestion and extended periods of watery saliva secretion in sieve tubes distant from previous feeding sites.^12–14 These contrasting observations were described to be independent of the aphid species.¹³ The question arises how aphids induce these seemingly opposite plant responses?The aphid stylet pushing forward through cortical and vascular tissue is surrounded by a sheath of gel saliva, secreted into the apoplast.^15,16 Gel saliva contains cellulase and pectinase that amongst others produce oligogalacturonides (OGs) along the stylet sheath by digestion of cell wall material.^17,18 Usually, OGs act as elicitors, triggering a variety of plant responses against pathogens and insects in which the activation of calcium channels is involved.^19,20 This seems to conflict with a suppression of resistance as observed for the impact of watery saliva in SEs.¹⁰ We will make an attempt to explain this paradoxon.OG induced defense responses may be triggered in all cell types adjacent to the salivary sheath (Fig. 1). Because watery saliva is only secreted briefly into these cells, which are punctured for orientation purposes (Hewer et al., unpublished), it seems unlikely that OG induced defense is suppressed here by saliva-mediated calcium binding.¹⁵ The diffusion range of OGs may be restricted to the close vicinity of the stylet sheath leading to an enhanced regional defense with a limited sphere of action (Fig. 1). Because the settling distance of aphids is restricted by their body size (1–10 mm),²¹ aphids feeding on the same leaf are probably hardly confronted with the regional defense induced by another aphid (Fig. 1). Otherwise, they would show an increased number of test probes before first phloem activity, as described for volatile mediated plant defense in cortex cells.¹³ Circumstantial support in favor of our hypothesis is provided by production of hydrogen peroxide in the apoplast,²² which is most likely associated with the action of OGs.²² Observations of hydrogen peroxide production during aphid (Macrosiphum euphorbiae) infestation of tomato in a limited area along the leaf veins, the preferred feeding sites of this species, indicate a locally restricted defense response (Fig. 1 and ​and22).⁴ The question arises why the cell signals are not spread via plasmodesmata to adjacent cells to induce resistance in a more extended leaf area? Dissemination of the signals may be prevented by closure of plasmodesmata (Fig. 1) through callose deposition,^23,24 which is most likely directly coupled with calcium influx induced by OGs,²⁵ by apoplastic hydrogen peroxide and to a minor extent by stylet puncture (Fig. 2).^7,26Open in a separate windowFigure 1Hypothetical model on how stylet penetration induces and suppresses plant defense. Sheath saliva (light blue) that envelopes the stylet during propagation through the apoplast contains cellulase and pectinase,^17,18 enzymes producing elicitors (e.g., oligogalacturonides (oGs)) by local cell wall digestion.¹⁹ Parenchyma cells adjacent to the sheath may develop a defense response owing to signaling cascades triggered by oG-mediated Ca²⁺-influx.¹⁹ Together with a Ca²⁺-dependent transient closure of plasmodesmata by callose (black crosses),^23,24 the focused production of oGs may cause a defense response with a limited sphere of action (red—strong, brown—light, green—none). This restricted domain of defense may not be perceived by other aphids, since the settling distance is limited by the aphid body size. Nearby aphids do not show any sign of defense perception in their probing and feeding behavior.¹⁴ Signaling cascade compounds may be channeled from parenchyma cells to CCs (dashed yellow arrows), where they are subsequently released into the SEs. There they may act as long-distance systemic defense components (grey arrows). In contrast to the parenchyma domain (where only minor amounts of watery saliva are secreted), Ca²⁺-mediated reactions such as defense cascades and sieve-plate (SP) occlusion are suppressed in SEs by large amounts of watery saliva. The left aphid penetrates an SE and injects watery saliva (red cloud; ws) that inhibits local sieve-plate occlusion and,¹⁰ most likely, is transported by mass flow (black arrow) to adjacent SEs,²⁷ where occlusion is impeded as well. A short-distance systemic spread over a few centimeters may explain local suppression of plant defense resulting in a higher rate of colonization. Salivary proteins or their degradation products may serve as systemic defense signals as well (grey arrows), but may also diffuse via the PPUs into CCs where additional systemic signals are induced (yellow arrows).Open in a separate windowFigure 2Hypothetical involvement of Ca²⁺-channels in aphid-induced cell defense (detail). During probing with its stylet the aphid secretes gel saliva as a lubrication substance (light blue) into the apoplast.¹⁵ on the way to the sieve tubes, aphids briefly puncture most non-phloem cells (red) after which the puncturing sites are sealed with gel saliva.^7,16 Gel saliva also most likely prevents the influx of apoplastic calcium into pierced sieve elements (green) by sealing the penetration site.⁷ Watery saliva (red cloud), injected into the SE lumen,⁹ contains proteins which bind calcium ions (marked by X) that enter the SE via e.g., mechano sensitive Ca²⁺-channels activated by stylet penetration (blue tons).¹⁰ In this way, aphids suppress SE occlusion and activation of local defense cascades. In the parenchyma cells around the gel saliva sheath, a small cylindrical zone of defense may be induced by oligogalacturonides (oGs; brown triangles) produced by cell wall (grey) digestion.^17–19 Perceived by unknown receptor proteins (R; e.g., a receptor like protein kinase)³⁴ and kinase mediation (black dotted and dashed arrows), oGs lead to a Ca²⁺-influx through kinase activated calcium channels (orange tons).²⁵ Around the probing site, aphids apparently induce the production of superoxide by Ca²⁺-induced activation of the NADPH oxidase (violet box) and its following conversion to hydrogen peroxide (red spots) is mediated by superoxide dismutase (SoD).⁴ Hydrogen peroxide activates Ca²⁺-channels (violet tons) and diffuses through plasma membrane (curled arrows) therefore potentially acting as a intracellular signal.²⁶By contrast, Ca²⁺-influx into SEs, induced by presence of OGs or stylet insertion (Fig. 2), is not expected to trigger local defense given the abundant excretion of Ca²⁺-binding watery saliva.^7,10,25 Watery saliva may spread to down-stream and adjacent SEs through transverse and lateral sieve plates (Fig. 1).^7,27 Aphids puncturing nearby SEs may therefore encounter less severe sieve-plate occlusion which results in facilitated settling and thus in increased population growth. Aggregation of feeding aphids would self-amplify population growth until a certain density is attained. Farther from the colonization site, this effect may be lost due to dilution. Stimulation of aphid feeding by aphid infestation was observed locally on potato by Myzus persicae and M. euphorbiae, respectively, 96 h after infestation.¹³ However, a similar effect was not observed for M. persicae on Arabidopsis thaliana where aphids induced premature leaf senescence and resistance 12 h after infestation,²⁸ possibly induced by OGs.¹⁹As a speculation, OG induced Ca²⁺-influx into parenchyma cells adjacent to the salivary sheath activate Ca²⁺-induced signaling cascades via CaM,^26,29 CDPKs,^30,31 MAPKinases and reactive oxygen species (Fig. 2).³² Systemic resistance, induced by aphid infestation,^12–14 is mediated by unknown compounds such as, e.g., salivary proteins, their degradation products, signal cascade products or volatiles.¹³ Compounds produced in CCs first have to pass the PPUs, while SE signaling elements can be directly transported via mass flow (Fig. 1).The question arises if aphids profit from induced resistance on local (cortex and parenchyma cells) and systemic (distant plant organs) levels as holds for suppression of defense in SEs. Possibly settling and subsequent spread of competing pathogens/herbivores (e.g., fungi or other piercing-sucking insects) are suppressed by induced defense. In this context it is intriguing to understand how aphids cope with the self-induced systemic resistance, which probably lasts over weeks.³³     

Putting the brakes on cancer cell migration: JAM-A restrains integrin activation

Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, downregulation of JAM-A enhances cancer cell migration and invasion. Further, ectopic expression of JAM-A in highly metastatic MDA-MB-231 cells attenuates cell migration, and downregulation of JAM-A in low-metastatic T47D cells enhance migration. Interestingly, JAM-A expression is greatly diminished as breast cancer disease progresses. The molecular mechanism of this function of JAM-A is beyond its well-characterized barrier function at the tight junction. Our results point out that JAM-A differentially regulates migration of endothelial and cancer cells.Key words: JAM-A, integrin, α_vβ₃, FGF-2, breast cancer, cell migration and invasion, T47D, MDA-MB-231, siRNAEndothelial and epithelial cells exhibit cell polarity and have characteristic tight junctions (TJs) that separate apical and basal surfaces. TJs are composed of both transmembrane and cytoplasmic proteins. The three major families of transmembrane proteins include claudins, occludin and JAM family members.^1–3 Additionally, interaction between the peripheral proteins such as PDS-95/Discs large/ZO family (PDZ) domain-containing proteins in TJs plays an important role in maintaining the junctional integrity.^2,4,5JAMs are type I membrane proteins (Fig. 1) predominately expressed in endothelial and epithelial cell TJs, platelets and some leukocytes.^6–8 The classical JAMs are JAM-A, JAM-B and JAM-C, which can all regulate leukocyte-endothelial cell interaction through their ability to undergo heterophilic binding with integrins α_Lβ₂ or α_vβ₃, α₄β₁ and α_Mβ₂ respectively. The cytoplasmic tail of JAMs contains a type II PDZ-domain-binding motif (Fig. 1) that can interact with the PDZ domain containing cytoplasmic molecules such as ZO-1, ASIP/PAR-3 or AF-6.^9,10 Additionally, consistant with their junctional localization and their tendency to be involved in homophilic interactions, JAMs have been shown to modulate paracellular permeability and thus may play an important role in regulating the epithelial and endothelial barrier.^11,12 In addition, ectopic expression of JAM-A in CHO cells promotes localization of ZO-1 and occludin at points of cell contacts, which suggests a role for JAM-A in TJ assembly.^10,13,14 Recently, it has been shown that JAM-A regulates epithelial cell morphology by modulating the activity of small GTPase Rap1 suggesting a role for JAM-A in intracellular signaling.¹⁵Open in a separate windowFigure 1Schematic representation of the domain structure of JAM family proteins. V, variable Ig domain; C2, constant type 2 Ig domain; TM, transmembrane domain; T-II, Type II PDZ-domain binding motif.We have previously shown that JAM-A is a positive regulator of fibroblast growth factor-2 (FGF-2) induced angiogenesis.¹⁶ Evidence was provided to support the notion that JAM-A forms a complex with integrin α_vβ₃ at the cell-cell junction in quiescent human umbilical cord vein endothelial cells (HUVECs) and FGF-2 dissociates this complex.¹⁶ It was further established that inhibition of JAM-A using a function-blocking antibody also inhibits FGF-2 induced HUVECs migration in vitro and angiogenesis in vivo. Overexpression of JAM-A induced a change in HUVECs morphology similar to that observed when treated with FGF-2.¹⁷ Furthermore, overexpression of JAM-A, but not its cytoplasmic domain deletion mutant, augmented cell migration in the absence of FGF-2.¹⁷ In addition, downregulation of JAM-A in HUVECs using specific siRNA, resulted in reduced FGF-2-induced cell migration and inhibition of mitogen activated protein (MAP) kinase activation.¹⁸ These findings clearly suggested that JAM-A positively regulates FGF-2-induced endothelial cell migration. This was further confirmed in vivo by using JAM-A null mouse in which FGF-2 failed to support angiogenesis.¹⁹It is known that JAM-C, a JAM family member, is involved in the process of tumor cell metastasis.²⁰ However, little is known about JAM-A''s role in cancer progression. We recently found that JAM-A is expressed in breast cancer tissues and cell lines.²¹ Based on our studies with endothelial cells it was felt that JAM-A expression in breast cancer cells may also enhance the migratory ability of these cells. Surprisingly, we found an inverse relation between the expression of JAM-A and the metastatic ability of breast cancer cells. T47D cells, which express high levels of JAM-A, are the least migratory; whereas MDA-MB-231 cells, which are highly migratory, are found to express the least amount of JAM-A.²¹ We also found that overexpression of JAM-A in MDA-MB-231 cells caused a change in cell morphology from spindle-like to rounded shape and formed cobblestone-like clusters.²¹ This is consistent with the previous report, that downregulation of JAM-A expression from epithelial cells using siRNA results in the change of epithelial cell morphology.¹⁵ This change in cell morphology by knockdown of JAM-A was attributed to the disruption of epithelial cell barrier function.¹⁵ It was further shown that knockdown of JAM-A affects epithelial cell morphology through reduction of β₁integrin expression due to decreased Rap1 activity.¹⁵ Our observed effect of JAM-A downregulation in T47D cells, however, is not due to downregulation of β₁integrin, since the level of this integrin was not affected in these cells. Interestingly, overexpression of JAM-A significantly affected both the cell migration and invasion of MDA-MB-231 cells. Furthermore, knockdown of JAM-A using siRNA enhanced invasiveness of MDA-MB-231 cells, as well as T47D cells.²¹ The ability of JAM-A to attenuate cell invasion was found to be due to the formation of functional tight junctions as observed by distinct accumulation of JAM-A and ZO-1 at the TJs and increased transepithelial resistance. These results identify, for the first time, a tight junctional cell adhesion protein as a key negative regulator of breast cancer cell migration and invasion.²¹JAM-A has been shown to be important in maintaining TJ integrity.^15,22–25 Disruption of TJs has been implicated to play a role in cancer cell metastasis by inducing epithelial mesenchymal transition.²⁶ Several laboratories, including ours, have shown that cytokines and growth factors redistribute JAM-A from TJs.^16,27,28 Consistent with this finding, it has been shown that hepatocyte growth factor (HGF) disrupts TJs in human breast cancer cells and downregulates expression of several TJ proteins.²⁹ It is therefore conceivable that the loss of JAM-A in highly metastatic cells is a consequence of disruption of TJs. This was further supported by the findings that overexpression of JAM-A forms functional TJs in MDA-MB-231 cells and attenuates their migratory behavior. Our result is the first report correlating an inverse relationship of JAM-A expression in breast cancer cells to their invasive ability.²¹Using cDNA microarray technology, it has been revealed how genes involved in cell-cell adhesion, including those of the TJ, are under or overexpressed in different carcinomas.^15,30 Cell-cell adhesion molecules have been well documented to regulate cancer cell motility and invasion. Of these, the cadherin family have been studied the most.^31,32 It was proposed that a cadherin switch, that is, the loss of E-cadherin and subsequent expression of N-cadherin, may be responsible for breast cancer cell invasion.^33,34 Although the role of cadherins is well-documented, it remains controversial since some breast cancer cell lines that do not express these proteins still posses highly invasive characteristics.^33,34 However, the observed effect of overexpression of JAM-A does not appear to be simply due to the formation of TJs, since individual cells that express increased JAM-A show reduced migration.²¹ This is not surprising, considering the fact that JAM-A in addition to its function of regulating TJ integrity is also shown to participate in intracellular signaling. JAM-A is capable of interacting homotypically as well as heterotypically on the cell surface.^35,36 It has also been shown that it interacts with several cytoplasmic proteins through its PDZ domain-binding motif and recruits signaling proteins at the TJs.³⁷ Recent findings using site-directed mutagenesis suggest that cis-dimerization of JAM-A is necessary for it to carry out its biological functions.³⁸ Our own observations suggest that a JAM-A function-blocking antibody inhibits focal adhesion formation in endothelial cells (unpublished data), whereas overexpresion of JAM-A in MDA-MB-231 cells show increased and stable focal adhesions.²¹ It is therefore conceivable that in quiescent endothelial/epithelial cells JAM-A associates with integrin to form an inactive complex at the TJ (Fig. 2). Growth factors such as FGF-2 signaling dissociates this complex thus allowing dimerization of JAM-A and activation of integrin augmenting cell migration (Fig. 2). On the contrary, in MDA-MB-231 cancer cells, which express low levels of JAM-A and do not form tight junctions, there may not be efficient inactive complex formation between JAM-A and integrin. Overexpression of JAM-A in these cells however, may promote such inactive complex formation leading to inhibition of integrin activation and JAM-A dimerization, both necessary events for cell migration. We are currently in the process of determining the specificity of interaction of JAM-A with integrins. Further experimentation is ongoing to determine the contribution of JAM-A dependent signaling in cell migration.Open in a separate windowFigure 2Schematic representation of JAM-A regulation of cell migration. JAM-A forms an inactive complex with the integrin and sequesters it at the TJs. Growth factor signaling dissociates this complex, promoting integrin activation and JAM-A dimerization leading to cell migration via MAP kinase activation. Ectopic expression of JAM-A in cancer cells may induce its association with integrin, forming an inactive complex and hence attenuation of migration.JAM-A differentially regulates cell migration in endothelial and cancer cells due to its ability to form inactive complex with integrin, making it a metastasis suppressor. The downregulation of JAM-A in carcinoma cells may be detrimental to the survival of breast cancer patients. It is therefore very important to determine the molecular determinants that are responsible for the downregulation of JAM-A during cancer progression. Thus, JAM-A, a molecule that dictates breast cancer cell invasion, could be used as a prognostic marker for metastatic breast cancer.     

Traffic jams in fish bones: ER-to-Golgi protein transport during zebrafish development

Extracellular matrix (ECM) proteins, cell adhesion molecules, cytokines, morphogens and membrane receptors are synthesized in the ER and transported through the Golgi complex to the cell surface and the extracellular space. The first leg in this journey from the ER to Golgi is facilitated by the coat protein II (COPII) vesicular carriers. Genetic defects in genes encoding various COPII components cause a broad spectrum of human diseases, from anemia to skeletal deformities. Here, we summarize our findings in zebrafish and discuss how mutations in COPII elements may cause specific cellular and developmental defects.Key words: Sec24D, Sec23A, ECM, COPII, craniofacial morphogenesisCOPII vesicle formation is initiated when the small, cytoplasmic GTPase Sar1 undergoes a conformational change upon GTP binding, exposing an amphipathic α-helix that allows Sar1 to associate with the ER membrane.^1–3 Sar1 then recruits the Sec23/Sec24 heterodimer to the ER surface, forming a “pre-budding complex.” Sec23 acts as a GTPase-activating protein for Sar1, whereas Sec24 plays a role in protein cargo selection.^4,5 These three proteins form the inner coat and are thought to impose the initial ER membrane deformation. Next, the COPII outer coat complex assembles by Sec13 and Sec31 heterotetramers, which form a cage that encompasses the pre-budding vesicle (Fig. 1A).^6,7Open in a separate windowFigure 1bulldog and crusher encode mutations in the COPII complex. (A) Graphic depicting the COPII inner coat bound to the ER membrane and a complete COPII vesicle. (B) Structure of human SEC24D and SEC23A and the truncation caused by bulldog and crusher mutations in zebrafish proteins as projected on human proteins. (C) Overlay of the structure of human SEC23A and SEC23B. Structures are based on known crystal structures by Mancias et al.⁵ with SEC23B (light blue) and unresolved loops modeled using Modeller.²⁷ Binding interfaces to other proteins are indicated by purple lines.COPII components are highly conserved throughout the plant and animal kingdoms. The yeast S. cerevisiae has one Sec23 gene and three Sec24 paralogs (Sec24, Lst1 and Iss), while vertebra genomes contain four Sec24 (A–D) and two Sec23 paralogs (A and B).^8,9 Although the yeast Sec23 and Sec24 are essential for survival, private variants in genes of COPII components in humans cause a broad spectrum of diseases with clinical manifestations as diverse as skeletal defects,¹⁰ anemia,¹¹ or lipid malabsorption.¹² The precise molecular and cellular mechanisms that lead to such outcomes are poorly understood, underscoring the importance of animal models to study these organ- and tissue-specific deficits.^11,13