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.     

Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Challenges to understand plant responses to wind

Understanding plant response to wind is complicated as this factor entails not only mechanical stress, but also affects leaf microclimate. In a recent study, we found that plant responses to mechanical stress (MS) may be different and even in the opposite direction to those of wind. MS-treated Plantago major plants produced thinner more elongated leaves while those in wind did the opposite. The latter can be associated with the drying effect of wind as is further supported by data on petiole anatomy presented here. These results indicate that plant responses to wind will depend on the extent of water stress. It should also be recognized that the responses to wind may differ between different parts of a plant and between plant species. Physiological research on wind responses should thus focus on the signal sensing and transduction of both the mechanical and drought signals associated with wind, and consider both plant size and architecture.Key words: biomechanics, leaf anatomy, phenotypic plasticity, plant architecture, signal transduction thigmomorphogenesis, windWind is one of the most ubiquitous environmental stresses, and can strongly affect development, growth and reproductive yield in terrestrial plants.^1–3 In spite of more than two centuries of research,⁴ plant responses to wind and their underlying mechanisms remain poorly understood. This is because plant responses to mechanical movement themselves are complicated and also because wind entails not only mechanical effects, but also changes in leaf gas and heat exchange.^5–7 Much research on wind has focused primarily on its mechanical effect. Notably, several studies that determine plant responses to mechanical treatments such as flexing, implicitly extrapolate their results to wind effects.^8–10 Our recent study¹¹ showed that this may lead to errors as responses to wind and mechanical stimuli (in our case brushing) can be different and even in the opposite direction. In this paper, we first separately discuss plant responses to mechanical stimuli, and other wind-associated effects, and then discuss future challenges for the understanding of plant responses to wind.It is often believed that responses to mechanical stress (thigmomorphogenesis) entail the production of thicker and stronger plant structures that resist larger forces. This may be true for continuous unidirectional forces such as gravity, however for variable external forces (such as wind loading or periodic flooding) avoiding such mechanical stress by flexible and easily reconfigurable structures can be an alternative strategy.^12–14 How plants adapt or acclimate to such variable external forces depends on the intensity and frequency of stress and also on plant structures. Reduced height growth is the most common response to mechanical stimuli.^15,16 This is partly because such short stature increases the ability of plants to both resist forces (e.g., real-locating biomass for radial growth rather than elongation growth), and because small plants experience smaller drag forces (Fig. 1). Some plant species show a resistance strategy in response to mechanical stress by increasing stem thickness^1,10 and tissue strength.⁷ But other species show an avoidance strategy by a reduction in stem or petiole thickness and flexural rigidity in response to MS.^11,15–18 These different strategies might be associated with plant size and structure. Stems of larger plants such as trees and tall herbs are restricted in the ability to bend as they carry heavy loads^7,10,19 (Fig. 1). Conversely short plants are less restricted in this respect and may also be prone to trampling for which stress-avoidance would be the only viable strategy.^18,20 Systematic understanding of these various responses to mechanical stress remains to be achieved.Open in a separate windowFigure 1A graphical representation of how wind effects can be considered to entail both a drying and a mechanical effect. Adaptation or acclimation to the latter can be through a force resistance strategy or a force avoidance strategy, the benefit of which may depend on the size and architecture of plants as well as the location of a given structure within a plant.Wind often enhances water stress by reducing leaf boundary layers and reduces plant temperature by transpiration cooling. The latter effect may be minor,¹¹ but the former could significantly affect plant development. Anten et al. (2010) compared phenotypic traits and growth of Plantago major that was grown under mechanical stimuli by brushing (MS) and wind in the factorial design. Both MS and wind treatments reduced growth and influenced allocation in a similar manner. MS plants, however, had more slender petioles and narrower leaf blades while wind exposed plants exhibited the opposite response having shorter and relatively thicker petioles and more round-shaped leaf blades. MS plants appeared to exhibit stress avoidance strategy while such responses could be compensated or overridden by water stress in wind exposure.¹¹ A further analysis of leaf petiole anatomy (Fig. 2) supports this view. The vascular fraction in the petiole cross-section was increased by wind but not by MS, suggesting that higher water transport was required under wind. Our results suggest that drying effect of wind can at least to some extent override its mechanical effect.Open in a separate windowFigure 2Representative images of petiole cross-sections of Plantago major grown in 45 days in continuous wind and/or mechanical stimuli (A–D). Petiole cross-section area (E) and vascular bundle fraction in the cross-section of petiole (F). mean + SD (n = 12) are shown. Significance levels of ANOVA; ***p < 0.001, **p < 0.01, *p < 0.05, ns p > 0.05.Physiological knowledge on plant mechanoreception and signal transduction has been greatly increased during the last decades. Plants sense mechanical stimuli through membrane strain with stretch activated channels²¹ and/or through some linker molecules connecting the cell wall, plasma membrane and cytoskeleton.^4,22,23 This leads to a ubiquitous increase in intracellular Ca²⁺ concentration. The increased Ca²⁺ concentration is sensed by touch induced genes (TCHs),^24,25 which activates downstream transduction machineries including a range of signaling molecules and phytohormones, consequently altering physiological and developmental processes.²⁶ Extending this knowledge to understand plant phenotypic responses to wind however remains a challenge. As responses to wind have been found to differ among parts of a plant (e.g., terminal vs. basal stem) and also across species, physiological studies should be extended to the whole-plant as integrated system rather than focusing on specific tissue level. Furthermore to understand the general mechanism across species, it is required to study different species from different environmental conditions. Advances in bioinformatics, molecular and physiological research will facilitate cross-disciplinary studies to disentangle the complicated responses of plants to wind.     

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.     

Reactive oxygen species in aerobic methane formation from vegetation

The first report of aerobic methane emissions from vegetation by an unknown mechanism¹ suggested that this potential new source may make a significant contribution to global methane emissions. We recently investigated possible mechanisms and reported^2,3 experiments in which UV-irradiation caused methane emissions from pectin, a major plant cell wall polysaccharide. Our findings also suggest that UV-generated reactive oxygen species (ROS) release methane from pectin. This has implications for all other, UV-independent processes which may generate ROS in or close to the plant cell wall and suggests a need to evaluate additional systems for ROS-generated methane emissions in leaves.Key words: methane, hydroxyl radicals, reactive oxygen species, UV, methyl esters, pectinUntil recently, the global methane budget was thought to be well understood, the only natural process for methane generation being an anaerobic microbial mechanism.⁴ However, observations by Keppler et al.¹ of aerobic methane emissions from vegetation caused controversy and called for a re-assessment of the natural sources of methane. While no mechanism was originally suggested, a putative source, the methyl ester groups of pectin, was proposed based on carbon isotope analyses.¹ We tested this hypothesis directly and reported that UV light could drive methane emissions from pectin in vitro under aerobic conditions.² While UV light was necessary for generation of methane from pectin, it is not tenable that UV was directly attacking pectic methyl ester groups since these do not absorb UV of the wavelengths used (280–400 nm). Instead, we proposed that the energy from the UV light was being absorbed by compounds such as phenolics, and that a reactive intermediary would be formed in the process. Importantly, our process had to be non-enzymic since no enzymes were present in either experimental system.^1,2 Following this hypothesis, we tested the effect of reactive oxygen species (ROS) on pectin in vitro and discovered that certain ROS cause production of methane: hydroxyl radicals (^•OH) and singlet oxygen were effective, but hydrogen peroxide and superoxide were not.³ Also, the addition of ROS-specific scavengers to pectin sheets stopped or severely reduced UV-induced methane emissions from pectin, suggesting that ROS are the intermediary in the mechanism of aerobic methane formation from pectin (Fig. 1). De-esterified pectin was produced by saponification and emitted only trace amounts of methane upon UV-irradiation, clearly establishing ester groups as the source of methane^2,3 and confirming findings of other research groups.^5,6 However, we also found that acetyl ester groups may contribute to methane emissions from pectin and should therefore be considered in future experiments attempting to identify methane sources. Interestingly, we also observed, for the first time, ethylene, ethane and CO₂ emissions from pectin upon UV-irradiation,² which corroborates the ROS hypothesis since ROS attack of methyl esters is likely to form methyl radicals, which can then either form methane or dimerise to form ethylene or ethane.Open in a separate windowFigure 1Proposed pathway for ^•OH-driven methane generation from pectin upon UV irradiation. The compound illustrated here, l-tryptophan, is merely an example of a possible photosensitiser. Hydroxyl radicals (^•OH) are shown to attack a methyl galacturonate residue of the homogalacturonan component of the pectin molecule since this is likely to be the most abundant source of methane, but the methyl esters found in xylogalacturonan domains and the acetyl esters found in homogalacturonan and rhamnogalacturonan domains are also possible methane sources. Note that only ∼70% of all galacturonic acid residues of the pectin backbone are methyl-esterified. Inset photograph shows experimental set-up during UV-irradiation of pectin.ROS are produced and destroyed constantly throughout the lifetime of plants. The generation of ROS in vivo can generally be linked to two sources: (i) a response to an external stimulus which may be perceived as a threat or (ii) a signaling process in the cell which may happen during growth, hormone action or programmed cell death.⁷ Our experiments showed that ROS could lead to methane formation from methyl ester groups; however, the origin of the ROS may not be important, only their nature. Indeed, hydrogen peroxide and superoxide, widely reported to be formed during an oxidative burst following a biotic stress,⁸ did not generate methane from pectin in vitro, and are therefore unlikely to do so in vivo. Only the hydroxyl radical (^•OH) and singlet oxygen generation led to methane formation, and therefore any process which generates them could also trigger UV-independent methane production. Abiotic stresses, such as drought, heat or salinity, which have been shown to lead to the production of ^•OH in vegetation,⁹ could therefore be processes leading to aerobic methane formation, as could exposure to elevated ozone concentrations.¹⁰ Indeed, physical injury (by cutting) of plant material has recently been demonstrated to cause methane emissions.¹¹The origin of the ROS may not be important, as long as their generation is in or close to the pectin of the plant cell wall, since ^•OH cannot travel far within a cell. Indeed, it is estimated that ^•OH typically reacts with organic matter within ∼1 nm of the site of radical production.¹² Processes such as growth^13,14 and calcium signaling,¹⁵ which both involve ROS production as an intermediary in the mechanism but are not necessarily due to external stress, may therefore have the potential to generate methane aerobically. Any process involved in the complicated pathways of ROS-regulation, for which 152 genes are responsible in Arabidopsis thaliana,¹⁶ could be involved in methane emission if the ROS generation is localised close to pectin or other potential substrates.In addition, hydrogen peroxide, which is generated in the cell walls of healthy plants,¹⁷ can be converted in the cell wall into ^•OH by processes such as the Fenton reaction,^18,19 especially in the presence of apoplastic ascorbate.^20,21 A complete analysis of the potential for ^•OH and singlet oxygen to be present in the plant cell wall is therefore necessary for a proper understanding of the different mechanisms that may drive aerobic methane generation. Further experiments into the effects of abiotic stresses other than UV on aerobic methane production from different types of vegetation are necessary in order that future in-vitro studies under simulated natural conditions can be carried out correctly. This type of study, in conjunction with direct in-vivo field studies and satellite observations, are essential to allow global estimates to be made accurately in the future and help us understand the significance of ROS-driven methane emission.     

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     

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.     

Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins

The conserved eukaryotic protein SGT1 (suppressor of G₂ allele of skp1) participates in diverse physiological processes such as cell cycle progression in yeast, plant immunity against pathogens and plant hormone signalling. Recent genetic and biochemical studies suggest that SGT1 functions as a novel co-chaperone for cytosolic/nuclear HSP90 and HSP70 molecular chaperones in the folding and maturation of substrate proteins. Since proteins containing the leucine-rich repeat (LRR) protein-protein interaction motif are overrepresented in SGT1-dependent phenomena, we consider whether LRR-containing proteins are preferential substrates of an SGT1/HSP70/HSP90 complex. Such a chaperone organisation is reminiscent of the HOP/HSP70/HSP90 machinery which controls maturation and activation of glucocorticoid receptors in animals. Drawing on this parallel, we discuss the possible contribution of an SGT1-chaperone complex in the folding and maturation of LRR-containing proteins and its evolutionary consequences for the emergence of novel LRR interaction surfaces.Key words: heat shock protein, SGT1, co-chaperone, HSP90, HSP70, leucine-rich repeat, LRR, resistance, SCF, ubiquitinThe proper folding and maturation of proteins is essential for cell viability during de novo protein synthesis, translocation, complex assembly or under denaturing stress conditions. A complex machinery composed of molecular chaperones (heat-shock proteins, HSPs) and their modulators known as co-chaperones, catalyzes these protein folding events.^1,2 In animals, defects in the chaperone machinery is implicated in an increasing number of diseases such as cancers, susceptibility to viruses, neurodegenerative disease and cystic fibrosis, and thus it has become a major pharmacological target.^3,4 In plants, molecular genetic studies have identified chaperones and co-chaperones as components of various physiological responses and are now starting to yield important information on how chaperones work. Notably, processes in plant innate immunity rely on the HSP70 and HSP90^5–7 chaperones as well as two recently characterised co-chaperones, RAR1 (required for Mla12 resistance) and SGT1 (suppressor of G₂ allele of skp1).^8–11SGT1 is a highly conserved and essential co-chaperone in eukaryotes and is organized into three structural domains: a tetratricopeptide repeat (TPR), a CHORD/SGT1 (CS) and an SGT1-specific (SGS) domain (Fig. 1A). SGT1 is involved in a number of apparently unrelated physiological responses ranging from cell cycle progression and adenylyl cyclase activity in yeast to plant immunity against pathogens, heat shock tolerance and plant hormone (auxin and jasmonic acid) signalling.^7–9,12,13 Because the SGT1 TPR domain is able to interact with Skp1, SGT1 was initially believed to be a component of SCF (Skp1/Cullin/F-box) E3 ubiquitin ligases that are important for auxin/JA signalling in plants and cell cycle progression in yeast.^13,14 However, mutagenesis of SGT1 revealed that the TPR domain is dispensable for plant immunity and auxin signalling.¹⁵ Also, SGT1-Skp1 interaction was not observed in Arabidopsis.¹³ More relevant to SGT1 functions appear to be the CS and SGS domains.¹⁶ The former is necessary and sufficient for RAR1 and HSP90 binding. The latter is the most conserved of all SGT1 domains and the site of numerous disabling mutations.^14,16,17Open in a separate windowFigure 1Model for SGT1/chaperone complex functions in the folding of LRR-containing proteins. (A) The structural domains of SGT1, their sites of action (above) and respective binding partners (below) are shown. N- and C-termini are indicated. TPR, tetratricopeptide repeat; CS, CHORD/SGT1; SGS, SGT1-specific. (B) Conceptual analogy between steroid receptor folding by the HOP/chaperone machinery and LRR protein folding by the SGT1/chaperone machinery. LRR motifs are overrepresented in processes requiring SGT1 such as plant immune receptor signalling, yeast adenylyl cyclase activity and plant or yeast SCF (Skp1/Cullin/F-box) E3 ubiquitin ligase activities. (C) Opposite forces drive LRR evolution. Structure of LRRs 16 to 18 of the F-box auxin receptor TIR1 is displayed as an illustration of the LRR folds.³⁰ Leucine/isoleucine residues (side chain displayed in yellow) are under strong purifying selection and build the hydrophobic LRR backbone (Left). By contrast, solvent-exposed residues of the β-strands define a polymorphic and hydrophilic binding surface conferring substrate specificity to the LRR (Right) and are often under diversifying selection.We recently demonstrated that Arabidopsis SGT1 interacts stably through its SGS domain with cytosolic/nuclear HSP70 chaperones.⁷ The SGS domain was both necessary and sufficient for HSP70 binding and mutations affecting SGT1-HSP70 interaction compromised JA/auxin signalling and immune responses. An independent in vitro study also found interaction between human SGT1 and HSP70.¹⁸ The finding that SGT1 protein interacts directly with two chaperones (HSP90/70) and one co-chaperone (RAR1) reinforces the notion that SGT1 behaves as a co-chaperone, nucleating a larger chaperone complex that is essential for eukaryotic physiology. A future challenge will be to dissect the chaperone network at the molecular and subcellular levels. In plant cells, SGT1 localization appears to be highly dynamic with conditional nuclear localization⁷ and its association with HSP90 was recently shown to be modulated in vitro by RAR1.¹⁶A co-chaperone function suits SGT1 diverse physiological roles better than a specific contribution to SCF ubiquitin E3 ligases. Because SGT1 does not affect HSP90 ATPase activity, SGT1 was proposed rather as a scaffold protein.^16,19 In the light of our findings and earlier studies,²⁰ SGT1 is reminiscent of HOP (Hsp70/Hsp90 organizing protein) which links HSP90 and HSP70 activities and mediates optimal substrate channelling between the two chaperones (Fig. 1B).²¹ While the contribution of the HSP70/HOP/HSP90 to the maturation of glucocorticoid receptors is well established,²¹ direct substrates of an HSP70/SGT1/HSP90 complex remain elusive.It is interesting that SGT1 appears to share a functional link with leucine-rich repeat- (LRR) containing proteins although LRR domains are not so widespread in eukaryotes. For example, plant SGT1 affects the activities of the SCF^TIR1 and SCF^COI1 E3 ligase complexes whose F-box proteins contain LRRs.¹³ Moreover, plant intracellular immune receptors comprise a large group of LRR proteins that recruit SGT1.^8,9 LRRs are also found in yeast adenylyl cyclase Cyr1p and the F-box protein Grr1p which is required for SGT1-dependent cyclin destruction during G₁/S transition.^12,14 Yeast 2-hybrid interaction assays also revealed that yeast and plant SGT1 tend to associate directly or indirectly with LRR proteins.^12,22,23 We speculate that SGT1 bridges the HSP90-HSC70 chaperone machinery with LRR proteins during complex maturation and/or activation. The only other structural motif linked to SGT1 are WD40 domains found in yeast Cdc4p F-box protein and SGT1 interactors identified in yeast two-hybrid screens.¹²What mechanisms underlie a preferential SGT1-LRR interaction? HSP70/SGT1/HSP90 may have co-evolved to assist specifically in folding and maturation of LRR proteins. Alternatively, LRR structures may have an intrinsically greater need for chaperoning activity to fold compared to other motifs. These two scenarios are not mutually exclusive. The LRR domain contains multiple 20 to 29 amino acid repeats, forming an α/β horseshoe fold.²⁴ Each repeat is rich in hydrophobic leucine/isoleucine residues which are buried inside the structure and form the structural backbone of the motif (Fig. 1C, left). Such residues are under strong purifying selection to preserve structure. These hydrophobic residues would render the LRR a possible HSP70 substrate.²⁵ By contrast, hydrophilic solvent- exposed residues of the β strands build a surface which confers ligand recognition specificity of the LRRs (Fig. 1C). In many plant immune receptors for instance, these residues are under diversifying selection that is likely to favour the emergence of novel pathogen recognition specificities in response to pathogen evolution.²⁶ The LRR domain of such a protein has to survive such antagonist selection forces and yet remain functional. Under strong selection pressure, LRR proteins might need to accommodate less stable LRRs because their recognition specificities are advantageous. This could be the point at which LRRs benefit most from a chaperoning machinery such as the HSP90/SGT1/HSP70 complex. This picture is reminiscent of the genetic buffering that HSP90 exerts on many traits to mask mutations that would normally be deleterious to protein folding and/or function, as revealed in Drosophila and Arabidopsis.²⁷ It will be interesting to test whether the HSP90/SGT1/HSP70 complex acts as a buffer for genetic variation, favouring the emergence of novel LRR recognition surfaces in, for example, highly co-evolved plant-pathogen interactions.^28,29     

Myosin light chain kinases: Division of work in cell migration

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.Key words: myosin, MLCK, ROK, MRCK, phosphorylation, cell migrationCell movement is a fundamental activity underlying many important biological events ranging from embryological development to immunological responses in the adult. A typical cell movement cycle entails polarization, membrane protrusion, formation of new adhesions, cell body translocation and finally rear retraction.¹ A precise temporal and spatial coordination of these separate steps that take place in different parts of the cell is important for rapid and efficient movement.²One major event during eukaryotic cell migration is the myosin II (MYO2)-mediated contraction that underlies cell protrusion, traction and retraction.^1,3 An emerging theme from collective findings is that there are distinct myosin contractile modules responsible for the different functions which are separately regulated by local myosin regulatory light chain (MLC) kinases. These kinases contribute to contractile forces that connect adhesion, protrusion and actin organization.² Unraveling the regulation of these contractile modules is therefore pivotal to a better understanding of the coordination mechanism.At the lamellipodium, the conventional calcium/calmodulin-dependent myosin light chain kinase (MLCK) has been shown to play an essential role in a Rac-dependent lamellipodial extension.⁴ Inhibition of calmodulin or MLCK activity by specific photoactivatable peptides in motile eosinophils effectively blocks lamellipodia extension and net movement.⁵ Furthermore, there is a strong correlation between activated MLCK and phosphorylated MLC within the lamellipodia of Ptk-2 cells as revealed by fluorescence resonance energy transfer (FRET) analysis.⁶ More recent studies showed MLCK to regulate the formation of focal complexes during lamellipodia extension.^7,8 Functionally, MLCK is thought to play a critical role in the environment-sensing mechanism that serves to guide membrane protrusion. It mediates contraction that exerts tension on integrin-extracellular matrix (ECM) interaction, which, depending on the rigidity of the substratum, will lead to either stabilization of adhesion resulting in protrusion or destabilization of attachment seen as membrane ruffling on non-permissive surfaces.^8,9As a Rho effector, Rho-associated kinase (ROK/ROCK/Rho-kinase) has been shown to regulate stress fibers and focal adhesion formation by activating myosin, an effect that can be blocked by the specific ROK inhibitor Y-27632.^10,11 Myosin activation by ROK is the effect of two phosphorylation events: the direct phosphorylation on MLC and the inhibition of myosin phosphatase through phosphorylation of its associated myosin-binding subunit (MBS).¹¹ Consistent with this notion of a localization-function relationship, ROK and MBS, which can interact simultaneously with activated RhoA,¹¹ have been shown to colocalize on stress fibers.^12,13 In migrating cells, Rho and ROK activities have been mostly associated with the regulation of tail retraction, as inhibition of their activities often results in trailing tails due to the loss of contractility specifically confined to the cell rear.^14,15 Tail retraction requires high contractile forces to overcome the strong integrin-mediated adhesion established at the rear end, an event which coincides with the strategic accumulation of highly stable and contractile stress fibers that assemble at the posterior region of migrating cells.MRCK was previously shown to phosphorylate MLC and promote Cdc42-mediated cell protrusion.¹⁶ More recently, it was found to colocalize extensively with and regulate the dynamics of a specific actomyosin network located in the lamella and cell center, in a Cdc42-dependent manner but independent of MLCK and ROK.¹⁷ The lamellar actomyosin network physically overlaps with, but is biochemically distinct from the lamellipodial actin meshwork.^9,18 The former network consists of an array of filaments assembled in an arrangement parallel to the leading edge, undergoing continuous retrograde flow across the lamella, with their disassembly occurring at the border of the cell body zone sitting in a deeper region.^17–19 Retrograde flow of the lamellar network plays a significant role in cell migration as it is responsible for generating contractile forces that support sustained membrane protrusion and cell body advancement.^17–19It is therefore conceivable that these three known MLC kinases are regulated by different signaling mechanisms at different locations and on different actomyosin contractile modules. The coordination of the various modules will ensure persistent directional migration (Figure 1). Phosphorylation of MLC by PAK and ZIP kinase has also been reported, but their exact roles in this event have yet to be determined.^20,21 It is also noteworthy that individual kinases can work independently of each other, as amply shown by evidence from inhibitor treatments. This is particularly true for MRCK in the lamella, whose activity on lamellar actomyosin flow is not affected by ML7 and Y-27632, the inhibitors of MLCK and ROK respectively.¹⁷ These findings further indicate that although both ROK and MRCK have been shown to upregulate phosphorylated MLC levels by inhibiting the myosins phosphatases,^11,22 they are likely to act as genuine MLC kinases themselves, without the need of MLCK as previously suggested.¹¹Open in a separate windowFigure 1Upper panel depicts a model for the specific activation of the different MLC kinases at various locations in the cell. In response to upstream signals, MLC kinases MLCK, MRCK and ROK are activated and localized to different regions. In the case of MRCK and ROK, the interaction of the GTP-bound Rho GTPase binding domain will determine the specific action of the downstream kinase, resulting in actomyosin contractility at different locations. The coordination of these signalling events is crucial for directional cell migration. Lower panel shows a typical front-rear location for Myosin 2A and 2B in a migrating U2OS cell.In conjunction with their differences in localization, the three MLC kinases show apparent individual preferences and specificity towards the MYO2 isoforms that they associate with. The two major MYO2 isoforms MYO2A and 2B are known to have distinct intracellular distributions that are linked to their individual functions (Figure 1).^23,24 In motile cells, MYO2A localization that is skewed towards the protruding cell front is consistent with it being the major myosin 2 component of the lamellar filaments regulated by MRCK as well as its regulation by MLCK in lamellipodial contraction.^8,17,19 In contrast, the enrichment of MYO2B at retracting cell rear conforms well with the requirement of thick and stable stress fibers capable of causing tail contraction and prevention of protrusion under the control of Rho/ROK signaling.^23,25 The selection for MYO2B filaments in the cell rear stems from their more contractile and stable nature compared with MYO2A, a consequence of their higher time-averaged association with actin.^26,27 Conversely, the lower tension property of MYO2A filaments suggests that they are more dynamic in nature,^26,27 a characteristic which fits well with the dynamic actomyosin activities at the leading edge and lamella that regulate protrusion.It deserves special mention that the three MLC kinases display subtle differences in their specificity towards MLC. While MLCK and MRCK phosphorylate only a single Ser19 site (monophosphorylation),^18,28 ROK is able to act on both Thr18 and Ser19 residues causing diphosphorylation of MLC,²⁹ MLCK only causes diphosphorylation when present at higher concentrations.³⁰ By further increasing its actin-activated ATPase activity, diphosphorylation of MLC has been shown to induce a higher myosin activation and filament stability.^30–32 The use of specific antibodies that can differentiate between the two populations of phosphorylated MLC has been instrumental in revealing their localization and correlation with the activity of the MLC kinases. The emerging picture from these experiments is that mono and diphosphorylated MLC exhibit distinct distributions in migrating cells, with the monophosphorylated MLC localized more towards the protrusive region, while the diphosphorylated form is more enriched at the posterior end.^21,33 Taking into account their biochemical properties, the polarized distributions of these differentially phosphorylated MLC coincide functionally with the segregation of the MYO2 isoforms and their corresponding regulators. These findings provide further support for the existence of segregated contractile modules in migrating cell and their distinctive regulation.The mechanisms that determine the specific segregation of the contractile modules and their regulation are unclear. However, some clues have emerged from recent studies. It has been shown that the C-terminal coiled-coil region of MYO2B is important for determining its localization in cell rear²⁵ and which requires Rho/ROK activity as their inhibition resulted in the loss of this specific localization.²³ Correspondingly, the inhibition of MRCK activity resulted in the loss of lamella-localized MYO2A.¹⁷ These findings suggest that activation of MYO2 filaments by their upstream regulators is important for their functional segregation and maintenance. It is noteworthy that both ROK and MRCK have distinct regulatory domains including the pleckstrin homology domains which have been shown to be essential for their localization, a process which may involve myosin interaction and lipid-dependent targeting as has been respectively shown for ROK and MRCK.^11,13,16 Further, the specificity of MRCK for lamellar actomyosin is believed to be largely determined by the two proteins it forms a complex with: the adaptor LRAP35a, and the MYO2-related MYO18A. Activation of MYO18A by MRCK, a process bridged by LRAP35a, is a crucial step which facilitates MRCK regulation on lamellar MYO2A.¹⁷The mechanisms responsible for segregating the contractile modules and their regulators may also comprise a pathway that parallels the microtubule-modulatory Par6/aPKC/GSK3β signalling pathway which regulates cellular polarization. This notion is supported by both Cdc42 and Rho being common upstream regulators of these two pathways.³⁴ GTPase activation may determine the localized activities of the separate contractile modules and create an actomyosin-based asymmetry across the cell body, which together with the microtubule-based activities, result in the formation of a front-back axis important for directional movement. The involvement of MRCK in MTOC reorientation and nuclear translocation events,³⁵ and our unpublished observation that LRAP35a has a GSK3β-dependent microtubule stabilizing function are supportive of a possible cross-talk between these two pathways.In conclusion, the complex regulation of contractility in cell migration emphasizes the importance of the localization, specificity and enzymatic properties of the different MLC kinases and myosin isoforms involved. The initial excitement and confusion caused by the emergence of the different MLC kinases are fading, being now overtaken by the curiosity about how they cooperate and are coordinated while promoting cell motility.