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.     

A new function for odorant receptors: MOR23 is necessary for normal tissue repair in skeletal muscle

Myofibers with an abnormal branching cytoarchitecture are commonly found in various neuromuscular diseases as well as after severe muscle injury. These aberrant myofibers are fragile and muscles containing a high percentage of these myofibers are weaker and more prone to injury. To date the mechanisms and molecules regulating myofiber branching have been obscure. Recent work analyzing the role of mouse odorant receptor 23 (MOR23) in muscle regeneration revealed that MOR23 is necessary for proper skeletal muscle regeneration in mice as loss of MOR23 leads to increased myofiber branching. Further studies demonstrated that MOR23 expression is induced when muscle cells were extensively fusing and plays an important role in controlling cell migration and adhesion. These data demonstrate a novel role for an odorant receptor in tissue repair and identify the first molecule with a functional role in myofiber branching.Key words: muscle regeneration, odorant receptor, olfactory receptor, MOR23, myofiber splitting, myofiber branching, myoblast, fusion, myotube, olfr16Skeletal muscle is characterized by an extensive ability to regenerate after injury due to trauma or disease. Muscle regeneration results from a finely orchestrated series of steps that are spatially and temporally regulated, many of which are not understood. Elucidation of the mechanisms that regulate muscle regeneration may be beneficial for enhancing the rate or extent of muscle regeneration in injury, disease or age.Skeletal muscle is composed of myofibers, which are long cylindrical cells containing hundreds of myonuclei in a common cytoplasm (Fig. 1). Each myofiber is surrounded by a basal lamina sheath; between the myofiber and the basal lamina lie myogenic stem cells called satellite cells. When muscle is injured, myofibers degenerate and satellite cells proliferate to give rise to progeny myoblasts. Myoblasts differentiate and undergo migration, adhesion and fusion to form regenerated myofibers and normal tissue architecture is restored. In many neuromuscular diseases muscle regeneration is aberrant and various abnormalities, such as variation in myofiber size, decreased myofiber number, fibrosis and branched myofibers, are observed. In the clinical literature, branched myofibers are more commonly referred to as “split myofibers.”Open in a separate windowFigure 1Myofiber growth during normal muscle regeneration. (A) Myofibers contain many myonuclei within a common cytoplasm and are surrounded by a basal lamina sheath. Underneath the basal lamina lie satellite cells, myogenic stem cells responsible for muscle regeneration. (B) Myofiber degeneration leads to activation of quiescent satellite cells and their reentry into the cell cycle. Their progeny myoblasts proliferate to yield a pool of progenitor cells. (C) Myoblasts differentiate and undergo migration, adhesion and fusion to form nascent myofibers within the original basal lamina sheath. Additional myoblasts fuse with these newly formed myofibers and the myofiber will continue to grow in size. (D) At later time points regenerated myofibers are similar in size to undamaged myofibers but contain centrally located myonuclei, a hallmark of a regenerated myofiber.Branched myofibers are malformed cells which, instead of having a normal cylindrical shape, contain one or more offshoots of small daughter myotubes contiguous with the parent myofiber (Fig. 2). Branched myofibers can be simple with only one branch (Fig. 2A) or complex with many anastomosing branches resembling a gnarled tree root (Fig. 2B).¹ In myofibers with complex cytoarchitecture, individual branches can persist up to hundreds of microns and then eventually recombine with the parent myofiber. Each daughter branch is enclosed in its own basal lamina, which is contiguous with the basal lamina of the parent myofiber.² The frequency of branched myofibers in rodent muscle under normal conditions is low, on the order of 0.003%.³ However, the frequency in both rodent and human muscle is increased in response to hypertrophy^4,5 as well as regeneration due to induced injury,^6–8 muscle transplantation,^9,10 or muscular dystrophy.^1,11–16 In mdx mice, a model of Duchenne Muscular Dystrophy, up to 65–90% of myofibers by 7 months and older display this abnormal branched morphology compared to 6–17% of myofibers at 1–3 months of age.^12–14 Branched myofibers display functional abnormalities such as alterations in myofiber calcium signaling.¹³ Additionally, isolated branched myofibers are more prone to rupture at branches during stimulation¹⁷ and mdx muscles containing a high percentage of branched myofibers are more vulnerable to contraction-induced injury.^12,14 Thus, decreasing the number of branched myofibers would likely be beneficial to muscle function.Open in a separate windowFigure 2Myofiber branching during aberrant muscle regeneration. (A) Phase contrast microscopy of a normal (left) and a branched (right) myofiber. The branched myofiber contains one branch at the end of the myofiber. (B) Schematic diagrams of myofibers with more complex patterns of branching than depicted in (A).Although branched myofibers have been reported in literature for over 100 years, the mechanism by which they arise is unknown and no causative molecules have been identified. The aberrant cytoarchitecture of branched myofibers likely arises from incomplete fusion of small myotubes during muscle regeneration⁸ though direct proof is lacking. Evidence in favor of these malformed myofibers arising from abortive regenerative processes includes the expression of neonatal myosin, a marker of early muscle regeneration, in the small branches.¹³ That these branches are newly formed is further suggested by the presence of centrally located nuclei,^13,18 a hallmark of regenerated myofibers. The observation that during muscle regeneration multiple small myotubes can form within the old basal lamina sheath^8,16,19 leads credence to the idea that aberrant fusion of such small myotubes underlies generation of branched myofibers. Indeed, electron microscopic studies support the ability of myotubes to fuse with one another in vivo;²⁰ myotubes readily fuse with one another in vitro.²¹ Recent studies of odorant receptor function during muscle regeneration in mice¹⁸ have identified the first molecule with a functional role in controlling myofiber branching and suggest that defects in muscle cell migration and/or adhesion may underlie the genesis of these branches.     

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?     

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.     

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.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

The pulling,pushing and fusing of lens fibers: A role for Rho GTPases

Lens development and differentiation are intricate and complex processes characterized by distinct molecular and morphological changes. The growth of a transparent lens involves proliferation of the epithelial cells and their subsequent differentiation into secondary fiber cells. Prior to differentiation, epithelial cells at the lens equator exit from the cell cycle and elongate into long, ribbon-like cells. Fiber cell elongation takes place bidirectionally as fiber tips migrate both anteriorly and posteriorly along the apical surface of the epithelium and inner surface of the capsule, respectively. The differentiating fiber cells move inward from the periphery to the center of the lens on a continuous basis as the lens grows throughout life. Finally, when fiber cells reach the center or suture line, their basal and apical tips detach from the epithelium and capsule, respectively, and interlock with cells from the opposite direction of the lens and form the suture line. Further, symmetric packing of fiber cells and degradation of most of the cellular organelle during fiber cell terminal differentiation are crucial for lens transparency. These sequential events are presumed to depend on cytoskeletal dynamics and cell adhesive interactions; however, our knowledge of regulation of lens fiber cell cytosketal reorganization, cell adhesive interactions and mechanotransduction, and their role in lens morphogenesis and function is limited at present. Recent biochemical and molecular studies have targeted cytoskeletal signaling proteins, including Rho GTPases, Abl kinase interacting proteins, cell adhesion molecules, myosin II, Src kinase and phosphoinositide 3-kinase in the developing chicken and mouse lens and characterized components of the fiber cell basal membrane complex. These studies have begun to unravel the vital role of cytoskeletal proteins and their regulatory pathways in control of lens morphogenesis, fiber cell elongation, migration, differentiation, survival and mechanical properties.Key words: lens, fiber cells, elongation, migration, adhesion, Rho GTPasesLens morphogenesis involves a complex network of regulatory genes and interplay between growth factor, mitogenic, cell adhesive and cytoskeletal signaling pathways. The lens originates from surface ectoderm near the optic vesicle and lens vesicle that is formed via invagination of lens placode differentiates into primary fibers (the posterior half ) and epithelial cells (the anterior half ). These changes in embryonic cells control the lens distinctive anterior-posterior polarity. Subsequently, the lens grows through the proliferation of epithelial cells and the differentiation of their progeny into secondary fiber cells.^1,2 The continuous addition of new fiber cells at the lens periphery leads to a gradual inward movement of older cells to the center of the lens. The ectodermal basement membrane that surrounds the lens vesicle thickens to form the lens capsule and is composed of mainly proteins of extracellular matrix.^2,3 Since the lens does not shed cells, they are retained throughout the lens''s life and are packed symmetrically within the lens⁴ (Fig. 1).Open in a separate windowFigure 1Diagram of organization of lens epithelial and differentiating fiber cells. The lens is enclosed by a thick capsule consisting of various extracellular matrix proteins. Lens epithelial cells at the equator divide and exit from the cell cycle, and as they exit from the cell cycle, they start to elongate bidirectionally by making apical (AMC) and basal (BMC) membrane complexes with epithelium and capsule, respectively. As fiber cells elongate, they are pushed down and migrate toward the center. As the fiber cells migrate toward the center, both the basal and apical membrane complexes are expected to undergo changes in a regulated manner to control fiber cell adhesive, protrusive and contractile activity. Finally, when the fiber cells reach the center or suture line, their basal and apical ends detach from the epithelium and capsule, respectively and interlock with cells from the opposite direction of the lens and form suture. During fiber cell elongation and differentiation, cell adhesive interactions are reorganized extensively, and terminally differentiated fiber cells exhibit loss of cellular organelle and extensive membrane remodeling with unique ball and socket interdigitations. Arrows indicate the direction of fiber cell movement. This schematic is a modified version of Figure 2 from Lovicu and McAvoy.¹Lens fiber cell elongation and differentiation is associated with a remarkable change in cell morphology, with the length of fiber cells increasing on the order of several hundredfold. These morphological changes are associated with extensive membrane and cortical cytoskeletal remodeling, actomyosin reorganization and cell adhesion turnover.^5–17 Additionally, the tips of the elongating fiber cells at both the anterior and posterior terminals slide along the lens epithelium and capsule, respectively, as these cells migrate inward, and finally detach at the suture, where they form contacts with their counterparts from the opposite side of the lens.^4,12 These cell movements are fundamental for maintaining distinct lens fiber cell polarity and are temporally and spatially regulated as the lens grows continuously throughout life.^1,2,12 Another unique feature of the lens is that during fiber cell terminal differentiation, all the cellular organelles, including nuclei, endoplasmic reticulum and mitochondria, are degraded in a programmed manner.¹⁸ It has been well documented that lens epithelial cell elongation and differentiation is associated with reorganization of actin cytoskeleton, increased ratio of G-actin to F-actin, integrin switching, formation of N-cadherin linked cell adhesions, and expression of actin capping protein tropomodulin.^{5,6,9,10,13,15,17,19–21} Importantly, disruption of actin cytoskeletal organization has been shown to impair lens epithelial differentiation and induce cataract formation, indicating the significance of actin cytoskeleton in lens differentiation and maintenance of lens optical quality.^14,22 Further, during accommodation, lens shape is changed in a reversible manner. Therefore, the tensional homeostasis between actomyosin inside the fiber cell and fiber cell adhesion on the inner side of the lens capsule is considered to be crucial for accommodation.¹²In the developing mouse and chicken lens, the tips of the fiber cells (both apical and basal) have been reported to cluster with different cytoskeletal proteins, including actin, myosin II, actin capping protein tropomodulin, and N-cadherins.^10,19,21 Similarly, adhesion regulating signaling molecules including integrins, focal adhesion kinase, Cdk5, abl kinase interacting protein (Abi-2), and Rho GTPases have been shown to localize to the fiber cell apical and basal tips.^20,23–26 Moreover, isolation and characterization of the fiber cell basal membrane complexes (BMCs) had revealed a symmetric organization of N-cadherin, myosin II, actin in association with myosin light chain kinase, focal adhesion kinase, β1 integrin and caldesmon.¹² The signaling activity, tensional property and dynamics of BMCs are thought to control the coordinated migration of fiber cells along the lens capsule, formation of lens suture line, and lens accommodation.¹² Additionally, the BMCs have been shown to undergo a characteristic regional rearrangement (including size and shape) during lens elongation and migration along the lens capsule.²⁷ Therefore, impaired fiber cell migration on the lens capsule is expected to induce cataractogenesis.²⁷ Taken together, these different observations convincingly indicate the importance of cytoskeleton and cell adhesion regulatory mechanisms in lens fiber cell elongation and migration.Although important insights have emerged regarding external cues controlling lens epithelial cell proliferation, elongation and differentiation, little is known regarding the specific signaling pathways that drive the processes culminating in fiber cell formation, migration, packing and maturation.^1,7,28 For example, growth factors are known to play key roles in influencing cell fates during development. Some of the major growth factor families, including FGFs and TGFβ/BMPs, have been shown to be involved in the regulation of lens developmental processes and primary fiber cell differentiation via ERK kinase activation.^1,28,29 However, the identity and role of signaling pathways acting downstream to growth factors regulating lens secondary fiber cell elongation, migration, adhesion, membrane remodeling and survival are poorly understood.^1,12,21,30 In particular, regulatory mechanisms involved in cytoskeletal reorganization, tensional force and cell adhesive interactions during these cellular processes have yet be identified and characterized.^{7,9,12,21,30–32}Our laboratory has been working on a broad hypothesis that the actin cytoskeletal and cell adhesive signaling mechanisms composed of Rho GTPases (Rho, Rac and Cdc42) and their effector molecules play a critical role in controlling lens growth and differentiation, and in maintaining lens integrity.⁷ The Rho family of small GTPases regulates morphogenesis, polarity, migration and cell adhesion.³³ These proteins bind GTP, exhibit GTPase activity, and cycle between an inactive GDP-bound form and an active GTP-bound form. This cycling is regulated by three groups of proteins: guanine-nucleotide exchange factors, which facilitate the exchange of GDP for GTP, thus rendering Rho GTPases active; GTPase-activating proteins, which regulate the inactivation of Rho by accelerating intrinsic GTPase activity and converting Rho GTPases back to their GDP-bound form; and GDP dissociation inhibitors (GDIs), which inhibit the dissociation of GDP bound to Rho GTPases.^33,34 The GTP-bound form of the Rho GTPases interact with downstream effectors, which include protein kinases (e.g., ROCK and PAK), regulators of actin polymerization (e.g., N-WASP/WAVE, PI3-kinase and mDia), and other proteins with adaptor functions.³³ The selective interaction of the different Rho GTPases with a variety of effectors determines the final outcome of their activation.³³ For example, during cell movement, Rac and Cdc42 stimulate formation of protrusions at the leading edges of cells, and RhoA induces retraction at the tail ends of cells. This coordinated cytoskeletal reorganization permits cells to move toward a target.³⁵ PI3-kinase and PI (3, 4, 5) P3 have also been widely implicated in controlling cell migration and polarity in a Rac GTPase-dependent manner.³⁵ Members of the Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin homologous protein (WAVE) families serve to link Rho GTPases signals to the ARP2/3 complex, leading to actin polymerization that is crucial for the reorganization of the actin cytoskeleton at the leading edge for processes such as cell movement and protrusions.³⁶ Importantly, all three Rho GTPases also regulate microtubule polymerization and assembly of adherens junctions to influence polarity and cell adhesion, respectively.^33,37Likewise, a tensional balance between cell adhesion on the outside and myosin II-based contractility on the inside of the cells is regulated by Rho GTPases.³⁸To explore the role of the Rho GTPases in lens morphogenesis and differentiation, we have targeted the lens Rho GTPases by overexpressing either the C3 exoenzyme (inactivator of RhoA and RhoB) or RhoGDIα (Rho GDP dissociation inhibitor) in a lens-specific manner in transgenic mice and followed their effects developmentally. These two transgenic mouse models exhibited ocular phenotype, including lens opacity (cataract) and microphthalmic eyes. Importantly, various histological, immunofluorescence and biochemical analyses performed in these developing transgenic mice have revealed defective lens morphogenesis, abnormal fiber cell migration, elongation, disrupted cytoskeletal organization and adhesive interactions, along with changes in proteins of the fiber cell gap junctions and water channels.^32,39 These lenses have also shown decreased ERM (ezrin, radixin, moesin) protein phosphorylation,⁴⁰ proteins that are involved in crosslinking of the plasma membrane with actin cytoskeleton,⁴¹ and increased apoptosis.³² Defective fiber cell migration has been found to be more notable in the Rho GDI overexpressing lenses than in the C3 exoenzyme expressing lenses (Fig. 2). The Rho GDI overexpressing lenses have shown a defective membrane localization of Rho, Rac and Cdc42 confirming their inactivation. These data, together with mechanistic studies performed using the lens epithelial cells and the noted effects on cell shape, actin polymerization, myosin phosphorylation and cell adhesive interactions, reveal the importance of Rho GTPase-dependent signaling pathways in processes underlying fiber cell migration, elongation, cytoskeletal and membrane organization and survival in the developing lens.⁷ Lens fiber cell BMC has been found to be localized intensely with Rac GTPase involved in cell migration (our unpublished work). Additionally, the Rho GDI transgenic lenses showed an impaired apical-apical cell-cell interactions between the fiber cells and epithelial cells.³² Moreover, the ruptured posterior capsule and disrupted suture lines in these lenses are indicative of defective BMC organization and activity.³²Open in a separate windowFigure 2Abnormal lens phenotype in the neonatal Rho GDIα overexpressing transgenic mouse. Hematoxylin and eosin-stained sagittal sections of P1 RhoGDIα transgenic eyes reveal abnormal migration and morphology of the posterior lens fibers as compared with the symmetric organization of lens fibers and their migration toward the lens suture in the wild type mouse (reproduced with permission from Maddala et al.)³².Further support for involvement of Rho GTPases in lens fiber cell differentiation and survival has come from studies conducted with chick lens epithelial explants and cultured epithelial cells. Inactivation of Rho kinase or Rac activation by PI3 kinase in chick lens epithelial cells has been reported to induce fiber cell differentiation and survival in association with distinct cortical actin cytoskeletal reorganization, indicating the significance of Rho GTPases in lens fiber cell differentiation and survival.^9,42 Additionally, lens fiber cell elongation and differentiation has been found to be associated with increased myosin light chain (MLC) phosphorylation, and inhibition of MLC phosphorylation regulated by MLC kinase and Rho kinase has induced lens opacity and disruption of cytoskeletal integrity, supporting the importance of myosin II activity in maintaining lens architecture and transparency.¹⁰ Importantly, various growth factors that regulate lens morphogenesis, fiber cell differentiation, and survival have been found to activate Rho and Rac GTPases and to induce MLC phosphorylation, actin cytoskeletal reorganization, and focal adhesion formation in lens epithelial cells.^7,30 In addition to Rho GTPases, inhibition of Src kinase has been shown to induce fiber cell differentiation in association with actin cytoskeletal reorganization and cell adhesive interactions.⁴³ Also, the expression and activation of focal adhesion kinase has been reported to increase in differentiating and migrating lens epithelial cells.⁴⁴ Both these molecules are well recognized to regulate cell migration by participating in the disassembly of cell adhesions at the front of migrating cells.³⁵Additional evidence for the participation of actin cytoskeletal organization and Rho GTPases in lens fiber cell migration and elongation has been derived from the studies of Abi-2 deficient mouse. Abl-interactor adaptor proteins Abi-1 and Abi-2 are linked to the Rac-WAVE-Arp2/3 signaling pathway and regulate actin polymerization and cell-cell adhesive interactions.⁴⁵ Homozygous deletion of Abi-2 in mice has been shown to exhibit ocular phenotype including microphthalmia and lens opacity similar to the Rho GDI overexpressing transgenic mouse eyes noted in previous studies.^23,32 In the absence of Abi-2, the secondary lens fiber orientation, migration and elongation were found to be defective, supporting the importance of Rac-WAVE-Arp2/3 signaling in lens fiber cell migration and cell adhesion.²³ Abi-2 has been shown to localize intensely to the both basal and apical regions of the fiber cells and adherens junctions, and suppression of Abi-2 expression in epithelial cells resulted in impaired adherens junctions and downregulation of actin nucleation promoting factors.²³ The significance of cytoskeletal signaling in lens has also been implicated in Lowe syndrome, a rare X-linked disorder characterized by congenital cataracts, results from mutations in the OCRL1 gene. The OCRL1 protein product (phosphatidylinositol 4, 5 bisphosphate 5-phosphatase) has been shown to participate in Rac GTPase regulated actin cytoskeletal organization, cell migration, and cell adhesion in various cell types.⁴⁶ Finally, Wnt/PCP signaling via activation of Rho GTPases has been suggested to control lens morphogenesis, fiber cell migration and differentiation.²⁶Importantly, given how the activity of the Rho GTPases is regulated by external cues and various effector proteins, a detailed understanding of the regulation of Rho GTPase signaling is necessary for a better appreciation of their role in lens morphogenesis, fiber cell elongation and differentiation, and tensional homeostasis. Further mechanistic studies are critical to unravel the specific role(s) of Rho GTPases and other cytoskeletal regulatory mechanisms involved in regulating the formation and disassembly of fiber cell basal and apical membrane complexes, fiber cell lateral membrane remodeling, and fiber cell-cell adhesive interactions during lens differentiation. Very little is known in terms of the assembly of different cell adhesive molecules at the apical-apical interface between the lens fibers and epithelial cells. We are only beginning to glimpse the regulatory networks involved in the regulation of fiber cell elongation, polarity, migration and adhesion. Many challenging questions remain: for example, how are the pathways regulating migration, basal and apical membrane complexes, and tensional homeostasis controlled by extracellular signals, and how are they integrated during fiber cell migration, suture formation, and packing? Novel insights into the molecular mechanisms regulating these cellular processes are expected to advance our understanding of lens morphogenesis, function and cataractogenesis.     

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