The AGC Kinase MtIRE: A Link to Phospholipid Signaling During Nodulation?

The development of nitrogen fixing root nodules is complex and involves an interplay of signaling processes. During maturation of plant host cells and their endocytosed rhizobia in symbiosomes, host cells and symbiosomes expand. This expansion is accompanied by a large quantity of membrane biogenesis. We recently characterized an AGC kinase gene, MtIRE, that could play a role in this expansion. MtIRE''s expression coincides with host cell and symbiosome expansion in the proximal side of the invasion zone in developing Medicago truncatula nodules. MtIRE''s closest homolog is the Arabidopsis AGC kinase family IRE gene, which regulates root hair elongation. AGC kinases are regulated by phospholipid signaling in animals and fungi as well as in the several instances where they have been studied in plants. Here we suggest that a phospholipid signaling pathway may also activate MtIRE activity and propose possible upstream activators of MtIRE protein''s presumed AGC kinase activity.Key Words: AGC kinase, nitrogen fixation, nodulation, Medicago truncatula, Sinorhizobium meliloti, infection zone, 3-phosphoinositide-dependent kinase, root hair elongationDuring symbiotic nitrogen-fixing nodule development, both plant cells and rhizobia undergo cell division and expansion.^1–3 In legume roots, nodule organogenesis is triggered by rhizobial Nod factor at the emerging root hair zone. In the indeterminate Medicago-Sinorhizobium symbiosis, inner cortical cell divisions form nodule primordia which emerge from the root and differentiate into complex nodule structures. Rhizobia enter the nodules through plant derived conduits, the infection threads (ITs). ITs begin in curled root hairs, grow through several cell layers and end at nodule primordia where rhizobia are deposited into host cell symbiosomes.² In mature nodules, the meristematic zone I at the nodule apex contains dividing cells. Rhizobia from ITs infect these cells as they exit zone I and enter the infection zone, zone II. The newly released rhizobia, now termed bacteroids, are rod-shaped. In the distal part of zone II, bacteroids divide along with the symbiosome membrane (also called the peribacteroid membrane) that contains them.⁴ As the plant cells with their internalized bacteroids progress toward the proximal end of zone II, bacteroid division ceases. Bacteroid elongation and expansion of the surrounding symbiosome space and membrane is a feature of the proximal side of zone II.⁴ Enormous membrane biogenesis accompanies progression through zone II. As the cells exit zone II, both host cells and bacteroids stop expanding. Interzone II-III is characterized by starch accumulation and zone III is where nitrogen fixation takes place.Members of the protein kinase AGC (for cAMP dependent, cGMP dependent, and protein kinase C) family have been shown to be important in yeast and mammalian signal transduction. The interaction of growth factors with their receptors leads to the activation of phosphatidylinositol (PtdIns) 3-kinase and the phosphorylation of PtdIns species.⁵ These then activate PDK1 enzymes, 3-phosphoinositide-dependent kinases, also AGC kinases,⁵ which then phosphorylate and activate downstream AGC kinases. Several plant AGC kinases have important roles in development and defense,^6–8 although most plant AGC kinases'' functions are still to be discovered.⁹ Two Arabidopsis AGC kinases, IRE and AGC2-1 have been shown to have roles regulating root hair elongation.^10,11We recently cloned and characterized a Medicago IRE-like AGC kinase gene MtIRE,¹² possibly orthologous to the Arabidopsis IRE gene, AtIRE.¹⁰ Because of MtIRE''s homology to AtIRE we thought it might function during infection, because infection threads can be viewed as inward root hair growth. However, MtIRE''s expression is novel. It is expressed only in nodules and flowers and not in roots or root hairs. During nodule development, its initial expression correlates with the onset of host cell and symbiosome expansion. Expression studies with nodulation mutants demonstrate that MtIRE expression correlates with mutant nodules'' abilities to support host cell and symbiosome expansion.¹² An MtIRE promoter-gusA reporter construct (Fig. 1A) shows expression in the proximal part of zone II, the site of continued host cell expansion and bacteroid and symbiosome elongation. RNA interference experiments were unfortunately inconclusive,¹² probably because of closely related more ubiquitously expressed IRE homologs.Open in a separate windowFigure 1(A) Localization of pMtIRE-gusA expression in wild-type nodulated roots. Composite M. truncatula plants with transgenic roots were grown in the presence of S. meliloti and stained with X-Gluc (blue) for the localization of MtIRE promoter activity. The arrow points to the X-Gluc staining in the proximal side of zone II in a 15 dpi nodule. The arrowhead points to root hairs in which no staining was observed. Bar = 100 µM. (B) Phospholipid signaling pathway that may activate MtIRE protein''s presumed kinase activity.We predict that MtIRE is part of a signal pathway regulating an aspect of host cell expansion or symbiosome elongation, or both. The CCS52A gene has a demonstrated role in host cell expansion, mediating endoreduplication.¹³ In contrast to MtIRE, its expression is found throughout zone II, as well as zone I, where it acts in cell division. One might expect other genes that regulate host cell expansion to also be expressed throughout zone II, which MtIRE is not. A unique feature of the region expressing MtIRE is symbiosome elongation.⁴ Because of MtIRE''s temporal and spatial expression patterns, we favor it having a role in symbiosome expansion, although we cannot rule out a role in the latter stages of host cell expansion.Signaling pathway for MtIRE activation is speculative (Fig. 1B) and based on AGC kinase signaling in other systems. AGC kinases are activated by phosphorylation by phosphoinositide-dependent kinase (PDK1) enzymes, also AGC kinases.⁹ We found 4 tentative consensus sequences (TCs) in the DFCI index (compbio.dfci.harvard.edu) that correspond to PDK1 genes of which 3, TC107355, TC94724 and TC94899, were isolated from expression libraries from roots with developing or mature nodules. PDKs are activated by interaction with lipids. The Arabidopsis PDK1 binds to several signaling lipids, including phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidic acid (PA).¹⁴ Phosphatidylinositol 3-kinase (PI3K) activity produces PtdIns3P and PI3K genes have been observed to be induced during nodule organogenesis in soybean¹⁵ and in M. truncatula.¹⁶ In soybean, two PI3K genes were identified with one specifically expressed during the early stages of nodulation when membrane biogenesis takes place. This gene''s predicted protein has potential phosphorylation sites for cAMP dependent kinases and Ca/calmodulin-dependent kinases.¹⁵ In soybean, PI3K enzymatic activity correlated with membrane proliferation during nodulation.¹⁵ More generally, PI3Ks are implicated in vesicular trafficking and cytoskeletal organization;¹⁷ both are required for host cell and symbiosome elongation. We suggest a model where MtIRE kinase activity is activated by PDK1, which is itself regulated by PI3K through the production of PtdIns3P. More speculatively, PI3K could be under the control of the Nod factor signaling pathway Ca/calmodulin-dependent kinase DMI3.^18,19 DMI3 is induced during nodulation, with highest expression levels found in the distal side of the infection zone,²⁰ before expression of MtIRE. Expression could persist to the proximal side of this zone, similar to the expression of another Nod factor signaling component, DMI2.²¹ Alternatively, MtIRE could be activated by PA in a PDK1-dependent manner similar to Arabidopsis AGC2-1.¹¹ PA can be produced by phospholipase C (PLC) or phospholipase D (PLD) pathways, both of which have been implicated in transducing Nod factor signals.^22–26 Either of these models includes Nod factor signaling in proximal zone II, which has not been well-studied. Expression of rhizobial nod genes has been observed in zone II,²⁷ making Nod factor signaling in this zone plausible. Further examination of zone II and predicted upstream regulators of MtIRE will address this model.     

Why have chloroplasts developed a unique motility system?

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

Structural basis of transmembrane domain interactions in integrin signaling

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

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     

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 chemical cross talk between rice and barnyardgrass

The chemical cross talk between rice and barnyardgrass which is one of the most noxious weeds in rice cultivation was investigated. Allelopathic activity of rice was increased by the presence of barnyardgrass seedlings or barnyardgrass root exudates. Rice allelochemical, momilactone B, concentration in rice seedlings and momilactone B secretion level from rice were also increased by the presence of barnyardgrass seedlings or barnyardgrass root exudates. As momilactone B possesses strong growth inhibitory activity and acts as an allelochemical, barnyardgrass-induced rice allelopathy may be due to the increased momilactone B secretion. These results suggest that rice may respond to the presence of neighboring barnyardgrass by sensing the chemical components in barnyardgrass root exudates and increase allelopathic activity by elevated production and secretion levels of momilactone B. Thus, rice allelopathy may be one of the inducible defense mechanisms by chemical-mediated plant interaction between rice and barnyardgrass and the induced-allelopathy may provide a competitive advantage for rice through suppression of the growth of barnyardgrass.Key words: allelopathy, Echinochloa, chemical interaction, induced-allelopathy, momilactone, Oryza sativaThe chemical cross talk between host and symbiotic or parasitic plants is an essential process for the development of physical connections in symbiosis and parasitism.^1–3 Barnyardgrass is one of the most common and noxious weeds in rice paddy fields.⁴ Although barnyardgrass is adapted rice production system due to its similarity in growth habit, the reason why barnyardgrass so often invades into the rice paddy fields is unknown. There might be some special interactions between both plant species.Plants are able to accumulate phytoalexins around infection sites of pathogens soon after sensing elicitors of pathogen origin. This accumulation of phytoalexins can protect the plants from further pathogen infection.^5,6 Plants are also able to activate defense mechanisms against attacking herbivores by sensing volatile compounds, such as methacrolein and methyl jasmonate, released by herbivore-attacked plant cells. The volatile-sensed plants increase the production of phenolics, alkaloids, terpenes and defense proteins, which reduce herbivory attacks.^7,8 Therefore, plants are able to elevate the defense mechanisms against several biotic stress conditions by detection of various compounds.Allelopathy is the direct influence of organic chemicals released from plants on the growth and development of other plants.^9–11 Allelochemicals are such organic chemicals involved in the allelopathy.^12,13 Allelochemicals can provide a competitive advantage for host-plants through suppression of soil microorganism and inhibition of the growth of competing plant species because of their antibacterial, antifungal and growth inhibitory activities.^3,14,15Rice has been extensively studied with respect to its allelopathy as part of a strategy for sustainable weed management, such as breeding allelopathic rice strains. A large number of rice varieties were found to inhibit the growth of several plant species when these rice varieties were grown together with these plants under the field or/and laboratory conditions.^16–20 These findings suggest that rice may produce and release allelochemicals into the neighboring environments and may inhibit the growth of the neighboring plants by the allelochemicals.Potent allelochemical, momilactone B, was isolated from rice root exudates.²¹ Momilactone B inhibits the growth of typical rice weeds like barnyardgrass and Echinochloa colonum at concentrations greater than 1 µM and the toxicity of momilactone B to rice itself was very low.²² In addition, rice plants secrete momilactone B from the roots into the rhizosphere over their entire life cycle.²² The observations suggest rice allelopathy may be primarily dependant on the secretion levels of momilactone B from the rice seedlings.^22,23Allelopathic activity of rice exhibited 5.3- to 6.3-fold increases when rice and barnyardgrass seedlings were grown together. Root exudates of barnyardgrass seedlings also increased allelopathic activity and momilactone B concentration in rice seedlings. The increasing the exudate concentration increased the allelopathic activity and momilactone B concentration in rice.²⁴ Thus, the chemical components in barnyardgrass root exudates may affect gene expressions involved in momilactone B biosynthesis. However, effects of the barnyardgrass root exudates on the secretion level of mimilactone B from rice has not yet reported.Rice seedlings were incubated in the medium containing barnyardgrass root exudates for 10 d, and secretion level of momilactone B by rice was determined (Fig. 1). The root exudates increased the secretion level significantly at concentrations greater than 30 mg/L of barnyardgrass root exudates, and increasing the concentration increased the secretion level. At concentrations of 300 mg/L of the root exudates, the secretion level was 10-fold greater than that in control (0 mg of root exudate). There was no significant difference in the osmotic potential between the medium contained barnyardgrass root exudates and control medium (all about 10 mmol/kg), and pH value of the medium was maintained at 6.0 throughout the experiments.²⁵ These results suggest that unknown chemical components in the barnyardgrass root exudates may induce the secretion of momilactone B from rice. As momilactone B possesses strong phytotoxic and allelopathic activities,^21–23,25 the elevated production and secretion of momilactone B in rice may provide a competitive advantage for root establishment through local suppression of pathogens and inhibition of the growth of competing plant species including barnyardgrass. Thus, barnyardgrass-induced rice allelopathy may be caused by the chemical components in the barnyardgrass root exudates.Open in a separate windowFigure 1Effects of barnyardgrass root exudates on momilactone B secretion level in rice. Rice seedlings were incubated in the medium containing barnyardgrass root exudates for 10 d, and secretion level of momilactone B was determined as described by Kato-Noguchi.²⁴ The experiment was repeated six times with three assays for each determination. Different letters show significant difference (p < 0.01) according to Tukey''s HSD test.Although mechanisms of the exudation are not well understood, it is suggested that plants are able to secrete a wide variety of compounds from root cells by plasmalemma-derived exudation, endoplasmic-derived exudation and proton-pumping mechanisms.^3,15 Through the root exudation of compounds, plants are able to regulate the soil microbial community in their immediate vicinity, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species.^3,14,15 The present research suggests that rice may be aware of the presence of neighboring barnyardgrass by detection of certain key in barnyardgrass root exudates, and this sensorial function may trigger a signal cascade resulting in increasing rice allelopathy through increasing production of momilactone B and secretion of momilactone B into the rhizosphere. Therefore, rice allelopathy may potentially be an inducible defense mechanism by chemical-mediated plant interactions between rice and barnyardgrass.     

Protein Misfolding and the Serpinopathies

The serpins are the largest superfamily of protease inhibitors. They are found in almost all branches of life including viruses, prokaryotes and eukaryotes. They inhibit their target protease by a unique mechanism that involves a large conformational transition and the translocation of the enzyme from the upper to the lower pole of the protein. This complex mechanism, and the involvement of serpins in important biological regulatory processes, makes them prone to mutation-related diseases. For example the polymerization of mutant α₁-antitrypsin leads to the accumulation of ordered polymers within the endoplasmic reticulum of hepatocytes in association with cirrhosis. An identical process in the neuron specific serpin, neuroserpin, results in the accumulation of polymers in neurons and the dementia FENIB. In both cases there is a clear correlation between the molecular instability, the rate of polymer formation and the severity of disease. A similar process underlies the hepatic retention and plasma deficiency of antithrombin, C1 inhibitor, α₁-antichymotrypsin and heparin co-factor II. The common mechanism of polymerization has allowed us to group these conditions together as a novel class of disease, the serpinopathies.Key Words: serpins, α1-antitrypsin, neuroserpin, polymerization, dementia, conformational disease, serpinopathiesSerpins (or serine protease inhibitors) are the largest family of protease inhibitors. They have been found in all major branches of life including viruses, prokaryotes and eukaryotes.^1–3 Despite their name there is increasing evidence that serpins can also inhibit other classes of proteases as demonstrated by the viral serpin CrmA and recently by a plant serpin, serpin1.^4,5 They can even play a non-inhibitory role in events as diverse as blood pressure regulation (angiotensinogen), chromatin condensation (MENT), tumor progression (maspin), protein folding (hsp47) and hormone transport (cortisol and thyroxine binding globulin).⁶One of the most important roles of serpins is the regulation of enzymes involved in proteolytic cascades. Among these serpins are α₁-antitrypsin, α₁-antichymotrypsin, C1 inhibitor, antithrombin and plasminogen activator inhibitor-1, which play an important role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways, respectively.^1,3 The serpin superfamily is characterised by more than 30% homology with the archetypal serpin α₁-antitrypsin and conservation of tertiary structure.^7,8 Serpins adopt a metastable conformation composed in most cases of 9 α-helices, three β-sheet (A to C) and an exposed mobile reactive centre loop (RCL). This flexible RCL typically contains 20 residues that act as a pseudo substrate for the target protease (Fig. 1A).^9–15 After formation of a Michaelis complex^16,17 the enzyme cleaves the P1-P1′ bond of the serpin, releasing the P1'' residue and forming an ester bond between the protease and the serpin.^18,19 This is then followed by a dramatic conformational transition from a stressed to relaxed conformation with the enzyme being pulled from the upper to the lower pole of the serpin and the insertion of the reactive loop as an extra strand in β-sheet A.^20–25 As a consequence of this conformational change the thermal stability of the serpin is greatly enhanced. Whereas a typical serpin in its native state exhibits a midpoint of thermal denaturation of around 50–60°C, a cleaved serpin with its RCL fully incorporated into β-sheet A denatures at temperatures >120°C.^9,26,27 Another consequence is the inactivation of the enzyme, stabilised at the acyl-intermediate and unable to proceed further to deacylation of the complex.^24,28 This serpin-protease complex then binds to members of the lipoprotein receptor family and is cleared from the circulation.^29–31Open in a separate windowFigure 1Inhibition of neutrophil elastase by α₁-antitrypsin and the structural basis of polymerization. (A) After docking (left) the neutrophil elastase (grey) is inactivated by movement from the upper to the lower pole of the protein (right). This is associated with the insertion of the RCL (red) as an extra strand into β-sheet A (green). (B) The structure of α₁-antitrypsin is centred on β-sheet A (green) and the mobile reactive centre loop (red). Polymer formation results from the Z variant of α₁-antitrypsin (Glu342Lys at P17; indicated by arrow) or mutations in the shutter domain (blue circle) that open β-sheet A to favour partial loop insertion and the formation of an unstable intermediate (M*). The patent β-sheet A then accepts the loop of another molecule to form a dimer (D), which then extends into polymers (P). The individual molecules of α₁-antitrypsin within the polymer, although identical, are coloured red, yellow and blue for clarity. Figure reproduced with permission from Lomas et al.⁹⁷Despite the evolutionary advantage conferred upon serpins by the remarkable mobility of the native state, their complexity is also their weak point.^19,32 Mutations affecting the serpins can lead to a variety of diseases, resulting from either a gain or loss of function.^6,19 For example mutations can cause aberrant conformational transitions that result in the retention of the serpin within the cell of synthesis. This will lead to either protein overload and death of the cell in which the serpin is synthesised, or disease as a consequence of the resulting plasma deficiency. Such a mechanism underlies diseases as diverse as cirrhosis, thrombosis, angio-oedema, emphysema and dementia. We review here the common mechanism underlying these diseases that we have grouped together as the serpinopathies.^33–35 The aggregation and accumulation of conformationally destabilized proteins is an important feature of many neurodegenerative diseases, including Alzheimer''s and Parkinson''s disease and the spongiform encephalopathies. Indeed we have used the serpinopathies as a paradigm for these other ‘conformational diseases’.³⁶     

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

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