Cell identity in the preimplantation mammalian embryo : an epigenetic perspective from the mouse The early preimplantation mouse embryo is a unique system where it is possible to explore the foundations of totipotency and differentiation . Following fertilization , a single cell , the zygote , will give rise to all tissues of the organism . The first signs of differentiation in the embryo are evident at the blastocyst stage with the formation of the trophectoderm , a differentiated tissue that envelopes the inner cell mass . The question of when and how the cells start to be different from each other in the embryo is central to developmental biology : as cell fate decisions are undertaken , loss of totipotency comes about . Although the blastomeres of the preimplantation embryo are totipotent , as the embryo develops some differences appear to develop between them which are , at least partially , related to the epigenetic information of each of these cells . The hypothesis of epigenetic asymmetries acting as driver for lineage allocation is presented . Although there are now some indications that epigenetic mechanisms are involved in cell fate determination , much work is needed to discover how such mechanisms are set in play upon fertilization and how they are transmitted through cell division . These considerations are further discussed in the context of preimplantation genetic diagnosis : does it matter to the embryo which cell is used for genetic diagnosis ? The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future . Embryonic development starts from a single cell , the zygote . In this cell , the two gametes convey and contribute information to start a new developmental programme . The formation of the newly fertilized zygote constitutes therefore the climax of totipotency because of the resulting zygote 's inherent ability to produce all cell types in a new organism . In the mammalian embryo , the first differentiative event occurs as inner cells form upon cell division at the 8-cell stage . As a result of this division , an ` inner ' and an ` outer ' population of cells can be distinguished in the 16-cell stage embryo , which will respectively occupy different positions in the morula -LRB- Johnson and Ziomek , 1981 -RRB- . Much work has been done in trying to understand how cell polarity develops in the inner and outer cells , but I will not deal with this topic here and instead will refer the reader to an excellent review published elsewhere -LRB- Johnson and McConnell , 2004 -RRB- . The inner cells will develop into the inner cell mass -LRB- ICM -RRB- and the outer layer of cells will differentiate into the trophectoderm -LRB- Tarkowski and Wroblewska , 1967 ; Ziomek and Johnson , 1982 -RRB- . Morphologically , however , the first overt signs of differentiation are evident only at the blastocyst stage , with the formation of the trophectoderm , which is the first differentiated tissue to form as an epithelial layer that envelops the ICM -LRB- Fig. 1A -RRB- . The latter , in contrast to the trophectoderm , retains its pluripotent character and the ability to self renew . While the ICM will give rise to the embryo proper , the trophectoderm will give rise to the extraembryonic tissues that will support development of the embryo during gestation . The ICM will also give rise to yet another extraembryonic tissue , the primitive endoderm , which is first visible as a cuboidal layer of cells lining the blastocoelic cavity on the fourth day of development . The trophectoderm and the ICM each display molecular identity , which is reflected in part by the expression of specific genes that are , for the ICM , involved in its specification and the maintenance of pluripotency -LRB- such as Nanog and Oct4 -RRB- or , for the trophectoderm , that are required for its differentiation -LRB- such as Cdx2 -RRB- -LRB- Palmieri et al. , 1994 ; Nichols et al. , 1998 ; Chambers et al. , 2003 ; Mitsui et al. , 2003 ; Strumpf et al. , 2005 -RRB- . The bifurcation of these two lineages is complete at the late blastocyst stage . At this stage , cells from the ICM do no longer have the potential to form trophectoderm derivates in vivo upon transplantation -LRB- Rossant and Lis , 1979 -RRB- , indicating that these cells have lost their totipotency and that lineage allocation has definitely occurred . Figure 1 : Cell lineages of the mammalian blastocyst and epigenetic marking -LRB- A -RRB- Representation of the lineages in the mammalian blastocyst on the third day of gestation -LRB- E3 .5 -RRB- . The blastocyst is composed of two distinct populations of cells : the trophectoderm -LRB- red -RRB- and the inner cell mass -LRB- ICM , green -RRB- , which display molecular identity and epigenetic asymmetries . The embryonic-abembryonic regions -LRB- dotted line -RRB- of the blastocyst are determined by the position of the ICM , which lies within the embryonic region of the blastocyst . -LRB- B -RRB- Diagram illustrating some of the epigenetic marks . The DNA wrapped around the nucleosome -LRB- beige cylinders -RRB- is shown as light blue . The DNA is subject to DNA methylation , which constitutes one of the main epigenetic players . The core histones -LRB- beige -RRB- that form the nucleosome can be covalently modified -LRB- by acetylation , methylation , phosphorylation -RRB- , particularly on their N-terminal tails . Each of these marks can have an effect on how the information contained in the DNA is read by modulating downstream events such as transcriptional activation or repression . For example , histone methylation -LRB- Me -RRB- can have a positive effect on transcription -LRB- green -RRB- or a repressive one -LRB- red -RRB- . The marks can be present in different combinations and may change during the cell cycle . -LRB- C -RRB- Model for epigenetic marking and lineage allocation . In this model , an epigenetic mark would be laid down in a given cell during development . There could be other epigenetic event -LRB- s -RRB- that reinforce and/or are influenced by the first marking event . Cumulatively , this could result in determination of the fate of that cell towards a lineage in the blastocyst . The lineage specific marks could stabilize such cell identity and might be necessary for further differentiation . Alternatively , the acquisition of these epigenetic marks could be the result of cell fate determination . One should also consider that not only the nature of the mark would be important , but also the different regions of the chromatin that would be affected by such marks . Because the cells in the preimplantation embryo are totipotent and because the chromatin will still need to be dynamically remodelled during subsequent development , flexibility should be an important component of epigenetic mechanisms taking place during early development . As cell fate decisions are taken , a concomitant loss of totipotency takes place Investigations during recent years have recreated an interest in whether the blastomeres of the mammalian embryo are truly alike throughout preimplantation development before the first differentiative division mentioned above . In other words , whether they acquire a ` fate ' or whether they start to differ from each other prior to their spatial ` inner/outer ' allocation within the embryo upon the formation of the morula . Of course , if this were to be the case , the big challenge would be to ascribe molecular mechanisms to these processes . The question of when and how the cells start to be different from each other is not a trivial one . In particular , because as the first cell fate decisions are undertaken , a concomitant loss of totipotency occurs . The developmental time window when this first cell fate decision occurs comprises a number of epigenetic events -LRB- Morgan et al. , 2005 ; Surani et al. , 2007 -RRB- . These events include the reprogramming of the parental chromatin . Whether such epigenetic events are the cause or the consequence of reprogramming remains an exciting open question , but it is probably a combination of the two . Moreover , the two lineages of the blastocyst exhibit some epigenetic asymmetries . This mini-review is divided in two parts , the first one will deal with the main epigenetic mechanisms that are known to occur during mammalian preimplantation development . The second one will give an overview on data obtained through experimental embryology manipulations and lineage tracing observations to study cell fate in the early mouse embryo . Epigenetic mechanisms in the preimplantation embryo In general terms , epigenetic mechanisms include DNA methylation , covalent modification of histones , chromatin remodelling and histone replacement through incorporation of the so-called histone variants -LRB- Fig. 1B -RRB- . Histone marks have emerged as one of the main players involved in epigenetic mechanisms -LRB- Kouzarides , 2007 -RRB- . Histone modifications can be highly dynamic , or have a function in epigenetic memory . Although it is still unclear whether they are the actors of the epigenetic information or the epigenetic information itself , it is evident that covalent modifications of histones are essential components of the epigenome . Histones can be modified by a number of enzymes that mediate methylation , acetylation , phosphorylation , ubiquitynation and ADP-ribosylation of specific amino acid residues -LRB- reviewed in Kouzarides , 2007 -RRB- . By and large , the highest density of modifications so far described occurs in histone H3 , particularly on its tail . The effects of these modifications on the chromatin and on cellular processes are very diverse , and a modification of the same residue can even have opposite effects depending on the type of modification . For example , trimethylation of H3K9 is considered as a repressive mark , whereas acetylation of the same lysine has a positive effect on transcription -LRB- Bannister and Kouzarides , 2005 -RRB- . Likewise , methylation of arginine residues can have a positive effect on transcription -LRB- Chen et al. , 1999 -RRB- or a repressive effect -LRB- Pal et al. , 2004 -RRB- , depending both on the targeted residue and on whether the methylation is symmetric or asymmetric . For some of the modified residues , there is a very clear view of the outcome of an eventual modification : H3K9me3 creates a specific docking site for the heterochromatin protein 1 -LRB- HP1 -RRB- , which subsequently recruits the H3K9 methyltransferase Su -LRB- var -RRB- 3-9 and reinforces an autoregulatory loop for heterochromatin formation and maintenance -LRB- Bannister et al. , 2001 ; Lachner et al. , 2001 ; Nakayama et al. , 2001 -RRB- . The levels of regulation of epigenetic events in the preimplantation mouse embryo are multiple . They include the regulation of the subcellular localization of DNA methyltransferase activity , highlighted by the cytoplasmic retention of Dnmt1o -LRB- Carlson et al. , 1992 -RRB- ; the exclusion of a particular histone modification from the chromatin , which is exemplified by the lack of detection of H3K9me3 in the paternal pronucleus after fertilization resulting in an asymmetry of histone marks between the two pronuclei -LRB- Arney et al. , 2002 ; Santos et al. , 2005 -RRB- , the differential incorporation of chaperons and histone variants in the parental chromatin -LRB- van der Heijden et al. , 2005 ; Torres-Padilla et al. , 2006 -RRB- and the acquisition of highly specific histone variants in the gametes -LRB- Clarke et al. , 1992 ; Tanaka et al. , 2001 ; Govin et al. , 2007 -RRB- . Further , the maternal and paternal pronuclei exhibit different patterns of global DNA methylation : while the paternal pronucleus is rapidly demethylated -- presumably through an active mechanism -- right after fertilization , the maternal pronucleus is only passively demethylated through the subsequent rounds of replication and cell division that follow the first mitosis of the embryo -LRB- Mayer et al. , 2000 -RRB- . Moreover , while the centromeric and pericentric paternal chromatin remain DNA methylated , the maternal DNA loses methylation in such regions -LRB- Rougier et al. , 1998 -RRB- . The changes in the levels of DNA methylation as development proceeds in the preimplantation embryo are dynamic . Global levels of DNA methylation have been analysed by immunofluorescence , bisulphate sequencing and restriction digestion -LRB- Rougier et al. , 1998 ; Mayer et al. , 2000 ; Santos et al. , 2002 ; Aranyi and Paldi , 2006 -RRB- . Bisulphate sequencing and restriction digestion have also been used to analyse the methylation status of repeat sequences -LRB- such as L1 and IAP repeats -RRB- and some single-copy sequences -LRB- such as actin -RRB- -LRB- Howlett and Reik , 1991 ; Oswald et al. , 2000 -RRB- . These studies have revealed that although global levels of DNA methylation decrease until the blastocyst stage , changes in DNA methylation do not occur to the same extent on all genes . Remarkably , imprinted genes -LRB- such as H19 -RRB- and some repeat sequences -LRB- such as IAPs -RRB- do not undergo demethylation -LRB- Tremblay et al. , 1997 -RRB- . During early stages of development , decisions involved in cell fate determination and pluripotency have to be assumed . These processes require the chromatin to be dynamically remodelled to ensure its plasticity . This implies that the mechanisms involved in regulation of chromatin structure need to ensure stability across generations and cell division , but they also need to be flexible -LRB- Reik , 2007 -RRB- . The double nature of a covalent modification either on histones and/or on the DNA as dynamic -LRB- because in principle it can be added and removed -RRB- and at the same time its potential ability to propagate a memory , fits well with these aforementioned needs . Moreover , in keeping with the importance of epigenetic mechanisms during early development , the possibility for an epigenetic mark -LRB- s -RRB- underlying these phenomena appears very attractive . Concerning the epigenetic asymmetries of the ICM and the trophectoderm , the ICM displays , in global terms , higher levels of DNA methylation compared with the trophectoderm -LRB- Dean et al. , 2001 ; Santos et al. , 2002 -RRB- . Specific histone marks such as trimethylation of lysines 9 and 27 of histone H3 -LRB- H3K9me3 and H3K27me3 , respectively -RRB- are enriched in the ICM compared with the trophectoderm -LRB- Erhardt et al. , 2003 -RRB- . Likewise , the trophectoderm retains an imprinted form of X inactivation , where the paternal X chromosome is silenced -LRB- Heard and Disteche , 2006 -RRB- . This is in contrast to the ICM , where there is reactivation of the inactive X chromosome and a subsequent round of inactivation occurs at random in which either the maternal or the paternal chromosome is inactivated -LRB- Mak et al. , 2004 ; Okamoto et al. , 2004 -RRB- . The aforementioned epigenetic asymmetries of the two lineages of the blastocyst are evident once lineage allocation has taken place and might reinforce their molecular identity . However , epigenetic asymmetry could also act as a driver for lineage allocation , in which case , the former would precede the latter -LRB- Fig. 1C -RRB- . This constitutes a fascinating current working hypothesis . Development of cell identity in the mouse embryo From experimental embryology , we have learnt from pioneer experiments performed in the 50 's that after mechanical separation of the blastomeres of a 2-cell stage embryo and transfer into foster mothers , each of these two cells gives rise to an adult mouse -LRB- Tarkowski , 1959 -RRB- . This indicates that mouse embryos are very flexible in what people have referred to as developmental potential . Derivation of twins from mouse blastomeres at later stages of development -LRB- e.g. 4-cell stage or later -RRB- has not been possible . Although this has been linked to the low number of cells present in the resulting embryos and hence their inability to form an ICM with a normal cell number , rather than to their developmental potential or identity -LRB- Tarkowski and Wroblewska , 1967 ; Rossant , 1976 -RRB- . Indeed , when random single 4 - and 8-cell stage blastomeres are aggregated with ` carrier ' blastomeres , their progeny is able to contribute to all the tissues of the embryo -LRB- Kelly , 1977 ; Tarkowski et al. , 2005 -RRB- , and in this sense , the blastomeres were considered to be totipotent . So , what are these carrier cells providing ? Is it a simple matter of cell number ? and/or of an appropriate environment ? It is also possible however that some subtle intrinsic differences of these blastomeres might be masked by the limitations of the outcome of transplantation procedures , given that the results of these studies are very often far from 100 % and the transplantation efficiency is never absolute . Whether this is solely related to technical difficulties linked to these challenging manipulations or to an intrinsic property of specific blastomeres of the embryo is impossible to ascertain . Despite a controversial viewpoint on whether there is any polarity in the early mouse embryo or not , most reports coincide with the interpretation that a blastomere at the 2-cell stage contributes to both the ICM and the trophectoderm -LRB- reviewed in Edwards and Beard , 1997 ; Zernicka-Goetz , 2006 -RRB- . The suggestion of a given blastomere at the 2-cell stage having a ` preferential ' fate towards either of the blastocyst lineage is not resolved and some researchers have suggested that a slight , but distinct difference in the fate of 2-cell stage blastomeres might be disturbed by experimental manipulations -LRB- Alarcon and Marikawa , 2005 ; Hiiragi et al. , 2006 -RRB- . The role of extrinsic factors to the embryo , such as the shape of the zona pellucida , in axis specification of the blastocyst is also a matter of controversy -LRB- Gardner , 2007 ; Kurotaki et al. , 2007 -RRB- . However , there are some indications that a bias for a blastomere to contribute to a given region of the embryo in the blastocyst could already exist at the 4-cell stage -LRB- Fujimori et al. , 2003 ; Piotrowska-Nitsche and Zernicka-Goetz , 2005 -RRB- . These conclusions are mostly based on lineage tracing experiments of labelled blastomeres , and their degree of invasiveness is debatable . Some groups have used the plane of division in relation to the animal -- vegetal axis of the embryo as a sort of guideline to distinguish and characterize blastomeres according to their cleavage plane . By convention , the animal pole is demarcated by the position of the second polar body -LRB- which is extruded after resumption of meiosis II upon fertilization -RRB- and hence the vegetal pole lies on the opposite side -LRB- Fig. 2A -RRB- . The division from the 2 - to the 4-cell stage would segregate for the first time the ` animal ' and the ` vegetal ' components of the zygote if it occurs equatorially , that is , perpendicular to the animal -- vegetal axis of the conceptus -LRB- Gardner , 2002 -RRB- . Thus , whereas a cell that derives from a meridional division -LRB- parallel to the animal -- vegetal axis -RRB- inherits both components , an equatorial division gives rise to an ` animal ' and a ` vegetal ' blastomere -LRB- Fig. 2B -RRB- . By looking into the plane of division and the order at which this division occurs from the 2 - to the 4-cell stage and subsequent lineage tracing , a subgroup of embryos was identified where it is possible , with a relatively high probability , to predict the future position of the blastomeres in the blastocyst -LRB- Piotrowska-Nitsche et al. , 2005 -RRB- . This group of embryos are referred to as ME embryos -LRB- for Meridional -- Equatorial , reflecting the type of cleavage plane and order of division that generated them -LRB- Fig. 2B -RRB- -LRB- Piotrowska-Nitsche et al. , 2005 ; Piotrowska-Nitsche and Zernicka-Goetz , 2005 -RRB- . Although the ME type embryos constitute only a small part -LRB- 20 % -RRB- of a complete litter , they provide a very nice system where it is possible to explore the foundations of differentiation in the embryo . Indeed , the ` vegetal ' blastomere would most often populate the abembryonic region of the blastocyst , which contains mainly mural trophectoderm . Figure 2 : Blastomere division planes according to the animal-vegetal axis in the embryo -LRB- A -RRB- The animal -- vegetal -LRB- A -- V -RRB- axis of the preimplantation embryo is demarcated , by convention , by the position of the second polar body , which marks the animal pole . The opposite side to the site of extrusion of the polar body is , by default , the vegetal pole of the embryo . -LRB- B -RRB- The division pattern from 2-to - 4-cell stage of a typical embryo that undergoes one meridional -LRB- M -RRB- and one equatorial -LRB- E -RRB- division -LRB- ME embryo -RRB- is represented . The cleavage plane is depicted by a red dashed line -LRB- embryo on the left -RRB- . The blastomere that divides earlier is represented on the left . A Meridional division has a cleavage plane that is parallel to the A -- V axis of the embryo and hence gives rise to two cells containing both ` animal ' and ` vegetal ' components -LRB- two cells with pink and yellow motifs on the embryo depicted on the right -RRB- . In contrast , when a 2-cell stage blastomere divides equatorially , a segregation of the ` animal ' and ` vegetal ' cytoplasm occurs and follows derivation of an ` animal ' -LRB- pink -RRB- and a ` vegetal ' -LRB- yellow -RRB- blastomere In looking for epigenetic marks that could be involved in an eventual cell fate decision of the blastomeres of these ME embryos , it was found that the ` vegetal ' blastomere displays the lowest levels of dimethylated arginine 26 of H3 -LRB- H3R26me2 -RRB- . If H3R26me2 participates in lineage allocation , one might predict that modulating the levels of histone arginine methylation , would have an effect over cell fate . Overexpression of the histone methyltransferase that methylates this residue on H3 , PRMT4/CARM1 , into individual blastomeres not only induced upregulation of Nanog and Sox2 , but also resulted in an almost complete allocation of these blastomeres into the ICM compartment -LRB- Torres-Padilla et al. , 2007 -RRB- . It is interesting to note that in the mouse , the differences described in histone H3 arginine methylation appear at the 4-cell stage -LRB- Torres-Padilla et al. , 2007 -RRB- , that is . as early as one cell cycle after the major wave of embryonic genome activation occurs -LRB- Schultz , 2002 ; Hamatani et al. , 2004 -RRB- , which suggests that these events might be , at least in part , linked to the transcriptional programme of the embryo . The developmental stage at which genome activation occurs in other mammalian species varies considerably : it takes place at the 1-to - 2-cell stage in mice , the 4-to - 8-cell stage in cows and humans , and the 8-to - 16-cell stage in sheep and rabbits -LRB- Schultz and Heyner , 1992 -RRB- . Would this anticipate a different timing for an eventual ` cell fate path ' for other species ? Normal fertile adults can be derived from single blastomeres from 2 - , 4 - and 8-cell stage embryos in the rabbit , sheep and cattle -LRB- Moore et al. , 1968 ; Willadsen , 1981 ; Willadsen and Polge , 1981 -RRB- . Thus , these species indeed support blastomere isolation and further development at later stages than the mouse does . Transplantation of isolated 4-cell blastomeres into morula stage embryos has demonstrated that the blastomeres at the 4-cell stage are totipotent -LRB- Kelly , 1977 -RRB- . In this context , it is important to note that aggregating the ` vegetal ' cell from ME embryos to form chimeric embryos , showed that this cell is able to contribute to all tissues in the embryo . However , aggregating the same blastomere with other ` vegetal ' blastomeres from ME embryos exclusively , results in a failure to proceed through development -LRB- Piotrowska-Nitsche et al. , 2005 -RRB- . Thus , the environment where the blastomeres develop is crucial for the success of the embryo throughout development , and in a ` normal ' situation , where the embryo has not been perturbed and a given cell develops in its niche , some differences appear to develop , which are , at least partially , related to the epigenetic information of each of them -LRB- Torres-Padilla et al. , 2007 -RRB- . If epigenetic asymmetries of the early embryo are related to lineage allocation , it is still uncertain whether they are a cause or a consequence for lineage choice . Also , it remains unknown whether such epigenetic asymmetries would affect only particular regions of the genome . For example , whether genes involved in specification of the ICM such as Sox2 and Nanog would all be targeted by the same epigenetic marks in the same blastomere or whether such marks would vary among genes and/or among blastomeres . Further , are ` inner ' and ` outer ' cells at the 16-cell stage distinguishable in terms of their chromatin landscapes ? The experiments showing that blastomeres are able to respond to the overexpression of a histone modifier and change their fate , indicate that these cells have not yet acquired a ` fix ' destiny , but that they can still be responsive to some kind of signals . These experiments have an important impact on showing that manipulating the epigenetic information can affect cell fate in the preimplantation embryo , in line with the importance of epigenetic mechanisms being crucial for early development . Moreover , these results do illustrate that such cells can still be flexible and accommodate themselves after a perturbing event -LRB- in this case , overexpression of a histone methyltransferase and the downstream effects on the information that is . imparted through specific histone modifications -RRB- . These studies have originated some interest from the part of the medical community , particularly , in the context of preimplantation genetic diagnosis -LRB- PGD -RRB- -LRB- Goldman , 2007 -RRB- . Does it matter to the embryo which cell is used for genetic diagnosis ? Might the death of one of these cells have an effect on subsequent development ? This question is equally valid on the impact of cell loss upon cryopreservation -LRB- Cohen et al. , 2007 -RRB- . Although these are very delicate questions with very likely no easy answer , from the perspective of the mouse embryo , at least four things are to be considered . A tendency for a blastomere of some 4-cell stage embryos to contribute to a given region of the embryo has been documented . Second , the blastomeres in the 4-cell stage show clear differences not only in the levels of histone methylation , but also in their transcriptional activiy when they develop without being perturbed . However -LRB- third -RRB- , the cells undergo a redirection of cell fate when a histone methyltransferase is overexpressed , indicating that they can readapt . Finally , the environment in which cells develop seems to be crucial for completing development and somehow the remaining cells in the embryo could compensate provided they are somehow different from each other . It is also important to note that a 4-cell stage mouse embryo might correspond to a very different developmental stage than a 4-cell embryo in other mammalian species , as illustrated by the differences in the onset of genome activation between them . Indeed , PGD is most often performed at the 8-cell stage and some reports document a better rate of development when 6-to - 9-cell stage embryos are diagnosed , as opposed to 3-to - 4-cell stage embryos -LRB- Wang et al. , 2007 -RRB- . Moreover , the effects of in vitro fertilization procedures and culture on embryonic development are also extremely important , as they have been shown to alter epigenetic information in the mouse -LRB- Li et al. , 2005 -RRB- . As a final consideration , I would like to leave the reader with an open perspective of some ongoing questions in the field . Although there are strong indications that epigenetic mechanisms are involved in cell fate determination , we are still far from establishing a direct link between an epigenetic mark -LRB- s -RRB- and the derivation of a particular cell lineage in the embryo . Much work is still to be done to determine how these mechanisms are set in play upon fertilization and how they are transmitted during subsequent cleavage stages . Also , what other epigenetic marks contribute to the inheritability of cell fate decisions ? How do these marks relate to and influence each other ? Are different lineage-specific genes marked by a different combination -LRB- s -RRB- of epigenetic marks ? Do the marking of these genes occur at different stages of development ? It is also tempting to expand these notions into the stem cell field and question whether these mechanisms would also underlie the intrinsic self renewal ability of adult stem cells and their potential to differentiate into other cell types . The exquisite complexity and richness of chromatin-regulated events in the early embryo will certainly be the subject of exciting research in the future . Funding The author acknowledges support from the PNRRE/INSERM .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cell fate
histone methylation
mouse embryo
pluripotency


Crystal structures of Lymnaea stagnalis AChBP in complex with neonicotinoid insecticides imidacloprid and clothianidin Neonicotinoid insecticides , which act on nicotinic acetylcholine receptors -LRB- nAChRs -RRB- in a variety of ways , have extremely low mammalian toxicity , yet the molecular basis of such actions is poorly understood . To elucidate the molecular basis for nAChR -- neonicotinoid interactions , a surrogate protein , acetylcholine binding protein from Lymnaea stagnalis -LRB- Ls-AChBP -RRB- was crystallized in complex with neonicotinoid insecticides imidacloprid -LRB- IMI -RRB- or clothianidin -LRB- CTD -RRB- . The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185 , while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55 . IMI showed higher binding affinity for Ls-AChBP than that of CTD , consistent with weaker CH -- π interactions in the Ls-AChBP -- CTD complex than in the Ls-AChBP -- IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD . Yet , the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143 , offering an explanation for the diverse actions of neonicotinoids on nAChRs . Introduction Nicotinic acetylcholine receptors -LRB- nAChRs -RRB- are ligand-gated ion channels that mediate fast-acting excitatory cholinergic neurotransmission in vertebrates and invertebrates . Each nAChR molecule consists of five subunits ; acetylcholine -LRB- ACh -RRB- binds at extracellular ligand-binding domain -LRB- LBD -RRB- at the subunit interfaces , and a central , cation-permeable ion channel opens transiently in response to ACh and agonists -LRB- Karlin 2002 ; Sine and Engel 2006 -RRB- . Each subunit possesses an N-terminal extracellular domain with a conserved di-cysteine loop , four transmembrane regions -LRB- TM1 -- TM4 of which TM2 provides most of the channel-lining residues -RRB- , and a large intracellular loop between TM3 and TM4 -LRB- Karlin 2002 -RRB- . Usually , nAChRs are made up of α and non-α subunits -LRB- Fig. 1a -RRB- , the α subunits being defined by adjacent cysteines in loop C of LBD . The ACh binding site is located at the LBD interfaces and formed by loops A , B and C of the α subunit and loops D , E and F which are normally located in non-α subunit -LRB- Corringer et al. 2000 ; Karlin 2002 ; Lindstrom 2003 -RRB- . However , α7 , α8 and α9 subunits can form functional homo-pentamers -LRB- Couturier et al. 1990 ; Elgoyhen et al. 1994 ; Gerzanich et al. 1994 -RRB- -LRB- Fig. 1a -RRB- , whereas α9 and α10 subunits form α hetero pentamers -LRB- Elgoyhen et al. 2001 -RRB- . When only α subunits are present , they donate not only loops A -- C , but also loops D -- F. Fig. 1Nicotinic acetylcholine receptors -LRB- nAChRs -RRB- and the acetylcholine binding protein -LRB- AChBP -RRB- . a Schematic representation of neuronal nicotinic acetylcholine receptors and acetylcholine binding protein . b Chemical structures of natural nicotinic ligands and neonicotinoids -LSB- imidacloprid -LRB- IMI -RRB- and clothianidin -LRB- CTD -RRB- -RSB- used in this study . c Multiple sequence alignment of AChBP with the ligand binding domain of nAChR subunits . Lymnaea stagnalis AChBP -LRB- Ls-AChBP -RRB- and N-terminus LBD sequences of insect -LRB- species names are colored magenta -RRB- and vertebrate -LRB- species names are colored black -RRB- nAChRs are aligned using ClustalW2 program -LRB- Larkin et al. 2007 -RRB- with default parameters and the details are manually adjusted . Secondary structure elements from the Ls-AchBP -- IMI complex are indicated above the multiple alignments . Amino acids involved in the interactions with neonicotinoids are highlighted in bold with yellow and cyan backgrounds in the principal -LRB- + -RRB- - and complementary -LRB- - -RRB- - sides , respectively . Amino acid residue number from methionine for each protein was indicated at the top of each sequence In mammals , nAChRs play central roles in neuromuscular and inter-neuronal cholinergic neurotransmission . Mutations in muscle nAChR subunits account for many cases of congenital myasthenia syndrome -LRB- Engel et al. 2003 -RRB- and also multiple pterygium -LRB- Morgan et al. 2006 -RRB- , whereas mutations in neuronal nAChR subunits -LRB- α4 , β2 -RRB- can produce epilepsies -LRB- Aridon et al. 2006 ; Steinlein 2004 -RRB- . It has also been shown that cholinergic neurotransmission is reduced in Alzheimer 's -LRB- Marutle et al. 1999 -RRB- and Parkinson 's diseases as well as in schizophrenia -LRB- Woodruff-Pak and Gould 2002 -RRB- . Further , in some cases of Alzheimer 's disease , loss of nAChRs has been reported -LRB- Court et al. 2001 -RRB- . Thus there is a growing interest in nAChRs as potential drug targets -LRB- Arneric et al. 2007 ; Dani and Bertrand 2007 -RRB- . In the insect nervous system , where ACh is the primary excitatory neurotransmitter , nAChRs are present at densities comparable to those in the electric organ of the electric fish , Electrophorus electricus -LRB- Sattelle 1980 -RRB- , and several classes of insecticides -LRB- cartap , bensultap , thiocyclam , spinosad and neonicotinoids -RRB- target insect nAChRs -LRB- Narahashi 2000 ; Raymond-Delpech et al. 2005 ; Millar and Denholm 2007 -RRB- . Of these , neonicotinoids -LRB- Fig. 1a -RRB- are increasingly used not only for controlling crop pests worldwide , but also for animal health application such as flea and louse controls . Imidacloprid -LRB- IMI ; Fig. 1b -RRB- , the first insecticide with billion dollars per annum sales , is a partial agonist of native -LRB- Deglise et al. 2002 ; Ihara et al. 2006 ; Tan et al. 2007 -RRB- as well as of recombinant -LRB- Ihara et al. 2003 -RRB- nAChRs expressed in Xenopuslaevis oocytes . On the other hand , clothianidin -LRB- CTD ; Fig. 1b -RRB- and its analog evoke supra-maximal responses -LRB- with reference to ACh responses -RRB- not only in the Drosophila Dα2/chicken β2 hybrid nAChRs -LRB- Ihara et al. 2004 -RRB- expressed in Xenopus oocytes , but also in native Drosophila central cholinergic neurons -LRB- Brown et al. 2006 -RRB- . Single channel recording has shown that a CTD analog induces the opening of the Drosophila nAChR channels at the largest conductance state more frequently than ACh , offering a possible explanation for its super-agonist action -LRB- Brown et al. 2006 -RRB- . Contrasting with these actions , some neonicotinoids -LRB- Ihara et al. 2006 ; Salgado and Saar 2004 -RRB- , and bis-neonicotinoids containing two neonicotinoid units joined together by an alkyl chain -LRB- Ihara et al. 2007a -RRB- , antagonize the acetylcholine-induced responses of native insect neurons . Neonicotinoids show higher affinity for insect nAChRs , accounting , at least in part , for their selective toxicity to insects over vertebrates -LRB- Matsuda et al. 2001 , 2005 ; Tomizawa and Casida 2005 -RRB- . Our studies using site-directed mutagenesis combined with two-electrode voltage-clamp electrophysiology have shown that the X residue in the YXCC motif of loop C -LRB- Shimomura et al. 2004 -RRB- , the region upstream of loop B -LRB- Shimomura et al. 2005 -RRB- and basic residues in loop D -LRB- Shimomura et al. 2002 , 2003 , 2006 -RRB- contribute to the high neonicotinoid sensitivity of insect nAChRs -LRB- see Fig. 1c for loop positions -RRB- . The acetylcholine binding protein -LRB- AChBP -RRB- from snail , Lymnaea stagnalis -LRB- Ls-AChBP -RRB- was discovered in glial cells as a water soluble protein modulating synaptic ACh concentration -LRB- Smit et al. 2001 -RRB- , and the X-ray crystal structure of the protein was described at the same time -LRB- Brejc et al. 2001 -RRB- . Subsequently , X-ray crystal structures of Ls-AChBP homologs from Aplysia californica -LRB- Ac-AChBP -RRB- -LRB- Bourne et al. 2005 -RRB- and Bulinus truncatus -LRB- Bt-AChBP -RRB- -LRB- Celie et al. 2005b -RRB- were reported . Both Ls - and Ac-AChBPs show similarities to the extracellular domain of neuronal α7 nAChRs and form homopentamers in solution -LRB- Smit et al. 2001 -RRB- . Notably , all the six regions -LRB- loops A -- F -RRB- that make up the ligand binding site are conserved in AChBPs . Thus AChBPs are considered as surrogates of the LBD in nAChRs and therefore have been employed to elucidate the mechanism for interactions with low molecular weight ligands as well as peptide toxin antagonists -LRB- Bourne et al. 2005 ; Celie et al. 2004 , 2005 ; Dutertre et al. 2007 ; Hansen et al. 2005 ; Hansen and Taylor 2007 ; Ulens et al. 2006 -RRB- . Employing the crystal structures of AChBPs , we have previously modeled LBDs in complex with IMI to show that basic residues in loop D may interact electrostatically with the nitro group of neonicotinoids -LRB- Shimomura et al. 2006 -RRB- . Yet , details of neonicotinoid -- nAChR interactions are poorly understood . Therefore , we have crystallized the Ls-AChBP in complex with two commercial neonicotinoids , IMI and CTD . Materials and methods Materials Lymnaea stagnalis derived from the stocks of Vrije Universeit -LRB- Amsterdam -RRB- was provided from Professor Sakakibara at Tokai University . IMI and CTD were gifts from BayerCropscience Co. in Japan . 1 - -LRB- 6-Bromopyridylmethyl -RRB- -2-nitroiminoimidazolidine -LRB- bromine-substituted imidacloprid ; Br-IMI -RRB- and N - -LRB- 2-bromo-thiazol-5-ylmethyl -RRB- - N ′ - methyl-N ′ ′ - nitroguanidine -LRB- bromine-substituted clothianidin ; Br-CTD -RRB- were synthesized according to the published methods -LRB- Maienfisch et al. 2000 ; Nishiwaki et al. 2000 -RRB- . Protein preparation The cDNA of Ls-AChBP was amplified by RT-PCR from L. stagnalis and was cloned into the pPICZα B vector -LRB- Invitrogen -RRB- at the Pst I site , with an L1A mutation introduced to facilitate cloning . The Ls-AChBPs were expressed in yeast , Pichia pastris X-33 using EasySelect Pichia Expression Kit -LRB- Invitrogen -RRB- . Secreted proteins were purified with Q-Sepharose column -LRB- GE Healthcare -RRB- immediately following concentration and buffer-exchange using Vivaflow200 -LRB- Zartorius -RRB- , and then treated with 50 U/mg protein of peptide-N-glycosidase F -LRB- Wako Pure Chemical Industries -RRB- at 37 °C for 24 h to remove the glycosyl-chain at Asn67 . The protein samples were subsequently purified with Mono Q and Superdex 200 columns -LRB- GE Healthcare -RRB- . Purified proteins were confirmed to be the samples of interest by N-terminal sequencing and mass spectrometry . Purified proteins were buffer-exchanged against 20 mM Tris -- HCl buffer with 0.02 % NaN3 -LRB- pH 8.0 -RRB- . Evaluation of equilibrium binding of neonicotinoids The equilibrium binding of neonicotinoids with Ls-AChBP was determined by quenching of intrinsic tryptophan fluorescence . Ls-AChBP at 600 nM in binding site concentration in 20 mM Tris -- HCl buffer with 0.02 % NaN3 -LRB- pH 8.0 -RRB- was incubated with each ligand at various concentrations on a 96-well plate . Ls-AChBP was excited at 280 nm , and emission intensity was recorded at 342 nm using Varioskan microplate reader -LRB- Thermo Fisher Scientific -RRB- at room temperature . Data were fitted by non-linear regression according to the following equation using Prism Software version 4.03 -LRB- GraphPad Software -RRB- : where ΔF and ΔFmax represent quenching of fluorescence at a ligand concentration X and maximum quenching of fluorescence at saturation , respectively . F0 and -LSB- Protein -RSB- are fluorescence measured in the absence of ligands and the concentration of Ls-AChBP , respectively . Crystallization and X-ray data collection Purified Ls-AChBPs -LRB- 6.0 mg/mL -RRB- were incubated with 0.5 mM neonicotinoids at 4 °C for 1 h prior to crystallization . Ls-AChBP-neonicotinoid complex crystals were obtained by vapor diffusion method at 20 °C with 1:1 ratio of protein to reservoir solution containing 0.2 M Na citrate pH 5.7 , 15 -- 22 % PEG3350 and about 0.5 mM of either IMI , CTD , Br-IMI or Br-CTD . The crystals were flash-cooled in liquid nitrogen after soaking into the cryoprotectant solutions containing 0.2 M Na citrate pH 5.7 , 25 % PEG3350 , 20 % glycerol and about 0.5 mM of each neonicotinoid . X-ray diffraction data sets were collected at 90 K using either Bruker AXS DIP6040 detector at BL44XU -LRB- Yoshimura et al. 2007 -RRB- or ADSC QUANTUM 210 detector at BL44B2 -LRB- Adachi et al. 2001 -RRB- beamlines in SPring-8 , and processed with Mosflm -LRB- Leslie 1992 -RRB- or HKL2000 -LRB- Otwinowski and Minor 1997 -RRB- . In order to identify the positions of the sulfur atom at the thiazole ring of CTD as well as the bromine atom of Br-IMI and Br-CTD , anomalous data from the crystals complexed with these neonicotinoids were collected at wavelength of 1.75 Å and 0.919 Å -LRB- Table 1 -RRB- , respectively . Table 1X-ray diffraction data collection and refinement statistics for Ls-AChBP -- neonicotinoid complexesIMICTDBr-IMIBr-CTDCTD -LRB- S ano -RRB- aData collection BeamlinebBL44XUBL44B2BL44B2BL44B2BL44B2 Wavelength -LRB- Å -RRB- 0.9000.9190.9190.9191.75 Space groupP65P65P65P65P65 Cell dimensions a , c -LRB- Å -RRB- 75.0 , 351.074.6 , 351.074.8 , 350.974.5 , 351.274.6 , 351.3 Resolution -LRB- Å -RRB- 15.8 -- 2.5650.0 -- 2.7050.0 -- 2.7050.0 -- 2.9050.0 -- 2.90 Rsym -LRB- % -RRB- c , d8 .0 -LRB- 41.3 -RRB- 8.5 -LRB- 38.9 -RRB- 9.7 -LRB- 34.1 -RRB- 11.8 -LRB- 34.1 -RRB- 8.7 -LRB- 39.5 -RRB- I/σc8 .1 -LRB- 1.7 -RRB- 20.7 -LRB- 4.0 -RRB- 47.0 -LRB- 10.7 -RRB- 45.5 -LRB- 10.9 -RRB- 60.8 -LRB- 9.32 -RRB- Completeness -LRB- % -RRB- c94 .8 -LRB- 96.7 -RRB- 100 -LRB- 100 -RRB- 100 -LRB- 100 -RRB- 100 -LRB- 100 -RRB- 99.5 -LRB- 96.2 -RRB- Redundancyc2 .6 -LRB- 2.6 -RRB- 5.7 -LRB- 5.7 -RRB- 23.0 -LRB- 23.3 -RRB- 22.5 -LRB- 23.2 -RRB- 21.6 -LRB- 20.5 -RRB- Refinement Resolution -LRB- Å -RRB- 15.8 -- 2.5850.0 -- 2.70 No . of reflections3303130121 R/Rfree -LRB- % -RRB- e20 .3 / 27.720.2 / 27.0 Bond length -LRB- Å -RRB- / angles -LRB- deg -RRB- f0 .008 / 1.30.008 / 1.3 Average B factor41 .841.6 PDB accession codeg2ZJU2ZJVSee text for abbreviations of the neonicotinoid namesaData set for detection of anomalous peaks at sulfur atomsbAll data sets were collected at SPring-8cValues in parentheses refer to data in the highest resolution shellsdRsym = Σ | I - < I > | / ΣI , where I is the observed intensity and <I> is the average intensity from multiple observations of the symmetry-related reflectionseR = Σ | | Fo | -- | Fc | | / Σ | Fo | . Rfree is an R factor of the CNS refinement evaluated for 5 % of reflections that were excluded from the refinementfRMS deviations from ideal values were calculatedgThe atomic coordinates and structure factors have been deposited in the Protein Data Bank Structure determination and refinement The structure of the IMI complex was solved by molecular replacement with Phaser -LRB- McCoy 2007 -RRB- using the coordinates of an Ls-AChBP in complex with nicotine -LRB- PDB entry : 1UW6 -RRB- -LRB- Celie et al. 2004 -RRB- as a search model . Subsequently , the refined coordinates of the IMI complex were used to solve the structure of the CTD complex . Refinements and manual model building were performed with CNS version 1.2 -LRB- Brünger et al. 1998 -RRB- and Coot -LRB- Emsley and Cowtan 2004 -RRB- , respectively . The non-crystallographic-symmetry restraints between the subunits were initially applied for the refinements and removed at later steps . The details of data collection and refinement statistics are provided in Table 1 . Figures are generated by PyMOL -LRB- DeLano Scientific LLC -RRB- , Bobscript -LRB- Esnouf 1999 -RRB- and Raster3D -LRB- Merritt and Bacon 1997 -RRB- . Results and discussion Structure analysis In this study , we have crystallized the Ls-AChBP in complex with IMI and CTD to elucidate the mechanism underpinning their selective and diverse actions on nAChRs . Both complexes were crystallized in the same space group with one pentamer in an asymmetric unit . Ligand-omit and anomalous electron density maps clearly indicated AChBP in the crystals bound neonicotinoids -LRB- see Fig. 3a , b -RRB- . The positions and orientations of the 2-chloropyridine ring in IMI and 2-chloro-1,3-thiazole ring in CTD were modeled to satisfy the observation of Br - and S-anomalous peak positions . The X-ray crystal structures of the Ls-AChBP complexed with IMI and CTD were refined at 2.58 and 2.70 Å resolutions , respectively -LRB- Table 1 -RRB- . The neonicotinoid-bound Ls-AChBPs form the same homopentameric structure -LRB- Fig. 2a , b -RRB- as those determined for the 4 - -LRB- 2-hydroxyethyl -RRB- -1-piperazineethanesulfonic acid -LRB- HEPES -RRB- - bound apo form -LRB- Brejc et al. 2001 -RRB- and the other ligand-bound forms -LRB- Celie et al. 2004 , 2005 ; Hansen et al. 2005 ; Ulens et al. 2006 -RRB- . Typically , each subunit has a secondary structure composed of an N-terminal 12-residue α-helix followed by β-strand-rich parts as shown previously for other Ls-AChBPs . Root mean square differences -LRB- rmsd -RRB- of Cα atoms between the IMI - or CTD-complex and the nicotine-complex -LRB- Celie et al. 2004 -RRB- were observed as 0.975 and 0.758 Å , respectively . In two structures determined in the present study , neonicotinoids occupied all five ligand-binding sites that are formed between the two adjacent subunits -LRB- Fig. 2a , b -RRB- . The neonicotinoids occupy broadly the same position as HEPES and -LRB- - -RRB- - nicotine within the pocket formed by `` A-F loop '' regions . Although `` loops A , B , D and E '' are mainly composed of β-strands and only loops C and F have no canonical secondary structure , we use these designations to facilitate comparison with previous studies on nAChRs -LRB- Corringer et al. 2000 ; Matsuda et al. 2001 , 2005 -RRB- . The principal side -LSB- -LRB- + -RRB- - chain -RSB- of the ligand-binding site possesses residues from loops A , B and C which are located in α subunits , whereas the complementary side -LSB- -LRB- - -RRB- - chain -RSB- contains loops D , E and F , which are donated by non-α subunits in α/non-α heteropentamers . Overall , the structures of the IMI and CTD complexes were very similar with Cα rmsd of 0.597 Å . However , the loop C region has a specific conformation in each complex . For example , compared with the IMI complex , loop C of the CTD complex takes a `` closed conformation '' , showing approximately 4 Å shift of the positions of the Cα atoms -LRB- Fig. 2c -RRB- . The variations in loop C conformation were often observed between different chains within the same Ls-AChBP pentamer in complex with neonicotinoids -LRB- e.g. subunits A and C of the CTD complex , data not shown -RRB- . Conformational change in loop C is evident when comparing structures with the apo form Ls-AChBP complexed with HEPES -LRB- PDB entry code : 1UX2 -RRB- -LRB- Fig. 2c -RRB- . The Thr155-Asp160 loop region of the -LRB- - -RRB- - chain upstream of loop F also indicates a conformational change in the ligand-binding site . These results suggest that induced-fit movement of loop regions is essential for recognition of ligands including neonicotinoids . The detailed binding interactions of neonicotinoids will be considered in relation to their key components , the aromatic ring and the guanidine/related moieties . Fig. 2Overall view of crystal structure of Ls-AChBP complexed with imidacloprid -LRB- IMI -RRB- . The pentameric structure of Ls-AChBP is viewed from the top -LRB- a -RRB- corresponding to an extracellular surface of nAChRs and from the side -LRB- b -RRB- . IMI molecules bound to Ls-AChBP are shown in space-filling model , whereas carbons , nitrogens and oxygens are colored green , blue and red , respectively . Two subunits , A and B chains , are colored by pink and blue , respectively . c The structure of A -- B domain boundary of the Ls-AChBP complexed with IMI -LRB- red -RRB- is compared with that of the Ls-AChBP complexed with CTD -LRB- blue -RRB- and the apo form -LRB- light brown -RRB- complexed with 4 - -LRB- 2-hydroxyethyl -RRB- -1-piperazineethanesulfonic acid -LRB- HEPES -RRB- -LRB- PDB entry code : 1UX2 -RRB- -LRB- Celie et al. 2004 -RRB- . Ligands are presented by stick models Interactions with aromatic rings of insecticides When complexed with Ls-AChBP , the pyridine ring of IMI was buried deep in the binding site , interacting with loop E segments from the -LRB- - -RRB- - chain -LRB- Fig. 3c -RRB- . Recently , Tomizawa et al. -LRB- 2008 -RRB- has reported that two azido-neonicotinoid probes containing the photolabile group in the pyridine ring labeled Tyr164 in loop F of the -LRB- - -RRB- - chain and Tyr192 in loop C of the -LRB- + -RRB- - chain in the Ls-AChBP . However , these two distinct binding modes were not observed in our models , neither in the IMI complex , nor in the CTD complex of the Ls-AChBP . The nitrogen atom of the pyridine ring forms a hydrogen bond with the amide group of Met114 and the carbonyl group of Leu102 in loop E via a water molecule , resembling the observations for both the Ls-AChBP-nicotine complex -LRB- Celie et al. 2004 -RRB- and Ac-AChBP-epibatidine complex -LRB- Hansen et al. 2005 -RRB- . Fig. 3Electron density maps of bound ligands and their interactions with the loop E region . The annealed Fo -- Fc omit maps -LRB- blue -RRB- for imidacloprid -LRB- IMI -RRB- -LRB- a -RRB- and clothianidin -LRB- CTD -RRB- -LRB- b -RRB- bound to the Ls-AChBP are contoured at 4σ and drawn with the final models of the native and non-Br-neonicotinoids . In addition , the anomalous maps of bromine -LRB- purple -RRB- from the Br-neonicotinoid-complexes and sulfur atoms -LRB- yellow -RRB- from the native-neonicotinoid-complexes for ligands , which are contoured at 5 and 1.5 σ , respectively , are overlaid . Interactions of the loop E region with pyridine moiety of IMI -LRB- c -RRB- and thiazole moiety of CTD -LRB- d -RRB- are shown in the binding site of the Ls-AChBP . In each ligand and amino acid , chlorine , nitrogen , oxygen and sulfur atoms are colored purple , blue , red and yellow , respectively . The hydrogen bonds are presented by orange broken lines The chlorine atom was located in the vicinity of a hydrogen bond bridge between the backbone C = O of Leu112 and the backbone amide NH of Arg104 . As a result , it made van der Waals contact the peptide backbones of these two amino acids . The aromatic ring of CTD also contacts with the loop E segment in a manner similar to IMI -LRB- Fig. 3d -RRB- . The nitrogen atom of the thiazole ring of CTD , in a similar fashion to the nitrogen atom of the pyridine ring of IMI , formed hydrogen bond with the backbone carbonyl of Leu102 and the backbone amide group of Met114 via water molecule -LRB- Fig. 3d -RRB- . Nicotine , which is active not only on insect , but also on vertebrate nAChRs , has also been shown to interact with the backbone of loop E in a similar manner to neonicotinoids -LRB- Celie et al. 2004 -RRB- . Therefore interactions with the backbone are insufficient to explain the selective actions of neonicotinoids . On the other hand , Met114 in loop E was located in the vicinity of the nitroimine group -LRB- Fig. 3c , d -RRB- which plays a key role in selective insect nAChR -- neonicotinoid interactions -LRB- Ihara et al. 2003 -RRB- . Some insect α subunits possess either basic or hydrogen bondable residues favorable for interactions with the nitro group such as those corresponding to Met114 -LRB- Fig. 1c -RRB- . Hence , if two adjacent α subunits form a ligand-binding site , such residues may contribute to the neonicotinoid sensitivity of insect nAChRs . Interactions with guanidine and related moieties The crystal structures of the Ls-AChBP bound with nicotine or carbamylcholine -LRB- Celie et al. 2004 -RRB- and Ac-AChBP in the presence of epibatidine -LRB- Hansen et al. 2005 -RRB- , as well as quantum chemical calculations -LRB- Cashin et al. 2005 -RRB- , suggested that the protonated nitrogen atom in nicotinic agonists undergoes cation -- π interactions with Trp143 in loop B . In line with this notion , it was earlier demonstrated that the electron-deficient guanidine moiety of neonicotinoids is likely to contact with Trp143 -LRB- Matsuda et al. 2005 ; Tomizawa and Casida 2005 -RRB- . However , the guanidine moiety of IMI and CTD was found to stack with the aromatic ring of Tyr185 in the crystal structures of CTD - as well as IMI-Ls-AChBP complexes -LRB- Fig. 4a , d -RRB- . Since Tyr185 is conserved throughout vertebrate and invertebrate nAChR α subunits -LRB- Fig. 1c -RRB- , this residue itself is not involved directly in determining the selective interactions with neonicotinoids . Yet the interactions with Tyr185 contribute to the affinity for neonicotinoids . The stacking level for CTD appears to be weaker than that for IMI -LRB- Fig. 4a , b -RRB- . In addition , the stacking interaction resulted in CH -- π contacts with Trp143 of the methylene -LRB- CH2 -- CH2 -RRB- bridge in IMI , which was not seen in CTD , accounting , at least in part , for the higher binding potency of IMI -LRB- Kd = 1.57 ± 0.21 μM , n = 3 -RRB- compared to that of CTD -LRB- Kd = 7.26 ± 1.13 μM , n = 3 -RRB- on the Ls-AChBP as evaluated by quenching of the protein fluorescence . Fig. 4Imidacloprid -LRB- IMI -RRB- and clothianidin -LRB- CTD -RRB- binding to Lymnaeastagnalis AChBP -LRB- Ls-AChBP -RRB- . a IMI -- Ls-AChBP complex . b CTD -- Ls-AChBP complex . The binding site is located at the interface between two adjacent subunits , as drawn in green for -LRB- + -RRB- - chains and in cyan for -LRB- - -RRB- - chain -LRB- see text -RRB- . The subunits EA and CD interfaces are shown for the binding sites for IMI -LRB- a -RRB- and CTD -LRB- b -RRB- , respectively . Orange broken lines depict hydrogen bonds . In each ligand and amino acid , chlorine , nitrogen , oxygen and sulfur atoms are colored purple , blue , red and yellow , respectively Another difference between CTD and IMI is that the former exposes a hydrogen bond donor NH at position 1 , whereas the latter lacks it . We suggested earlier that this NH may form a hydrogen bond with the backbone carbonyl of Trp143 in loop B -LRB- Ihara et al. 2007b -RRB- . As suggested , the NH of CTD made a hydrogen bond with the backbone carbonyl of Trp143 from the -LRB- + -RRB- - chain in the crystal structures of the Ls-AChBP -LRB- Fig. 4b -RRB- , although it was observed at only 2 of 5 interfaces . Such hydrogen bond formation may assist CTD in keeping contact with nAChRs when large conformational changes occur in LBD to open the central channel -LRB- Miyazawa et al. 2003 ; Unwin 2005 -RRB- , accounting , at least in part , for higher agonist efficacy of CTD compared to IMI and ACh in certain native and recombinant nAChRs -LRB- Brown et al. 2006 ; Ihara et al. 2004 -RRB- . Interactions with loop D and selectivity of neonicotinoids We have previously shown that substitution of a glutamate for the glutamine corresponding to Gln55 of Ls-AChBP -LRB- Fig. 1c -RRB- markedly reduced the maximum response to IMI and nitenpyram of the chicken α7 nAChR , whereas mutation of the same glutamine to arginine enhanced it , slightly shifting the concentration -- response curve to the left -LRB- lower concentrations -RRB- . In complete contrast , the same mutation reduced the -LRB- - -RRB- - nicotine sensitivity of the α7 , whereas substitution with a glutamate enhanced it -LRB- Shimomura et al. 2002 -RRB- . Therefore , it was postulated that this glutamine residue is likely to be located in close proximity to the nitro group of neonicotinoids , and that marked changes in the α7 response to neonicotinoids as well as -LRB- - -RRB- - nicotine are the results of electrostatic interactions between the added residues and the nitro group . Consistent with this hypothesis , Gln55 faced the nitro groups of IMI and CTD in the crystal structure of the Ls-AChBP within a hydrogen bondable distance in the AChBP -- IMI complex -LRB- < 3.3 Å in 2 of 5 interfaces in the crystal structure of the Ls-AChBP -RRB- -LRB- Fig. 4 -RRB- . For CTD , however , the distance between the Gln55 and the nitro group was longer than 3.5 Å , excluding or minimizing a contribution of hydrogen bonding to the binding potency of CTD . All vertebrate nAChRs , perhaps apart from those containing either human or rat β4 subunit , are likely to show reduced sensitivity to neonicotinoids due to the lack of the basic residues in loop D . The human β4 subunit -LRB- Fig. 1c -RRB- , as well as rat β4 subunit -LRB- sequence not shown -RRB- , possesses a lysine as residue corresponding to Gln55 of Ls-AChBP . However , the vertebrate β4 subunits possess a glutamate residue as that corresponding to Thr57 in Ls-AChBP which is located in the vicinity of Gln55 -LRB- Fig. 5 -RRB- . This glutamate residue is postulated to interfere electrostatically with the neighboring basic residue -- neonicotinoid interactions , thereby reducing the neonicotinoid sensitivity of nAChRs containing the β4 subunits . Fig. 5Interactions between loops C , D and F. IMI-binding site of Ls-AChBP is presented by especially focusing on loops C , D and F . The binding site at the interface of the subunits C and D is drawn in green for -LRB- + -RRB- - chains and in cyan for -LRB- - -RRB- - chain -LRB- see text -RRB- . In each ligand and amino acid , chlorine , nitrogen , oxygen and sulfur atoms are colored purple , blue , red and yellow , respectively . Orange broken lines depict hydrogen bonds Role of loops C and F in their interactions with neonicotinoids We have found that replacement by glutamate of the proline corresponding to Ser186 in the YXCC motif in loop C of the Drosophila Dα2 subunit reduces the IMI sensitivity of the Dα2β2 hybrid nAChR , whereas its reverse mutation in the α4β2 nAChR enhanced sensitivity -LRB- Shimomura et al. 2004 -RRB- , suggesting a contribution of the loop C residue to the neonicotinoid sensitivity of nAChRs . However , another view has been proposed in which the serine or threonine residue corresponding to Ser186 from -LRB- + -RRB- - chain of Ls-AChBP in loop C of insect nAChRs -LRB- see Fig. 1c -RRB- forms a hydrogen bond with the nitro group of neonicotinoids , thereby enhancing the binding potency of neonicotinoids -LRB- Tomizawa et al. 2007 -RRB- . In the crystal structures of Ls-AChBP , Ser186 made hydrogen bonds with Glu163 and Tyr164 in loop F of -LRB- - -RRB- - chain but not with the nitro group of IMI -LRB- Fig. 5 -RRB- . This hydrogen bond web , as well as the basic residues in loop D , seems to play an important role in determining the neonicotinoid sensitivity of nAChRs . In most heteromeric vertebrate nAChRs , α subunits possess acidic residues as those corresponding to Ser186 in loop C , resulting in electrostatic repulsion of the acidic residues in loop F of β subunits corresponding to Glu163 in Ls-AChBP -LRB- Fig. 1c -RRB- . On the other hand , insect nAChR α subunits do not possess such an acidic residue in loop C -LRB- Fig. 1c -RRB- . Hence , it is conceivable that amino acids not only from α subunits , but also from non-α subunits of insect nAChRs , contact more effectively with neonicotinoids than those of vertebrate nAChRs . In this context , Tyr164 may also contribute indirectly to the neonicotinoid sensitivity of insect nAChRs because vertebrate non-α subunits possess phenylalanine residue incapable of assisting the formation of the hydrogen-bond-network at this position . In conclusion , we have for the first time elucidated the crystal structures of the Ls-AChBP in complex with two commercial neonicotinoids IMI and CTD . Met114 in loop E and Gln55 in Loop D closest to the nitro group in neonicotinoids , suggesting that corresponding residues in insect nAChRs may play important roles in determining neonicotinoid sensitivity . Ser186 in loop C was found to form hydrogen bonds with Gln163 as well as Tyr164 in loop F , suggesting that these hydrogen bondable residues can also indirectly influence the selective interactions with neonicotinoids . CTD forms hydrogen bond with the backbone carbonyl of Trp143 in loop B and shows weaker hydrophobic contacts with Trp143 compared with other neonicotinoids tested , which may altogether lead to its unique actions on nAChRs . Although there are sequence and structural similarities between AChBPs and nAChRs , there are also important differences . Future structural studies on AChBP mutants engineered to resemble more closely insect or human nAChRs could help to enhance even further our understanding of neonicotinoid actions and selectivity . Nevertheless , we have provided new insights into the molecular basis of differential actions of neonicotinoid molecules . These results may be of value in the design of even safer new crop protection agents .

B I O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O 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B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

crystal structures
neonicotinoids
nicotinic acetylcholine receptors
ion channels

acetylcholine binding protein (lymnaea stagnalis)