brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.     

Single-molecule Study on the Temperature-sensitive Reaction of F1-ATPase with a Hybrid F1 Carrying a Single ��(E190D)

F₁-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α₃β₃ cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F₁-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F₁(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F₁, carrying a single copy of βE190D, was observed at 18 °C. The hybrid F₁ showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding.F₁-ATPase (F₁)² is an ATP-driven rotary motor protein. The subunit composition of the bacterial F₁-ATPase is α₃β₃γδϵ, and the minimum complex of F₁-ATPase as a rotary motor is α₃β₃γ subcomplex. This motor protein forms the F_oF₁-ATP synthase complex by binding to another rotary motor, namely, F_o, which is driven by the proton flux resulting from the proton motive force across the membranes (1–4). Under physiological conditions, where the proton motive force is sufficiently large, F_o forcibly rotates F₁-ATPase in the reverse direction of F₁-ATPase, leading the reverse reaction of ATP hydrolysis, i.e. ATP synthesis from ADP and inorganic phosphate (P_i). When the proton motive force diminishes or F₁ is isolated from F_o, F₁-ATPase hydrolyzes ATP to rotate the γ-subunit against the α₃β₃ stator ring in the counterclockwise direction as viewed from the F_o side (5). The catalytic sites are located at the interface of the α- and β-subunits, predominantly on the β-subunit (6). Each β-subunit carries out a single turnover of ATP hydrolysis during the γ-rotation of 360° following the common catalytic reaction pathway, whereas they are 120° different in the catalytic phase. In this manner, the three β-subunits undergo different reaction steps of ATP hydrolysis upon each rotational step. The rotary motion of the γ-subunit has been demonstrated by biochemical (7) and spectroscopic methods (8) and directly proved in single-molecule observation studies (5).Since the establishment of the single-molecule rotation assay, the chemomechanical coupling scheme of F₁ has been studied extensively by resolving the rotation into discrete steps. The stepping rotation was first observed under an ATP-limiting condition where F₁ makes discrete 120° steps upon ATP binding (9). Then, high speed imaging of the rotation with a small probe of low friction was performed, which revealed that the 120° step comprises 80 and 40° substeps, each initiated by ATP binding, and two unknown consecutive reactions, respectively (10). This finding necessitated the identification of the two reactions that trigger the 40° substep. Hence, the rotation assay was performed using a mutant, namely F₁(βE190D), and a slowly hydrolyzed ATP analog, namely ATPγS (11). Glutamate 190 of the β-subunit of F₁, derived from thermophilic Bacillus PS3 and the corresponding glutamates from other F₁-ATPases (Glu-181 of F₁ from Escherichia coli and Glu-188 of F₁ from bovine mitochondria), has been identified as one of the most critical catalytic residues for ATP hydrolysis (6, 12–15). When this glutamate was substituted with aspartic acid, which has a shorter side chain than that of glutamate, the ATP cleavage step of F₁ was drastically slowed. In the rotation assay, this mutant showed a distinct long pause before the 40° substep. ATPγS also caused a long pause before the 40° substep. These observations established that the 40° substep is initiated by hydrolysis. Accordingly, the pause angles before the 80 and 40° substeps are referred to as to the binding angle and the catalytic angle, respectively. Then, the rotation assay was performed in the presence of a high amount of P_i in the solution. It was shown that P_i rebinding caused the long pause at the catalytic angle, suggesting that P_i is released before the 40° substep (16).However, the reaction scheme of F₁ cannot be established by simply assigning each reaction step to either the binding angle or the catalytic angle, because each reaction step must be assigned to one of the three binding or catalytic angles when considering the 360° cyclic reaction scheme of each β-subunit. Direct information about the timing of ADP release was obtained by simultaneous imaging of fluorescently labeled nucleotides and γ rotation, which showed that each β retains ADP until the γ rotates 240° after binding of the nucleotide as ATP and releases ADP between 240 and 320° (16, 17). Another powerful approach is the use of a hybrid F₁ carrying a mutant β that causes a characteristic pause during the rotation. In a previous study, the hybrid F₁ carrying a single copy of β(E190D), α₃β₂β(E190D)γ, showed a distinct pause caused by the slow hydrolysis of β(E190D) at +200° from the ATP binding angle of the mutant β (18). From this observation, it was confirmed that each β executes the chemical cleavage of the bound ATP at +200° from the angle where the ATP binds to β. The asymmetric feature of the pause of the hybrid F₁ was also utilized in other experiments as a marker in the rotational trajectory to correlate the rotational angle and the conformational state of β (19) or to determine the state of F₁ in the crystal structures as the pausing state at catalytic angle (20).Recently, we have found a new reaction intermediate of F₁ rotation as a clear intervening pause before the 80° substep in the rotation assay below 9 °C (21). Furuike et al. (22) also observed the TS reaction in a high speed imaging experiment. The rate constant of this reaction was remarkably sensitive to temperature, giving a Q₁₀ factor around 19. When ADP was added to solution, the pause before the 80° substep was prolonged, whereas the solution P_i caused a longer pause before the 40° substep (21). Although this result can be explained by assuming that the temperature-sensitive (TS) reaction is ADP release, it was not decisive for the identification of the TS reaction.In this study, we found that the mutant F₁(βE190D) also exhibits the distinct pause of the TS reaction but at a higher temperature than for the wild-type F₁, i.e. at 18 °C. This feature was advantageous in identifying the angle position of the TS reaction in the catalytic cycle for each β-subunit coupled with the 360° rotation. Taking advantage of the feature of the hybrid F₁, we analyzed the rotational behavior of the hybrid F₁ at 18 °C in order to assign the angle position of the TS reaction in the catalytic cycle of the 360° rotation, and we have shown that the TS reaction is not directly involved in the ADP release but in some conformational rearrangement before or after ATP binding step.     

Cross-linking of two beta subunits in the closed conformation in F1-ATPase

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.     

Rotation of Escherichia coli F(1)-ATPase.

By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase. The torque value and the direction of the rotation were the same as those observed for TF(1). F(1)-ATPases seem to share common properties of rotation irrespective of the sources.     

Hypotonic buffer induces meiosis and formation of anucleate cytoplasmic islands in the egg of the two-spotted cricket Gryllus bimaculatus

In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus.     

piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus

Transgenic insects have been artificially produced to study functions of interesting developmental genes, using insect transposons such as piggyBac. In the case of the cricket, however, transgenic animals have not yet been successfully artificially produced. In the present study, we examined whether the piggyBac transposon functions as a tool for gene delivery in embryos of Gryllus bimaculatus. We used either a piggyBac helper plasmid or a helper RNA synthesized in vitro as a transposase source. An excision assay revealed that the helper RNA was more effective in early Gryllus eggs to transpose a marker gene of eGFP than the helper plasmid containing the piggyBac transposase gene driven by the G. bimaculatus actin3/4 promoter. Further, only when the helper RNA was used, somatic transformation of the embryo with the eGFP gene was observed. These results suggest that the piggyBac system with the helper RNA may be effective for making transgenic crickets.     

Fibroblast growth factor 10 is required for proper development of the mouse whiskers

Fibroblast Growth Factor (FGF) signaling is known to play an important role during cutaneous development. To elucidate the role of FGF10 during whisker formation, we examined the expression of Fgf10 in normal developing whiskers and phenotypes of Fgf10-deficient whiskers. Fgf10 is first expressed in the maxillary process, lateral and medial nasal processes, then in the mesenchymal cells underneath the future whisker placodes, and in the surrounding mesenchyme of developing whiskers. Fgf10-null whiskers exhibit a significant decrease in number and their structure is disorganized as revealed by scanning electron microscopy. Hair follicle marker genes such as Sonic hedgehog, Patched, and Patched 2 are aberrantly expressed in the mutant whiskers. Thus, FGF10 is required for proper whisker development mediated by SHH signaling in the mouse.     

Origin of apparent negative cooperativity of F(1)-ATPase

In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.     

F1-ATPase changes its conformations upon phosphate release

Motor proteins, myosin, and kinesin have gamma-phosphate sensors in the switch II loop that play key roles in conformational changes that support motility. Here we report that a rotary motor, F1-ATPase, also changes its conformations upon phosphate release. The tryptophan mutation was introduced into Arg-333 in the beta subunit of F1-ATPase from thermophilic Bacillus PS3 as a probe of conformational changes. This residue interacts with the switch II loop (residues 308-315) of the beta subunit in a nucleotide-bound conformation. The addition of ATP to the mutant F1 subcomplex alpha3beta(R333W)3gamma caused transient increase and subsequent decay of the Trp fluorescence. The increase was caused by conformational changes on ATP binding. The rate of decay agreed well with that of phosphate release monitored by phosphate-binding protein assays. This is the first evidence that the beta subunit changes its conformation upon phosphate release, which may share a common mechanism of exerting motility with other motor proteins.