The metaphase spindle is organized for accurate chromosome segregation. One of the fundamental features of the spindle across the species is its symmetrical shape; the spindle consists of two polar arrays of microtubules at both ends. Although it has been suggested that the formation of the bipolar shape requires force balance coordination by molecular motors, i.e., kinesins and dyneins, quantitative analysis for the pole mechanics has not been conducted. Here, we demonstrate that it is not only the shape but also the stiffness and microtubule density of the pairs of pole regions are symmetrically balanced in single spindles self-assembled in Xenopus egg extracts. We found that the inhibition of dynein functions dramatically reduced the stiffness and microtubule density in the pole region. By contrast, the inhibition of one of the kinesins, Eg5, which is the antagonistic motor protein of dynein, increased the value of these parameters. Moreover, the inhibition of both dynein and Eg5 recovered these parameter values to those of non-treated spindle poles. We also found that, when one pole structure was held widened with the use of two glass microneedles, the opposite pole structure spontaneously widened, resulting in the formation of the barrel-like shaped spindle. The values of stiffness and microtubule density in the manipulated pole region decreased, following the spontaneous decrement of those in the paired unmanipulated pole region. These results suggest that the spindle possesses a mechanism to dynamically maintain its symmetry in mechanical properties.
Channelrhodopsin (ChR)-1 and ChR2 were the first-identified members of ChRs which are a growing subfamily of microbial-type rhodopsins. Light absorption drives the generation of a photocurrent in cell membranes expressing ChR2. However, the photocurrent amplitude attenuates and becomes steady-state during prolonged irradiation. This process, called desensitization or inactivation, has been attributed to the accumulation of intermediates less conductive to cations. Here we provided evidence that the dark-adapted (DA) photocurrent before desensitization is kinetically different from the light-adapted (LA) one after desensitization, that is, the deceleration of both basal-to-conductive and conductive-to-basal transitions. When the kinetics were compared between the DA and LA photocurrents for the ChR1/2 chimeras, the transmembrane helices, TM1 and TM2, were the determinants of both basal-to-conductive and conductive-to-basal transitions, whereas TM4 may contribute to the basal-to-conductive transitions and TM5 may contribute to the conductive-to-basal transitions, respectively. The fact that the desensitization-dependent decrease of the basal-to-conductive and conductive-to-basal transitions was facilitated by the TM1 exchange from ChR2 to ChR1 and reversed by the further TM2 exchange suggests that the conformation change for the channel gating is predominantly regulated by the interaction between TM1 and TM2. Although the exchange of TM1 from ChR2 to ChR1 showed no obvious influence on the spectral sensitivity, this exchange significantly induced the desensitization-dependent blue shift. Therefore, the TM1 and 2 are the main structures involved in two features of the desensitization, the stabilization of protein conformation and the charge distribution around the retinal-Schiff base (RSB+).
In vitro display technologies such as mRNA and cDNA display are powerful tools to create and select functional peptides. However, in some cases, efficiency of mRNA-protein fusion is very low, which results in decreased library size and lower chance of successful selection. In this study, to improve mRNA-protein fusion efficiency, we prepared an mRNA display library of a protein with random N- and C-terminal coding regions consisting of 12 nucleotides (i.e. four amino acids), and performed an electrophoresis mobility shift assay (EMSA)-based selection of successfully formed mRNA display molecules. A single-domain antibody (Nanobody, or VHH) was used as a model protein, and as a result, a pair of sequences was identified that increased mRNA-protein fusion efficiency of this protein by approximately 20%. Interestingly, enhancement of the fusion efficiency induced by the identified sequences was protein-specific, and different results were obtained for other proteins including VHHs with different CDRs. The results suggested that conformation of mRNA as a whole, rather than the amino acid sequence of the translated peptide, is an important factor to determine mRNA-protein fusion efficiency.
The functions of intracellular signal transduction systems are determined by the temporal behavior of intracellular molecules and their interactions. Of the many dynamical properties of the system, the relationship between the dynamics of upstream molecules and downstream molecules is particularly important. A useful tool in understanding this relationship is a methodology to control the dynamics of intracellular molecules with an extracellular stimulus. However, this is a difficult task because the relationship between the levels of upstream molecules and those of downstream molecules is often not only stochastic, but also time-inhomogeneous, nonlinear, and not one-to-one. In this paper, we present an easy-to-implement model-based control method that makes the target downstream molecule to trace a desired time course by changing the concentration of a controllable upstream molecule. Our method uses predictions from Monte Carlo simulations of the model to decide the strength of the stimulus, while using a particle-based approach to make inferences regarding unobservable states. We applied our method to in silico control problems of insulin-dependent AKT pathway model and EGF-dependent Akt pathway model with system noise. We show that our method can robustly control the dynamics of the intracellular molecules against unknown system noise of various strengths, even in the absence of complete knowledge of the true model of the target system.
The Fo-a subunit of the Na+-transporting FoF1 ATP synthase from Propionigenium modestum plays a key role in Na+ transport. It forms half channels that allow Na+ to enter and leave the buried carboxyl group on Fo-c subunits. The essential Arg residue R226, which faces the carboxyl group of Fo-c subunits in the middle of transmembrane helix 5 of the Fo-a subunit, separates the cytoplasmic side and periplasmic half-channels. To elucidate contributions of other amino acid residues of transmembrane helix 5 using hybrid FoF1 (Fo from P. modestum and F1 from thermophilic Bacillus PS3), 25 residues were individually mutated to Cys, and effects of modification with the SH-modifying agent N-ethylmaleimide (NEM) on ATP synthesis and hydrolysis activity were analyzed. NEM significantly inhibited ATP synthesis and hydrolysis as well as proton pumping activities of A214C, G215C, A218C, I223C (cytoplasmic side from R226), and N230C (periplasmic side from R226) mutants and inhibited ATP synthesis activity of the K219C mutant (cytoplasmic side from R226). Thus, these residues contribute to the integrity of the Na+ half channel, and both half channels are present in the Fo-a subunit.
A spectrally silent change is often observed in the photocycle of microbial rhodopsins. Here, we suggest the presence of two O intermediates in the photocycle of Acetabularia rhodopsin II (ARII or also called Ace2), a light-driven algal proton pump from Acetabularia acetabulum. ARII exhibits a photocycle including a quasi-equilibrium state of M, N, and O (M<=>N<=>O→) at near neutral and above pH values. However, acidification of the medium below pH ~5.5 causes no accumulation of N, resulting in that the photocycle of ARII can be described as an irreversible scheme (M→O→). This may facilitate the investigation of the latter part of the photocycle, especially the rise and decay of O, during which molecular events have not been sufficiently understood. Thus we analyzed the photocycle under acidic conditions (pH ≤ 5.5). Analysis of the absorbance change at 610 nm, which mainly monitors the fractional concentration changes of K and O, was performed and revealed a photocycle scheme containing two sequential O-states with the different molar extinction coefficients. These photoproducts, termed O1 and O2, may be even produced at physiological pH, although they are not clearly observed under this condition due to the existence of a long M-N-O equilibrium.
Microbial rhodopsins are membrane proteins found widely in archaea, eubacteria and eukaryotes (fungal and algal species). They have various functions, such as light-driven ion pumps, light-gated ion channels, light sensors and light-activated enzymes. A light-driven proton pump bacteriorhodopsin (BR) contains a DTD motif at positions 85, 89, and 96, which is unique to archaeal proton pumps. Recently, channelrhodopsins (ChRs) containing the DTD motif, whose sequential identity is ~20% similar to BR and to cation ChRs in Chlamydomonas reinhardtii (CrCCRs), were found. While extensive studies on ChRs have been performed with CrCCR2, the molecular properties of DTD ChRs remain an intrigue. In this paper, we studied a DTD rhodopsin from G. theta (GtCCR4) using electrophysiological measurements, flash photolysis, and low-temperature difference FTIR spectroscopy. Electrophysiological measurements clearly showed that GtCCR4 functions as a light-gated cation channel, similar to other G. theta DTD ChRs (GtCCR1-3). Light-driven proton pump activity was also suggested for GtCCR4. Both electrophysiological and flash photolysis experiments showed that channel closing occurs upon reprotonation of the Schiff base, suggesting that the dynamics of retinal and channels are tightly coupled in GtCCR4. From Fourier transform infrared (FTIR) spectroscopy at 77 K, we found that the primary reaction is an all-trans to a 13-cis photoisomerization, like other microbial rhodopsins, although perturbations in the secondary structure were much smaller in GtCCR4 than in CrCCR2.
The myosin II SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain and possibly plays a key role in the arrangement of the converter/lever arm. Several point mutations within the SH1 helix in human myosin IIs have been shown to cause diseases. To reveal whether these SH1 helix mutations affect not only motile activities but also thermal properties of myosin II, here we introduced the E683K or R686C point mutation into the SH1 helix in Dictyostelium myosin II. Thermal inactivation as well as thermal aggregation rates of these mutant proteins demonstrated that these mutations decreased the thermal stability of myosin II. Temperature dependence of sliding velocities of actin filaments showed that these mutations also reduced the activation energy of a rate-limiting process involved in actin movement. Given that these mutations are likely to alter coupling between the subdomains, and thus their thermal fluctuations, we propose that the SH1 helix is a key structural element that determines the flexibility and thermal properties of the myosin motor. These characteristics of the SH1 helix may contribute to the pathogenesis of the human diseases caused by mutations within this structural element.
RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras–Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS–Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP–Ras/RalGDS clusters, to induce the full activation of Ral.
Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.
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