The SYP123-VAMP727 SNARE complex delivers secondary cell wall components for root hair shank hardening in Arabidopsis.

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

Structural Dynamics of Amyloid-ß Protofibrils and Actions of Anti-Amyloid-ß Antibodies as Observed by High-Speed Atomic Force Microscopy.

Amyloid-ß (Aß) aggregation intermediates, including oligomers and protofibrils (PFs), have attracted attention as neurotoxic aggregates in Alzheimer's disease. However, due to the complexity of the aggregation pathway, the structural dynamics of aggregation intermediates and how drugs act on them have not been clarified. Here we used high-speed atomic force microscopy to observe the structural dynamics of Aß42 PF at the single-molecule level and the effect of lecanemab, an anti-Aß PF antibody with the positive results from Phase 3 Clarity AD. PF was found to be a curved nodal structure with stable binding angle between individual nodes. PF was also a dynamic structure that associates with other PF molecules and undergoes intramolecular cleavage. Lecanemab remained stable in binding to PFs and to globular oligomers, inhibiting the formation of large aggregates. These results provide direct evidence for a mechanism by which antibody drugs interfere with the Aß aggregation process.

Elucidation of the Binding Mechanism of Anionic Phospholipids to Antioxidant Protein Peroxiredoxin 2.

Peroxiredoxins (Prxs) belong to a family of ubiquitously expressed peroxidases that detoxify reactive oxygen species. In addition to their enzymatic function, Prxs also function as molecular chaperones. This functional switch is related to their degree of oligomerization. We have previously revealed that Prx2 interacts with anionic phospholipids and that the anionic phospholipid-containing Prx2 oligomer forms a high molecular weight (HMW) complex in a nucleotide-dependent manner. However, the detailed mechanism of the oligomer and HMW complex formation remains unclear. In this study, we investigated the anionic phospholipid binding site in Prx2 using site-directed mutagenesis to understand the mechanism of the oligomer formation. Our findings demonstrated that six binding site residues in Prx2 are important for the binding of anionic phospholipids.

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy.

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-ß structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-ß structures gradually occur.

Computational see-through screen camera based on a holographic waveguide device.

This study proposes a novel computational imaging system that integrates a see-through screen (STS) with volume holographic optical elements (vHOEs) and a digital camera unit. Because of the unique features of the vHOE, the STS can function as a holographic waveguide device (HWD) and enable the camera to capture the frontal image when the user gazes at the screen. This system not only provides an innovative solution to a high-quality video communication system by realizing eye-contact but also contributes to other visual applications due to its refined structure. However, there is a dilemma in the proposed imaging system: for a wider field of view, a larger vHOE is necessary. If the size of the vHOE is larger, the light rays from the same object point are diffracted at different Bragg conditions and reflect a different number of times, which causes blurring of the captured image. The system imaging process is analyzed by ray tracing, and a digital image reconstruction method was employed to obtain a clear picture in this study. Optical experiments confirmed the effectiveness of the proposed HWD-STS camera.

Unraveling the Host-Selective Toxic Interaction of Cassiicolin with Lipid Membranes and Its Cytotoxicity.

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for Corynespora leaf fall disease in susceptible rubber trees. Currently, the molecular mechanisms of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more sensitive to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Mature Cas1 and Cas2 play an essential role in causing membrane disruption. Cytotoxicity tests on rubber leaves of Rubber Research Institute of Vietnam (RRIV) 1, RRIV 4, and Prang Besar (PB) 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryogenic scanning electron microscopy analyses of necrotic leaf tissues treated with Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and to disrupt specific membranes.

Discrete Fourier Transform Radar in the Terahertz-Wave Range Based on a Resonant-Tunneling-Diode Oscillator.

We used a resonant-tunneling-diode (RTD) oscillator as the source of a terahertz-wave radar based on the principle of the swept-source optical coherence tomography (SS-OCT). Unlike similar reports in the terahertz range, we apply the stepwise frequency modulation to a subcarrier obtained by amplitude modulation instead of tuning the terahertz carrier frequency. Additionally, we replace the usual optical interference with electrical mixing and, by using a quadrature mixer, we can discriminate between negative and positive optical path differences, which doubles the measurement range without increasing the measurement time. To measure the distance to multiple targets simultaneously, the terahertz wave is modulated in amplitude at a series of frequencies; the signal returning from the target is detected and homodyne mixed with the original modulation signal. A series of voltages is obtained; by Fourier transformation the distance to each target is retrieved. Experimental results on one and two targets are shown.

Ubiquitin chain specificities of E6AP E3 ligase and its HECT domain.

Ubiquitination of target proteins is accomplished by isopeptide bond formation between the carboxy group of the C-terminal glycine (Gly) residue of ubiquitin (Ub) and the ɛ-amino group of lysine (Lys) on the target proteins. The formation of an isopeptide bond between Ubs that gives rise to a poly-Ub chain on the target proteins and the types of poly-Ub chains formed depend on which of the seven Lys residues or N-terminal methionine (Met) residue on Ub is used for chain elongation. To understand the linkage specificity mechanism of Ub chains on E3, the previous study established an assay to monitor the formation of a free diubiquitin chain (Ub2 chain synthesis assay) by HECT type E3 ligase. In this study, we investigated Ub2 chain specificity using E6AP HECT domain. We here demonstrate the importance of the N-terminal domain of full length E6AP for Ub2 chain specificity.

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.

Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

Redox regulation of CF1-ATPase involves interplay between the γ-subunit neck region and the turn region of the ßDELSEED-loop.

The soluble F1 complex of ATP synthase (FoF1) is capable of ATP hydrolysis, accomplished by the minimum catalytic core subunits α3ß3γ. A special feature of cyanobacterial F1 and chloroplast F1 (CF1) is an amino acid sequence inserted in the γ-subunit. The insertion is extended slightly into the CF1 enzyme containing two additional cysteines for regulation of ATPase activity via thiol modulation. This molecular switch was transferred to a chimeric F1 by inserting the cysteine-containing fragment from spinach CF1 into a cyanobacterial γ-subunit [Y. Kim et al., redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch, J Biol Chem, 286 (2011) 9071-9078]. Under oxidizing conditions, the obtained F1 tends to lapse into an ADP-inhibited state, a common regulation mechanism to prevent wasteful ATP hydrolysis under unfavorable circumstances. However, the information flow between thiol modulation sites on the γ-subunit and catalytic sites on the ß-subunits remains unclear. Here, we clarified a possible interplay for the CF1-ATPase redox regulation between structural elements of the ßDELSEED-loop and the γ-subunit neck region, i.e., the most convex part of the α-helical γ-termini. Critical residues were assigned on the ß-subunit, which received the conformation change signal produced by disulfide/dithiol formation on the γ-subunit. Mutant response to the ATPase redox regulation ranged from lost to hypersensitive. Furthermore, mutant cross-link experiments and inversion of redox regulation indicated that the γ-redox state might modulate the subunit interface via reorientation of the ßDELSEED motif region.

Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca(2+) influx through transient receptor potential V1 (TRPV1) channels.

Non-selective transient receptor potential vanilloid (TRPV) cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM) is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1) and the Ca(2+)-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 µM) and pH 6.5 buffer elicited steep increases in the intracellular Ca(2+) concentration ([Ca(2+)]i), while treatment with THAM (pH 8.5) alone had no effect. However, treatment with THAM (pH 8.5) following capsaicin application elicited a profound, long-lasting increase in [Ca(2+)]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca(2+)]i increases, which could be a mechanism underlying pain induced by basic pH.

Distinct redox behaviors of chloroplast thiol enzymes and their relationships with photosynthetic electron transport in Arabidopsis thaliana.

The thiol/disulfide redox network mediated by the thioredoxin (Trx) system in chloroplasts ensures light-responsive control of diverse crucial functions. Despite the suggested importance of this system, the working dynamics against changing light environments remains largely unknown. Thus, we directly assessed the in vivo redox behavior of chloroplast Trx-targeted thiol enzymes in Arabidopsis thaliana. In a time-course analysis throughout a day period that was artificially mimicked to natural light conditions, thiol enzymes showed a light-dependent shift in redox state, but the patterns were distinct among thiol enzymes. Notably, the ATP synthase CF(1-γ) subunit was rapidly reduced even under low-light conditions, whereas the stromal thiol enzymes fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and NADP-malate dehydrogenase were gradually reduced/re-oxidized along with the increase/decrease in light intensity. Photo-reduction of thiol enzymes was suppressed by the impairment of photosynthetic linear electron transport using DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but sensitivity to the impairment was uneven between CF(1-γ) and other stromal thiol enzymes. These different dependencies of photo-reduction on electron transport, rather than the redox state of Trx and the circadian clock, could readily explain the distinct diurnal redox behaviors of thiol enzymes. In addition, our results indicate that the cyclic electron transport around PSI is also involved in redox regulation of some thiol enzymes. Based on these findings, we propose an in vivo working model of the redox regulation system in chloroplasts.

Reversible control of F(1)-ATPase rotational motion using a photochromic ATP analog at the single molecule level.

Motor enzymes such as F1-ATPase and kinesin utilize energy from ATP for their motion. Molecular motions of these enzymes are critical to their catalytic mechanisms and were analyzed thoroughly using a single molecule observation technique. As a tool to analyze and control the ATP-driven motor enzyme motion, we recently synthesized a photoresponsive ATP analog with a p-tert-butylazobenzene tethered to the 2' position of the ribose ring. Using cis/trans isomerization of the azobenzene moiety, we achieved a successful reversible photochromic control over a kinesin-microtubule system in an in vitro motility assay. Here we succeeded to control the hydrolytic activity and rotation of the rotary motor enzyme, F1-ATPase, using this photosensitive ATP analog. Subsequent single molecule observations indicated a unique pause occurring at the ATP binding angle position in the presence of cis form of the analog.

Super-enhancer trapping by the nuclear pore via intrinsically disordered regions of proteins in squamous cell carcinoma cells.

Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.

A conformational change of the γ subunit indirectly regulates the activity of cyanobacterial F1-ATPase.

The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30-40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α(3)ß(3)γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ε inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ε inhibition.

Redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch.

F(1)-ATP synthase (F(1)-ATPase) is equipped with a special mechanism that prevents the wasteful reverse reaction, ATP hydrolysis, when there is insufficient proton motive force to drive ATP synthesis. Chloroplast F(1)-ATPase is subject to redox regulation, whereby ATP hydrolysis activity is regulated by formation and reduction of the disulfide bond located on the γ subunit. To understand the molecular mechanism of this redox regulation, we constructed a chimeric F(1) complex (α(3)ß(3)γ(redox)) using cyanobacterial F(1), which mimics the regulatory properties of the chloroplast F(1)-ATPase, allowing the study of its regulation at the single molecule level. The redox state of the γ subunit did not affect the ATP binding rate to the catalytic site(s) and the torque for rotation. However, the long pauses caused by ADP inhibition were frequently observed in the oxidized state. In addition, the duration of continuous rotation was relatively shorter in the oxidized α(3)ß(3)γ(redox) complex. These findings lead us to conclude that redox regulation of CF(1)-ATPase is achieved by controlling the probability of ADP inhibition via the γ subunit inserted region, a sequence feature observed in both cyanobacterial and chloroplast ATPase γ subunits, which is important for ADP inhibition (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865).

Characterization of the relationship between ADP- and epsilon-induced inhibition in cyanobacterial F1-ATPase.

The ATPase activity of chloroplast and bacterial F(1)-ATPase is strongly inhibited by both the endogenous inhibitor ε and tightly bound ADP. Although the physiological significance of these inhibitory mechanisms is not very well known for the membrane-bound F(0)F(1), these are very likely to be important in avoiding the futile ATP hydrolysis reaction and ensuring efficient ATP synthesis in vivo. In a previous study using the α(3)ß(3)γ complex of F(1) obtained from the thermophilic cyanobacteria, Thermosynechococcus elongatus BP-1, we succeeded in determining the discrete stop position, ∼80° forward from the pause position for ATP binding, caused by ε-induced inhibition (ε-inhibition) during γ rotation (Konno, H., Murakami-Fuse, T., Fujii, F., Koyama, F., Ueoka-Nakanishi, H., Pack, C. G., Kinjo, M., and Hisabori, T. (2006) EMBO J. 25, 4596-4604). Because γ in ADP-inhibited F(1) also pauses at the same position, ADP-induced inhibition (ADP-inhibition) was assumed to be linked to ε-inhibition. However, ADP-inhibition and ε-inhibition should be independent phenomena from each other because the ATPase core complex, α(3)ß(3)γ, also lapses into the ADP-inhibition state. By way of thorough biophysical and biochemical analyses, we determined that the ε subunit inhibition mechanism does not directly correlate with ADP-inhibition. We suggest here that the cyanobacterial ATP synthase ε subunit carries out an important regulatory role in acting as an independent "braking system" for the physiologically unfavorable ATP hydrolysis reaction.

Regulation of F0F1-ATPase from Synechocystis sp. PCC 6803 by gamma and epsilon subunits is significant for light/dark adaptation.

The γ and ε subunits of F(0)F(1)-ATP synthase from photosynthetic organisms display unique properties not found in other organisms. Although the γ subunit of both chloroplast and cyanobacterial F(0)F(1) contains an extra amino acid segment whose deletion results in a high ATP hydrolysis activity (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855-865), its ε subunit strongly inhibits ATP hydrolysis activity. To understand the physiological significance of these phenomena, we studied mutant strains with (i) a C-terminally truncated ε (ε(ΔC)), (ii) γ lacking the inserted sequence (γ(Δ198-222)), and (iii) a double mutation of (i) and (ii) in Synechocystis sp. PCC 6803. Although thylakoid membranes from the ε(ΔC) strain showed higher ATP hydrolysis and lower ATP synthesis activities than those of the wild type, no significant difference was observed in growth rate and in intracellular ATP level both under light conditions and during light-dark cycles. However, both the ε(ΔC) and γ(Δ198-222) and the double mutant strains showed a lower intracellular ATP level and lower cell viability under prolonged dark incubation compared with the wild type. These data suggest that internal inhibition of ATP hydrolysis activity is very important for cyanobacteria that are exposed to prolonged dark adaptation and, in general, for the survival of photosynthetic organisms in an ever-changing environment.

Thiol modulation of the chloroplast ATP synthase is dependent on the energization of thylakoid membranes.

Thiol modulation of the chloroplast ATP synthase γ subunit has been recognized as an important regulatory system for the activation of ATP hydrolysis activity, although the physiological significance of this regulation system remains poorly characterized. Since the membrane potential required by this enzyme to initiate ATP synthesis for the reduced enzyme is lower than that needed for the oxidized form, reduction of this enzyme was interpreted as effective regulation for efficient photophosphorylation. However, no concrete evidence has been obtained to date relating to the timing and mode of chloroplast ATP synthase reduction and oxidation in green plants. In this study, thorough analysis of the redox state of regulatory cysteines of the chloroplast ATP synthase γ subunit in intact chloroplasts and leaves shows that thiol modulation of this enzyme is pivotal in prohibiting futile ATP hydrolysis activity in the dark. However, the physiological importance of efficient ATP synthesis driven by the reduced enzyme in the light could not be demonstrated. In addition, we investigated the significance of the electrochemical proton gradient in reducing the γ subunit by the reduced form of thioredoxin in chloroplasts, providing strong insights into the molecular mechanisms underlying the formation and reduction of the disulfide bond on the γ subunit in vivo.