Podcasts about f1 atpase

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.03.535492v1?rss=1 Authors: Manandhar, B., Pandzic, E., Deshpande, N., Chen, S.-Y., Wasinger, V., Kockx, M., Glaros, E. N., Ong, K. L., Thomas, S. R., Wilkins, M., Whan, R., Cochran, B. J., Rye, K.-A. Abstract: Objective: High cholesterol levels in pancreatic {beta}-cells cause oxidative stress and decrease insulin secretion. {beta}-cells can internalize apolipoprotein (apo) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves {beta}-cell insulin secretion by reducing oxidative stress. Approach: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-{beta}-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mM glucose was quantified by radioimmunoassay. Internalization of fluorescently labelled apoA-I by {beta}-cells was monitored by flow cytometry. The effects of apoA-I internalization on {beta}-cell gene expression was evaluated by RNA sequencing. ApoA-I binding partners on the {beta}-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in {beta}-cells and isolated islets with MitoSOX and confocal microscopy. Results: An F1-ATPase {beta}-subunit on the {beta}-cell surface was identified as the main apoA-I binding partner. {beta}-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol- and F1-ATPase {beta}-subunit-dependent. {beta}-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase {beta}-subunit levels than {beta}-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I co-localized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The ATPase inhibitory factor 1, IF1, attenuated apoA-I internalization and increased oxidative stress in Ins-1E {beta}-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion and mitochondrial function. Conclusions: These results establish that {beta}-cells are functionally heterogeneous and apoA-I restores insulin secretion in ?-cells with elevated cholesterol levels by improving mitochondrial redox balance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

マックスプランクフロリダの安田涼平さん（@Ryohei_Neuro）をゲストに、１分子イメージングから１スパイン・分子活性のイメージングへと分野を変えていった時に考えていたこと、Karel Svobodaのラボでポスドク時代に学んだこと、その後の独立、マネジメント哲学、最近の研究とこれからの新しい展開などについて伺いました（6/5収録） Show Notes: 安田さんラボHP 安田さんブログ 安田さんHP記事 アメリカでラボを持ちたい！ マックスプランクフロリダ (Max Planck Institute Florida Institute for Neuroscience) ATP合成酵素が回転するところをアクチンつけて観た仕事 東工大（当時）の野地さん：現在東大で主催しているラボ 木下研HP（早稲田へ） カレル：Karel Svobodaのこと。安田さんのポスドク時代のボスであり、萩原の現・ボス。 Karelが見つけたキネシンのステップ 120°ずつ回転、仕事効率ほぼ100% Winfried Denk (カレルのベル研での直接のメンターはおそらくTank。Denkはおそらく横にいて顕微鏡を提供した？Denkは最初に2光子顕微鏡作った人) KarelのラボからPIになった人…の参考図（NeuroTree） カレルがGCaMPに着手したタイミング GCaMP2はこの論文 佐藤 隆さん@MUSC (萩原は学部時代に佐藤さんの実験@Tübingenを手伝っていた) GCaMP6の論文 (現状カレルの論文の中で最も引用される論文となった) 顕微鏡に関する投稿：ポスドクを終えてDukeで独立する辺りは2005年～ Karelラボの２番目のプロジェクト（Rasの分子活性） 独立した後のCaMK2の仕事 林康紀さん が当時やってた（Camuiα） FLIM: ライフタイムイメージングのこと（PDF） 谷口さん 村越さん（ラボ） の仕事 安田さんのトークで最後に出てくる絵 (by R, Iyenger) 三國さん 西山さん MPFIのマシンショップ Michael Ehlers Fitzpatrickラボ 結局一番楽しかったタイミングはF1-ATPaseの回転が見えた時…の記事 安田さんのライフタイムイメージングの会社 Editorial Notes: 実は当初MPFIのFitzpatrickラボで博士をやる予定でした。いろいろあってvisionのdevelopmentから分野を変えたのは結果的にはよかったのではないかな〜と。安田さんをはじめ、成功してる研究者には楽観的な人が多いような気がしてる今日この頃、辛気臭くない感じで生きていきたいなと思ってます (萩原) 研究所のDirectorという雲の上の存在でありながら、我々のようなポスドクにフランクに接してくださってくださったこと、そしてpoliticalな感じが全然しなくて技術開発・現象探求にフォーカスしてらっしゃる様子が印象的でした(宮脇) これからを担う若い研究者のお二人とお話できて楽しい時間を過ごしました！ぜひミーティングなどでまたお会いしたいと思いました(安田）

The scope of this work is to gain insight and a deeper understanding of exploring and controlling molecular devices like proteins and rotors by fine tuned manipulation via mechanical or electrical energies. I focus on three main topics. First, I investigate vectorial forces as a tool to explore the energy landscape of protein complexes. Second, I apply this method to a biologically important force transduction complex, the integrin-talin complex. Third, I use Terahertz electric fields to manipulate the energy landscape of a molecular rotor on a gold surface and drive their effective rotation bidirectionally. Force is by nature a vector and depends on its three parameters: magnitude, direction and attachment point. Here, the impact of different force protocols varying these parameters is shown for an antibody-antigen complex and the ribonuclease-inhibitor complex barnase-barstar. Antibodies are essential for our adaptive immune system in their function to bind specific antigens. Here, the binding of an antibody to a peptide is probed with varying attachment points. Different attachment points clearly change the dissociation pathways. The barriers identified using experimental atomic force microscopy (AFM) and molecular dynamics (MD) simulations are in excellent agreement. I determine the molecular interactions of two main barriers for each setup. This results in a common outer barrier of the complex and different inner barriers probed by AFM. The ribonuclease barnase and its inhibitor barstar form an evolutionary optimized complex. Different force protocols are shown to determine the hierarchy of relative stability within a protein complex. For the barnase-barstar complex, the internal fold of the barstar is identified to be less stable than the barnase-barstar binding interaction. High velocities probe the lability or barriers of the system while low velocities probe the stability or energy wells of this system. Forces impact biological life on totally different length scales which range from whole organisms to individual proteins. Integrins are the major cell adhesion receptors binding to the extracellular matrix and talin. Talin activates the integrins and creates the initial connection to the actin cytoskeleton of the cell. Here, I have chosen to investigate the integrin-talin complex as a biologically important force transduction complex. The force dependence of the system is probed by constant force MD simulations. The two main results include the activation of the complex and its force response. I demonstrate, that the binding of talin to integrin does not disrupt the integrin's transmembrane helix interactions sterically. Since, this disruption is necessary for integrin activation, a modified activation mechanism requiring a small force application is proposed. The response of the integrin-talin complex normal and parallel to the cell membrane is analyzed. The complete dissociation pathways generated for both directions identify a force-induced formation of a stabilizing beta strand between integrin and talin only for normal forces. Furthermore, the complex tries to rotate such that the external force aligns with the more force resistant axis of the complex. In nature, molecular rotors are essential building blocks of many molecular machines and brownian motors like the F1-ATPase or the flagellum of a bacterium. The direction of rotation often steers different processes in clockwise and counterclockwise directions. Rotation on the nanoscopic level in artificial devices is still very limited and requires a deeper understanding. In my last project, I study the switching and driving of a molecular diethylsulfid rotor on a gold (111) surface by Terahertz electric fields. The response of the rotational energy landscape to static and oscillation electric fields is analyzed. Varying the Terahertz driving frequency, the rotation direction and frequency are controlled. A theoretical framework is presented to describe the behavior of the molecular rotor. This can be seen as the first step into the direction of man-made controllable nano-devices driven and controlled by energy from the electric wall-socket.

We model the dynamics of a processive or rotary molecular motor using a renewal processes, in line with the work initiated by Svoboda, Mitra and Block. We apply a functional technique to compute different types of multiple-time correlation functions of the renewal process, which have applications to bead-assay experiments performed both with processive molecular motors, such as myosin V and kinesin, and rotary motors, such as F1-ATPase

The protein translocation machineries of the outer and inner mitochondrial membranes usually act in concert during translocation of matrix and inner membrane proteins. We considered whether the two machineries can function independently of each other in a sequential reaction. Fusion proteins (pF-CCHL) were constructed which contained dual targeting information, one for the intermembrane space present in cytochrome c heme lyase (CCHL) and the other for the matrix space contained in the signal sequence of the precursor of F1-ATPase beta-subunit (pF1 beta). In the absence of a membrane potential, delta psi, the fusion proteins moved into the intermembrane space using the CCHL pathway. In contrast, in the presence of delta psi they followed the pF1 beta pathway and eventually were translocated into the matrix. The fusion protein pF51-CCHL containing 51 amino acids of pF1 beta, once transported into the intermembrane space in the absence of a membrane potential, could be further chased into the matrix upon re-establishing delta psi. The sequential and independent movement of the fusion protein across the two membranes demonstrates that the translocation machineries act as distinct entities. Our results support a model in which the two translocation machineries can function independently of each other, but generally interact in a dynamic fashion to achieve simultaneous translocation across both membranes. In addition, the results provide information about the targeting sequences within CCHL. The protein does not contain a signal for retention in the intermembrane space; rather, it lacks matrix targeting information, and therefore is unable to undergo delta psi-dependent interaction with the protein translocation apparatus in the inner membrane.

Sat, 25 Aug 1990 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7603/1/Neupert_Walter_7603.pdf Tropschug, Maximilian; Neupert, Walter; Müller, Harald A.; Harmey, Matthew A.; Rassow, Joachim

We have identified a 19 kd protein of the mitochondrial outer membrane (MOM19). Monospecific IgG and Fab fragments directed against MOM19 inhibit import of precursor proteins destined for the various mitochondrial subcompartments, including porin, cytochrome c1, Fe/S protein, F0 ATPase subunit 9, and F1 ATPase subunit β. Inhibition occurs at the level of high affinity binding of precursors to mitochondria. Consistent with previous functional studies that suggested the existence of distinct import sites for ADP/ATP carrier and cytochrome c, we find that import of those precursors is not inhibited. We conclude that MOM19 is identical to, or closely associated with, a specific mitochondrial import receptor.

The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1- complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1- ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease- sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.

The precursors of the mitochondrial proteins ADP/ATP carrier (AAC) and F1-ATPase subunit β (F1β) were accumulated at the stages of binding to receptor sites on the mitochondrial outer membrane, or in contact sites between outer and inner membranes. Specific antibodies raised against the mature proteins were added to the isolated mitochondria and efficiently bound to these translocation intermediates. Further movement of the precursors to consecutive steps along their import pathway was thereby inhibited. Controls showed that precursor proteins which were inserted into or translocated across the outer membrane were not recognized by the antibodies unless the mitochondrial membranes were disrupted. We conclude that the trapped translocation intermediates have antigenic sites exposed to the outside of the outer membrane.

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit β(F1β) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1β translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.

The role of nucleoside triphosphates (NTPs) in mitochondrial protein import was investigated with the precursors of N. crassa ADP/ATP carrier, F1-ATPase subunit β, F0-ATPase subunit 9, and fusion proteins between subunit 9 and mouse dihydrofolate reductase. NTPs were necessary for the initial interaction of precursors with the mitochondria and for the completion of translocation of precursors from the mitochondrial surface into the mitochondria. Higher levels of NTPs were required for the latter reactions as compared with the early stages of import. Import of precursors having identical presequences but different mature protein parts required different levels of NTPs. The sensitivity of precursors in reticulocyte lysate to proteases was decreased by removal of NTPs and increased by their readdition. We suggest that the hydrolysis of NTPs is involved in modulating the folding state of precursors in the cytosol, thereby conferring import competence.

Transport of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane requires a mitochondrial membrane potential. We have studied whether additional energy sources are also necessary for protein translocation. Reticulocyte lysate (containing radiolabelled precursor proteins) and mitochondria were depleted of ATP by pre-incubation with apyrase. A membrane potential was then established by the addition of substrates of the electron transport chain. Oligomycin was included to prevent dissipation of Δψ by the action of the F0F1-ATPase. Under these conditions, import of subunit β of F1-ATPase (F1β) was inhibited. Addition of ATP or GTP restored import. When the membrane potential was destroyed, however, the import of F1β was completely inhibited even in the presence of ATP. We therefore conclude that the import of F1β depends on both nucleoside triphosphates and a membrane potential.

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m- chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.