Cells require adenosine triphosphate (ATP) to drive the myriad processes associated with growth, replication, and homeostasis. Eukaryotic cells rely on mitochondria to produce the vast majority of their ATP. Mitochondria consist of a relatively smooth outer mitochondrial membrane (OMM) and a highly complex inner mitochondrial membrane (IMM), containing numerous invaginations, called cristae, which house the molecular machinery of oxidative phosphorylation (OXPHOS). Although mitochondrial form and function are intimately connected, limitations in the resolution of live-cell imaging have hindered the ability to directly visualize the relationship between the architecture of the IMM and its associated bioenergetic properties. Using advanced imaging technologies, including Airyscan, stimulated emission depletion (STED), and structured illumination microscopy (SIM), we developed an approach to image the IMM in living cells. Staining mitochondria with various ΔΨm-dependent dyes, we found that the fluorescence pattern along the IMM was heterogeneous, with cristae possessing a significantly greater fluorescence intensity than the contiguous inner boundary membrane (IBM). Applying the Nernst equation, we determined that the ΔΨm of cristae is approximately 12 mV stronger than that of IBM, indicating that the electrochemical gradient that drives ATP synthesis is compartmentalized in cristae membranes. Notably, deletion of key components of the mitochondrial contact site and cristae organizing system (MICOS), as well as OPA1, which regulate crista junctions (CJs), decreased ΔΨm heterogeneity. Complementing our super-resolution imaging of cristae in living cells, we also developed a machine-learning protocol to quantify IMM architecture. Tracking real-time changes in cristae density, size, and shape, we determined that cristae dynamically remodel on a scale of seconds. Furthermore, we found that cristae move away from sites of mitochondrial fission, and, prior to mitochondrial fusion, the IMM forms finger-like protrusions bridging the membranes of the fusing organelles. Lastly, we investigated the role of the motor adaptor protein, Milton1/TRAK1, in mitochondrial dynamics. Patient-derived Milton1-null fibroblasts not only had impaired mitochondrial motility but exhibited fragmentation corresponding to a roughly 40% decrease in mitochondrial aspect ratio and a 17% increase in circularity, associated with increased DRP1 activity. Conversely, we found that overexpression of Milton1 led to mitochondrial hyperfusion, decreased DRP1 activity, and aberrant clustering of mtDNA. Overall, our studies directly demonstrate that maintaining mitochondrial architecture is essential for preserving the functionality of mitochondria, the hubs of eukaryotic metabolism.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23048号 / 理博第4725号 / 新制||理||1677(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 准教授 竹中 瑞樹, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Chapter 1: “Role of cyclic electron transport around photosystem I in the regulation of proton motive force” is based on the following paper. Caijuan Wang, Hiroshi Yamamoto, Toshiharu Shikanai, Role of cyclic electron transport around photosystem I in regulating proton motive force, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1847, Issue 9, September 2015, Pages 931-938, ISSN 0005-2728, http://dx.doi.org/10.1016/j.bbabio.2014.11.013.(http://www.sciencedirect.com/science/article/pii/S0005272814006586) / 京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19535号 / 理博第4195号 / 新制||理||1602(附属図書館) / 32571 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 鹿内 利治, 教授 西村 いくこ, 教授 長谷 あきら / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

AcrA is the periplasmic component of an efflux system AcrA-AcrB-TolC, which can expel different classes of antibiotics. AcrB is the inner membrane (IM) pump that utilizes proton-motive force for the active transport, TolC is the outer membrane (OM) channel, and AcrA coordinates the actions of AcrB and TolC, so that substrates are expelled across the two membranes, bypassing the periplasm. It has been proposed that AcrA either provides a static seamless link between AcrB and TolC, or acts like its analogous viral membrane fusion protein (MFP) and actively brings the IM and OM closer for substrate transfer. To better understand the role of AcrA in the efflux mechanism, site-directed spin labeling (SDSL)/EPR (electron paramagnetic resonance) spectroscopy is used to investigate the structure and dynamics of AcrA in solution. My results demonstrated that AcrA is a dynamic protein that undergoes pH-dependent and reversible conformational changes. AcrA contains an interrupted alpha-helical, coiled-coil domain flanked by a pair of beta-stranded lipoyl motifs, and my SDSL/EPR analysis revealed that the pH-induced conformation change mainly involves the coiled-coil and the lipoyl domains. In addition, I found that each AcrA monomer folds into an intra-molecular hairpin and AcrA monomers oligomerize with their coiled-coil hairpins aligned in parallel. Unlike the pH-induced conformational rearrangement of a viral MFP, change in pH alters both intra- and inter-molecular interaction along the coiled-coil of AcrA without rearranging the hairpin fold. The organization of AcrA protomers and its pH-induced conformational switching are, however, congruent with the TolC coiled-coil hairpins in the iris-like opening of the TolC channel. Together, my studies suggest that rather than being a passive structural linkage between AcrB and TolC, AcrA plays an active role mediating the drug efflux. The reported AcrA dynamics provides new insights into the AcrA-TolC interactions for the channel opening during the efflux process.

AcrA is the periplasmic component of an efflux system AcrA-AcrB-TolC, which can expel different classes of antibiotics. AcrB is the inner membrane (IM) pump that utilizes proton-motive force for the active transport, TolC is the outer membrane (OM) channel, and AcrA coordinates the actions of AcrB and TolC, so that substrates are expelled across the two membranes, bypassing the periplasm. It has been proposed that AcrA either provides a static seamless link between AcrB and TolC, or acts like its analogous viral membrane fusion protein (MFP) and actively brings the IM and OM closer for substrate transfer. To better understand the role of AcrA in the efflux mechanism, site-directed spin labeling (SDSL)/EPR (electron paramagnetic resonance) spectroscopy is used to investigate the structure and dynamics of AcrA in solution. My results demonstrated that AcrA is a dynamic protein that undergoes pH-dependent and reversible conformational changes. AcrA contains an interrupted alpha-helical, coiled-coil domain flanked by a pair of beta-stranded lipoyl motifs, and my SDSL/EPR analysis revealed that the pH-induced conformation change mainly involves the coiled-coil and the lipoyl domains. In addition, I found that each AcrA monomer folds into an intra-molecular hairpin and AcrA monomers oligomerize with their coiled-coil hairpins aligned in parallel. Unlike the pH-induced conformational rearrangement of a viral MFP, change in pH alters both intra- and inter-molecular interaction along the coiled-coil of AcrA without rearranging the hairpin fold. The organization of AcrA protomers and its pH-induced conformational switching are, however, congruent with the TolC coiled-coil hairpins in the iris-like opening of the TolC channel. Together, my studies suggest that rather than being a passive structural linkage between AcrB and TolC, AcrA plays an active role mediating the drug efflux. The reported AcrA dynamics provides new insights into the AcrA-TolC interactions for the channel opening during the efflux process.

No description available.

Les bactériocines de classe IIa présentent une activité antimicrobienne résultant d'un mécanisme d'action ciblant les membranes des bactéries à Gram positif. Cette activité est modulée par différentes caractéristiques des surfaces bactériennes. Les propriétés physico-chimiques de surface de dix-huit souches bactériennes ont été déterminées afin d'étudier le lien entre ces propriétés et les phénotypes de résistance/sensibilité à Cbn BM1. Les résultats obtenus indiquent une grande diversité des propriétés physico-chimiques des surfaces analysées, sans cependant permettre d’établir un lien entre celles-ci et le phénotype de sensibilité/résistance à CbnBM1. Les mécanismes d'action de Cbn BM1 ont ensuite été étudiés sur Carnobacterium maltaromaticum DSM20730 et Listeria monocytogenes EGDe. L'atteinte de l'intégrité physique des membranes plasmiques par l'action de Cbn BM1 montre une hétérogénéité de réponse des populations bactériennes. Ce résultat a été confirmé par microscopie de force atomique in vivo à haute résolution. L'interaction de Cbn BM1 avec les membranes a été mise en évidence par mesure de l'anisotropie de fluorescence. Cette approche a révélé que Cbn BM1 présente des degrés de pénétration différents dans la membrane de C. maltaromaticum DSM20730 par rapport à L. monocytogenes EGDe. L'action de Cbn BM1 conduit cependant, pour les deux souches, à la modification de la force protomotrice membranaire. Ces différentes approches retenues pour l'étude des mécanismes d'action ont révélé que C. maltaromaticum DSM20730 et L. monocytogenes EGDe présentent une sensibilité à Cbn BM1 uniquement lorsque les cellules sont en phase exponentielle de croissance. / The antimicrobial activity of class IIa bacteriocins toward Gram positive bacteria relies on their membrane targeting mechanisms of action. These mechanisms are modulated by the bacterial surface properties. The physico-chemical surface properties of eighteen bacterial strains were determined to link these properties to the resistance/sensitivity to Cbn BM1 of the bacterial strains. In this way, two approaches were undertaken : the microbial adhesion to solvents and electrophoretic mobility measurements. The results show a large diversity of the determined properties among the strains but without establishing a direct link between the surface properties and the resistance/sensitivity phenotypes. Mechanisms of action of the bacteriocin Cbn BM1 on Carnobacterium maltaromaticum DSM20730 and Listeria monocytogenes EGDe were determined. Syto9® and propidium iodide allowed to show the heterogeneity of the bacterial populations toward the alteration of the membrane integrity. The interaction of Cbn BM1 with the bacterial membrane was studied by monitoring the fluorescence anisotropy of DPH and TMA-DPH. The results highlight a difference between the mechanism of action of Cbn BM1 on C. maltaromaticum DSM20730 and on L. monocytogenes EGDe. However, a treatment by Cbn BM1 leads to a perturbation of the component of the proton-motive force of the membrane for both strains. These approaches revealed that these bacterial strains exhibit a sensitivity to Cbn BM1 only when treated in log growth phase. Modification of nano-mechanical properties of C. maltaromaticum DSM20730 after a treatment by Cbn BM1 were assessed by an atomic force microscopy approach.