Reactive oxygen species (ROS) oxidize surrounding molecules and thus impair their functions. Since mitochondria are a major source of ROS, suppression of ROS overproduction in the mitochondria is important for cells. Spontaneous transient depolarization of individual mitochondria is a physiological phenomenon widely observed from plants to mammals. Mitochondrial uncoupling can reduce ROS production; therefore, it is conceivable that transient depolarization could reduce ROS production. However, transient depolarization has been observed with increased ROS production. Therefore, the exact contribution of transient depolarization to ROS production has not been elucidated. In this study, we examined how the spontaneous transient depolarization occurring in individual mitochondria affected ROS production. When the matrix pH increased after the addition of malate or exposure of the isolated mitochondria to a high-pH buffer, transient depolarization was stimulated. Similar stimulation by an increased matrix pH was also observed in the mitochondria in intact H9c2 cells. Modifying the mitochondrial membrane potential and matrix pH by adding K+ in the presence of valinomycin, a K+ ionophore, clarified that an increase in the matrix pH is a major cause of ROS generation. When we added ADP in the presence of oligomycin to suppress the transient depolarization without decreasing the matrix pH, we observed the suppression of mitochondrial respiration, increased matrix pH, and enhanced ROS production. Based on these results, we propose a model where spontaneous transient depolarization occurs during increased proton influx through proton channels opened by increased matrix pH, leading to the suppression of ROS production. This study improves our understanding of mitochondrial behavior.
A mitochondrion has a pH gradient between the two sides of its inner membrane in order to produce adenosine triphosphate (ATP). Because ATP depletion causes numerous diseases, the measurement of the pH value around the mitochondrion is expected to clarify the mechanism of these diseases. In this study, a dual-wavelength pH-sensitive dye was excited by two-photon absorption initiated using a femtosecond pulse laser. In addition, fluorescence from the dye was directly collected from the fluorescent point using the collection-mode probe of a scanning near-field optical microscope. By this proposed method, a pH calibration curve was obtained from the fluorescent intensity ratio of the dye solution, and temporal pH variations with 0.1 s time resolution following the addition of acid were observed. Moreover, mitochondrial activity on the basis of the pH changes was successfully observed in three different mitochondrial densities.
ConstruLtmg in vitro neuronal crrcultb has been an ]rnportant method te andiTyze neural activities We propose a method to compose a neuronal circurt with yng]e neurens preuscly using microfabncated mebilL plates Conventional dpproaches in constructmg m vi tro neuronal circuits ha-L a dltfiLulty in forrning exact netuork structLrre as-c want beLduse neurons do not al"ays adhere prLcisely on piedesigned geometries andonLc they dire cultured they can notbc hand]ed anvmore In this study "e make it possiblc to haiidle nying]e neurons and rearrange circuit geomeLrles m a Lulture dtsh We ± irst fabricated microp]ates which had neurite growth direction guldcpaths by standard ]ithoglaphy We underlaid gelatine which dissolved in 37 degrees C to make the microp]ates mosable and oiercoated protcins for cell sLafto]ds on the miLroplates Second we culturcd neurens and guided their ncumte growth direction along the paths by patteniing protcm rLsistallt materials Finally wL handted the microplatLs alld brought them into contaLt We succesgfutly assembled microptates wrth slnglc neurons into a netwoTk suuLtuie by manipuldtmg them wtth a glass capillary emid also rearranged thcii neL"erk geometry m cultuiedishes We be]ieve our inethod will be uHefu] fordegigning precise smglc ccll level neuional circuits and analymng neurd] Lircu!t act-itieg spaual distribution ofa fluoregcent dyc ofa speufiL antibody marker by phase contrasVfluorLsLellLe miLroscopy and image pieccsslng whiLh has not been iealized ugmg cenLentional ditfraLtion based cell sortmg systcms First we compaiLd the differences of micrescopic lmages (shapes) of neurons and g]ia ce]]g and found that only neurons have neuntes extendlng lrom the Lell body WL also tound that the smooth surlaLL sh ipe indicdteny neurons andi the Tough surfacL shapc indiLates gha ceTls Afiel plLkmg the neuron cells manually chosen by obsL-mg their ghapeg as desciibed abow vve confirmed that the puritied neurons can bc culuvated and can keep their elcctrophysiologiLdl funcuons on the Lhip even after the puntiLation procedure The results indicate tlie potcntial ot a nonlabe] noninvasivc on chip cell sorting procedure for neurons usmg micrograph imageg for an on ehip ultralngh speed camera baged imdigmg cett serter This Technique allows us to apply to various typL ofcells such as primary cells of cardiomyec}te and fibroblc]sts 3F1358 1ti-wtS;xlWfinAChRa)ajuadiAtiffofstMU Smgle moLecular rotational motion of membrane
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