BacFlash signals acid-resistance gene expression in bacteria.

Intracellular pH (pHi) homeostasis is crucial for cellular functions and signal transduction across all kingdoms of life. In particular, bacterial pHi homeostasis is important for physiology, ecology, and pathogenesis. Here we report an exquisite bacterial acid-resistance (AR) mechanism in which proton leak elicits a pre-emptive AR response. A single bacterial cell undergoes quantal electrochemical excitation, termed "BacFlash", which consists of membrane depolarization, transient pHi rise, and bursting production of reactive oxygen species. BacFlash ignition is dictated by acid stress in the form of proton leak across the plasma membrane and the rate of BacFlash occurrence is reversely correlated with the pHi buffering capacity. Through genome-wide screening, we further identify the ATP synthase Fo complex subunit a as the putative proton sensor for BacFlash biogenesis. Importantly, persistent BacFlash hyperactivity activates transcription of a panel of key AR genes and predisposes the cells to survive imminent extreme acid stress. These findings demonstrate a prototypical coupling between electrochemical excitation and nucleoid gene expression in prokaryotes.

Temperature dependence of mitoflash biogenesis in cardiac mitochondria.

Mitochondrial flashes (mitoflashes) represent fundamental biochemical and biophysical dynamics of the organelle, involving sudden depolarization of mitochondrial membrane potential (ΔΨm), bursting production of reactive oxygen species (ROS), and accelerated extrusion of matrix protons. Here we investigated temperature dependence of mitoflash biogenesis as well as ΔΨm oscillations, a subset of which overlapping with mitoflashes, in both cardiac myocytes and isolated respiring cardiac mitochondria. Unexpectedly, we found that mitoflash biogenesis was essentially temperature-independent in intact cardiac myocytes, evidenced by the constancy of frequency as well as amplitude and rise speed over 5 °C-40 °C. Moderate temperature dependence was found in single mitochondria charged by respiratory substrates, where mitoflash frequency was decreased over 5 °C-20 °C with Q10 of 0.74 for Complex I substrates and 0.83 for Complex II substrate. In contrast, ΔΨm oscillation frequency displayed a negative temperature dependence at 5 °C-20 °C with Q10 of 0.82 in intact cells, but a positive temperature dependence at 25 °C - 40 °C with Q10 of 1.62 in isolated mitochondria charged with either Complex I or Complex II substrates. Moreover, the recovery speed of individual mitoflashes exhibited mild temperature dependence (Q10 = 1.14-1.22). These results suggest a temperature compensation of mitoflash frequency at both the mitochondrial and extra-organelle levels, and underscore that mitoflashes and ΔΨm oscillations are related but distinctly different mitochondrial functional dynamics.

Mitochondrial flashes regulate ATP homeostasis in the heart.

The maintenance of a constant ATP level ('set-point') is a vital homeostatic function shared by eukaryotic cells. In particular, mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload. However, the exact mechanisms underlying this regulation of ATP homeostasis remain elusive. Here we show mitochondrial flashes (mitoflashes), recently discovered dynamic activity of mitochondria, play an essential role for the auto-regulation of ATP set-point in the heart. Specifically, mitoflashes negatively regulate ATP production in isolated respiring mitochondria and, their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes. Moreover, manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point. Mechanistically, the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation. These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart.

Regulation of Mitoflash Biogenesis and Signaling by Mitochondrial Dynamics.

Mitochondria are highly dynamic organelles undergoing constant network reorganization and exhibiting stochastic signaling events in the form of mitochondrial flashes (mitoflashes). Here we investigate whether and how mitochondrial network dynamics regulate mitoflash biogenesis and signaling. We found that mitoflash frequency was largely invariant when network fragmentized or redistributed in the absence of mitofusin (Mfn) 1, Mfn2, or Kif5b. However, Opa1 deficiency decreased spontaneous mitoflash frequency due to superimposing changes in respiratory function, whereas mitoflash response to non-metabolic stimulation was unchanged despite network fragmentation. In Drp1- or Mff-deficient cells whose mitochondria hyperfused into a single whole-cell reticulum, the frequency of mitoflashes of regular amplitude and duration was again unaltered, although brief and low-amplitude "miniflashes" emerged because of improved detection ability. As the network reorganized, however, the signal mass of mitoflash signaling was dynamically regulated in accordance with the degree of network connectivity. These findings demonstrate a novel functional role of mitochondrial network dynamics and uncover a magnitude- rather than frequency-modulatory mechanism in the regulation of mitoflash signaling. In addition, our data support a stochastic trigger model for the ignition of mitoflashes.