Temporal Partitioning of the Yeast Cellular Network - Systems Biology of Metabolic and Signaling Networks - page 329

transcription (Fig. 12.5 ) indicates mitochondrial activity must be tightly integrated
with the oscillatory dynamics.
The high amplitude excursions of dissolved O 2 (80-170 M) during the respira-

tory oscillations are accompanied by an in-phase modulation of NAD(P)H directly
as continuously monitored in the fermenter by fluorimetry (Murray et al. 1999 ;
Lloyd et al. 2002b ). This key indicator of intracellular redox state (Chance
et al. 2005 ) is pivotal at the core of the entire cellular network (Lloyd 2003 ;
Lloyd et al. 2003 ; Lloyd and Murray 2005 , 2006 ; Murray and Lloyd 2006 ). Electron
micrographs of thin sections of yeasts rapidly fixed at different stages of the 40-min
cycle showed marked ultra-structural changes in the mitochondria (Lloyd
et al. 2002a , b ; Sasidharan et al. 2012 ). The extremes of conformational state
correspond to those originally described for liver mitochondria (Hackenbrock
1966 , 1968 ) as “orthodox” or “condensed”. In the former, a relatively large matrix
volume with the inner membrane is closely opposed to the outer membrane: this
state was identified at high levels of dissolved O 2 (when respiration rate was low).
In the condensed form, the cristae became more clearly defined as the inter-
membrane compartment was larger. This corresponds to the energised state
(Chance and Williams 1955 ). It has been understood for many years that massive
and rapid changes in ion concentrations between the two mitochondrial
compartments and the cytosol accompany or drive these changes and
protonophores and ionophores that uncouple mitochondrial energy conservation
from electron transport by collapse of inner membrane electrochemical membrane
potential perturb mitochondrial structure (Hackenbrock 1968 ; Mitchell and Moyle
1969 ). The determination of total mitochondrial content of cytochromes b, c 1 ,c
and aa 3 as measured in difference spectra at 77K showed that any changes were
below the levels of detectability. However, the physiological redox states of
cytochromes c and aa 3 in vivo indicated that high respiration was associated with
elevated reduction of these two redox components. Effects of two protonophores
(m-chlorocarbonylcyanide phenylhydrazone, CCCP, and 5-chloro-t-butyl-2 0 -
chloro-4 0 -nitrosalicylanilide, S-13) (Fig. 12.6a, b ) were dramatic and similar.
The well-established acceleration of respiratory rates on addition of uncouplers
rapidly drives down the dissolved O 2 in the fermenter. At increased concentrations,
the subsequent respiratory cycle was prolonged, and more than five cycles were
required for recovery to the normal cycle time. At higher concentrations both
uncouplers produced very interesting effects that provided new insights (Lloyd
2003 ). For instance, at 10

μ

M CCCP the uncoupling effect was more evident and
the dissolved O 2 remains low for more than 5 h, during which no oscillation was
observed. Recovery to normal amplitudes required more than 20 h, although the
respiratory oscillations were restored before this, albeit with greatly diminished
amplitude. Another very significant observation was that uncoupler treatment gives
a lasting complex waveform where the 40-min oscillation had an 8-h envelope
waveform, most likely stemming from cell-division cycle dynamics (Fig. 12.6c, d ).
Thus, interference with mitochondrial energy generation can induce an alignment
of cell division cycle controls with ultradian clock control (Fig. 12.7 ). Further

μ