Supplemental data

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The associated Goehring is a time lapse of an imaging experiment corresponding to that depicted in Fig. 4a. Pyruvate-induced p oscillations in the presence of 2.8mM glucose. INS-1 832/13 cells were preincubated in buffer A in the presence of 2.8mM glucose. Where indicated 10mM Na-pyruvate was added, followed by 640ng/ml oligomycin and KCl to 25mM. The time-course is accelerated 40-fold: the 30s video corresponds to an actual duration of 20min.
TMRM and PMPI Simulations
The Excel spreadsheet originally published (and currently available) as a supplementary file to Nicholls, D.G. (2006) Simultaneous Monitoring of Ionophore- and Inhibitor-mediated Plasma and Mitochondrial Membrane Potential Changes in Cultured Neurons. J Biol. Chem. 281:14864-14874 allows the approximate relationship between dynamic changes in ∆ψm and ∆ψp and the fluorescence of PMPI and TMRM to be predicted for a wide range of conditions. For the present paper, starting conditions appropriate for INS-1 832/13 cells under the conditions relevant to this paper were employed.

Mitochondrial volume fraction in the cell (4.6%), external TMRM concentration (100nM, quench mode), rate constants for equilibration of TMRM and PMPI across the plasma membrane (0.001sec-1 and 0.1sec-1 respectively), quench limit at which TMRM begins to aggregate in the matrix (25µM). Initial values under low substrate conditions of 80mV for ∆ψp and 110mV for ∆ψm were assumed.

Figure S1: Simulated PMPI and TMRM fluorescence of cells during a simulated addition of 10mM pyruvate to depolarize p from 72 to 62mV followed by three brief depolarizing spikes of respectively 8, 16 and 32mV and finally addition of KCl to 25mM to lower p to 40mV. A constant m of 110mV (50) was assumed in this simulation to confirm that changes in p do not measurably affect the TMRM response over this short time frame.

Figure S2: Sensitivity of whole-cell TMRM signals to changes in m. The whole cell TMRM response was simulated for an initial hyperpolarization of m following addition of glucose or pyruvate. Starting conditions were 80mV for p and 110mV for m. The graph was used to calibrate Fig. 6.

Figure S3: Sensitivity of cytoplasmic TMRM signals to changes in m. The cytoplasmic TMRM response (mitochondria-poor nuclear region) is a more sensitive means of detecting the TMRM response to small changes in m. Values appropriate to Fig. 7 were taken. Starting conditions: p 75mV, m 115mV (0.4mM pyruvate) or p 66V, m 124mV (10mM pyruvate). Values were used to calibrate Fig. 7.

Figure S4: Extent to which a PMPI depolarizing spike is predicted to influence the TMRM signal. Because of the small size of the TMRM transient associated with a plasma membrane depolarizing (and [Ca2+]c elevating) spike, it is important to determine whether the brief change in p will affect the TMRM distribution. Conditions: p 66mV, m124mV, 32mV depolarizing p spike.

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