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. 2007 Dec 1;93(11):4031-40.
doi: 10.1529/biophysj.107.109629. Epub 2007 Aug 17.

Live-cell transforms between Ca2+ transients and FRET responses for a troponin-C-based Ca2+ sensor

Lai Hock Tay  1 , Oliver Griesbeck, David T Yue
Affiliations

Live-cell transforms between Ca2+ transients and FRET responses for a troponin-C-based Ca2+ sensor

Lai Hock Tay et al. Biophys J. .

Abstract

Genetically encoded Ca(2+) sensors promise sustained in vivo detection of Ca(2+) signals. However, these sensors are sometimes challenged by inconsistent performance and slow/uncertain kinetic responsiveness. The former challenge may arise because most sensors employ calmodulin (CaM) as the Ca(2+)-sensing module, such that interference via endogenous CaM may result. One class of sensors that could minimize this concern utilizes troponin C as the Ca(2+) sensor. Here, we therefore probed the reliability and kinetics of one representative of this class (cyan fluorescence protein/yellow fluorescent protein-fluorescence resonance energy transfer (FRET) sensor TN-L15) within cardiac ventricular myocytes. These cells furnished a pertinent live-cell test environment, given substantial endogenous CaM levels and fast reproducible Ca(2+) transients for testing sensor kinetics. TN-L15 was virally expressed within myocytes, and Indo-1 acutely loaded to monitor "true" Ca(2+) transients. This configuration permitted independent and simultaneous detection of TN-L15 and Indo-1 signals within individual cells. The relation between TN-L15 FRET responses and Indo-1 Ca(2+) transients appeared reproducible, though FRET signals were delayed compared to Ca(2+) transients. Nonetheless, a three-state mechanism sufficed to map between measured Ca(2+) transients and actual TN-L15 outputs. Overall, reproducibility of TN-L15 dynamics, coupled with algorithmic transforms between FRET and Ca(2+) signals, renders these sensors promising for quantitative estimation of Ca(2+) dynamics in vivo.

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Figures

FIGURE 1
FIGURE 1
Distinctive sensor spectra permitted independent detection of Indo-1 and TN-L15 in the same cells. (A) Data points represent HEK293 cells, loaded with Indo-1 and lacking TN-L15, that were imaged under CFP (bottom panel) and FRET (top panel) cubes. The gray box represents the working range of TN-L15 (y axis) and Indo-1 (x axis) signals in cardiac myocytes between resting and contracting conditions. Line of data regression falls near zero. (B) Data points represent HEK293 cells, expressing TN-L15 and lacking Indo-1, that were imaged under Indo405 (top panel) and Indo485 (bottom panel) cubes. Format similar to panel A. (C) Myocytes loaded with Indo-1 and lacking TN-L15, were stimulated to contract and imaged under Indo405, Indo485, CFP, and FRET cubes. Average of 10 traces for each cube were shown. (D) Myocytes expressing TN-L15 and lacking Indo-1 were imaged. Procedure same as panel C.
FIGURE 2
FIGURE 2
Simultaneous detection of TN-L15 and Indo-1 signals from single contractions in cardiac myocytes. (A) Cardiac myocytes expressing TN-L15 were simultaneously loaded with Indo-1 and imaged under Indo405, Indo485, CFP, and FRET cubes. (B) Exemplar traces showing the simultaneous rise of Indo405 signal (SIndo405) (top panel) and the fall of Indo485 signal (SIndo485) (second panel) when myocytes were field-stimulated at time 0. The Indo-1 ratio, RIndo (third panel) (RIndo = SIndo405/SIndo485) was converted into free intracellular Ca2+ concentration, [Ca2+] (bottom panel), according to Eq. 1. Average of 10 traces for each cube were shown. (C) Exemplar traces showing the simultaneous rise of FRET signal (SFRET) (top panel) and fall of CFP signal (SCFP) (second panel) at time 0. The TN-L15 ratio, RTNL,expt = SFRET/SCFP (third panel, black). RTNL,sim (third panel, gray) was obtained using our “forward transform”. The average percentage error between the RTNL,expt and RTNL,sim (error normalized to the peak of RTNL,sim) (n = 7) was plotted with the standard deviations shown as thin black lines (bottom panel).
FIGURE 3
FIGURE 3
TN-L15 mechanism for mapping between [Ca2+] and RTNL, and the steady-state profile of this mapping function. (A) Three-state mechanism mapping [Ca2+] into RTNL, with parameters shown in Table 2. (B) Model prediction of steady-state TN-L15 response. The black curve is the TN-L15 steady-state response predicted using our model parameters according to Eq. 5. The black data points were obtained from Heim et al. (12) but with the [Ca2+] divided by TN-L15 α (estimated to be 1.47). The scaling was necessary because the data points were plotted using the apparent TN-L15 Kd, which is the product of TN-L15 α and the true TN-L15 Kd, whereas the black curve was plotted using our true TN-L15 Kd. The results of scaling were the black data points plotted in Fig. 3 B.
FIGURE 4
FIGURE 4
Simultaneous detection of TN-L15 and Indo-1 signals from fused double contractions in cardiac myocytes. Format as in Fig. 2. Average of six traces for each cube were shown.
FIGURE 5
FIGURE 5
TN-L15 model back-calculates rapid Ca2+ transients based only on the slow TN-L15 response. (A) The TN-L15 ratio, RTNL,expt (top panel, black) for single-pulse contraction was fitted with a smooth function to give RTNL,fit (top panel, gray). [Ca2+] was calculated from RTNL,fit using our “backward transform” to give [Ca2+]back (t) (middle panel, gray). Indo-predicted [Ca2+] (middle panel, noisy) is shown for comparison. The average percentage error between [Ca2+]back (t) and Indo-predicted [Ca2+] (error normalized to the peak of [Ca2+]back) (n = 7) was plotted with the standard deviations shown as thin black lines (bottom panel). (B) Back-calculation for double-pulse contractions (format same as panel A).
FIGURE 6
FIGURE 6
Hypothesized full-bore Ca2+ response of TN-L15 Ca2+ sensor. (A) FRET ratio (RTNL) change as a function of steady-state Ca2+ concentration, reproduced from fit in Fig. 3 B. (B) Schematic of Ca2+ occupancy of key N-terminal lobe Ca2+ site as a function of Ca2+ concentration. This occupancy is the “rate-limiting step” that specifies FRET changes; hence, the shape of this curve corresponds closely to that in panel A. (C) Hypothetical Ca2+ occupancy of C-terminal lobe Ca2+ sites as a function of Ca2+ concentration. This occupancy might be obligatory for subsequent FRET changes controlled by N-terminal lobe sites, but C-terminal lobe occupancy may by itself be insufficient to drive FRET changes.
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