Table 1.
Experimental data and BD modeling results.
| −CAP | +CAP | |||||||
|---|---|---|---|---|---|---|---|---|
| Cell line | AcGFP1 | AcGFP12 | AcGFP13 | AcGFP14 | AcGFP1 | AcGFP12 | AcGFP13 | AcGFP14 |
| MW (kDa) a | 29.155 | 57.586 | 87.705 | 115.337 | 29.155 | 57.586 | 87.705 | 115.337 |
| LOG10 (MW) | 1.4641 | 1.7603 | 1.9430 | 2.06197 | 1.4641 | 1.7603 | 1.9430 | 2.06197 |
| Mitochondrial length, diameter and radius determined using confocal laser scanning microscopy (CLSM) b | ||||||||
| Lmito (μm) | 3.42 (N = 55) | 3.42 (N = 30) | 3.42 (N = 37) | 3.42 (N = 68) | 3.40 (N = 31) | ND | ND | 3.42 (N = 26) |
| Dmito (μm) | 0.562 ± 0.025 (N = 26) | 0.524 ± 0.012 (N = 41) | 0.549 ± 0.020 (N = 33) | 0.525 ± 0.017 (N = 29) | 0.552 ± 0.015 (N = 24) | ND | ND | 0.546 ± 0.017 (N = 20) |
| Rmito (μm) | 0.281 | 0.262 | 0.275 | 0.263 | 0.276 | ND | ND | 0.273 |
| Vmito (μm3) | 0.848 | 0.738 | 0.813 | 0.743 | 0.819 | ND | ND | 0.801 |
| Vmito (l) | 0.848·10−15 | 0.738·10−15 | 0.813·10−15 | 0.743·10−15 | 0.819·10−15 | ND | ND | 0.801·10−15 |
| Mitochondrial diameter, radius and cristae per mitochondrion determined using electron microscopy (EM) c | ||||||||
| Dmito (μm) | 0.510 ± 0.019 (N = 52) | ND | ND | 0.504 ± 0.015 (N = 59) | 0.490 ± 0.017 (N = 51) | ND | ND | 0.494 ± 0.018 (N = 53) |
| Rmito (μm) | 0.255 | ND | ND | 0.252 | 0.245 | ND | ND | 0.247 |
| ncristae (1/μm) | 5.620 ± 0.155 (N = 244) | ND | ND | 5.462 ± 0.174 (N = 314) | 2.705 ± 0.130 (N = 295) | ND | ND | 3.50 ± 0.145 (N = 260) |
| ncristae/mito | 19 | ND | ND | 19 | 9 | ND | ND | 12 |
| Mono‐exponential fitting parameters of the fluorescence recovery after photobleaching (FRAP) curve d | ||||||||
| N | 76 | 30 | 37 | 68 | 40 | 24 | 26 | 33 |
| R 2 | 0.922 | 0.971 | 0.932 | 0.921 | 0.962 | 0.947 | 0.780 | 0.951 |
| y0 | −80.0 ± 5.85 | −0.677 ± 2.42 | 24.7 ± 2.29 | 30.7 ± 2.17 | 39.1 ± 1.20 | 30.9 ± 0.800 | 44.2 ± 0.920 | 47.5 ± 0.767 |
| Amono | 178 ± 5.84 | 98.9 ± 2.40 | 68.6 ± 2.26 | 60.5 ± 2.14 | 55.4 ± 1.17 | 45.1 ± 0.667 | 23.6 ± 0.763 | 39.0 ± 0.706 |
| Tmono (s) | 0.789 ± 0.015 | 1.51 ± 0.031 | 2.01 ± 0.065 | 2.05 ± 0.066 | 2.75 ± 0.099 | 8.76 ± 0.357 | 9.31 ± 0.883 | 5.43 ± 0.168 |
| Tmono‐corr (s) | 2.78 | |||||||
| F0 | 41.4 ± 1.61% | 37.7 ± 2.40% | 42.0 ± 1.80% | 43.6 ± 1.22% | 42.0 ± 2.60% | 22.7 ± 3.38% | 40.2 ± 4.51% | 42.6 ± 2.50% |
| F∞ | 98.8 ± 11.7% | 98.2 ± 4.82% | 93.3 ± 4.55% | 91.2 ± 4.31% | 94.5 ± 2.37% | 76.0 ± 1.47% | 67.8 ± 1.68% | 86.5 ± 1.47% |
| Fm | 0.979 | 0.972 | 0.885 | 0.844 | 0.904 | 0.689 | 0.462 | 0.765 |
| Parameters and results regarding the BD model e | ||||||||
| 1. Simulation parameters for Dsolvent prediction | ||||||||
| Identical for all simulations | Lmito = 3.42 μm; Rmito = 0.270 μm; SFRAP = 1.4 μm; CBA = 0.95 | |||||||
| ncristae | 19 | 19 | 19 | 19 | 9 | ND | ND | 12 |
| 2. Predicted Dsolvent | ||||||||
| Dsolvent (μm2/s) | 23.9 | 11.8 | 8.59 | 6.02 | 2.91 | NA | NA | NA |
| LOG10 (Dsolvent) | 1.378 | 1.072 | 0.9340 | 0.7796 | 0.464 | NA | NA | NA |
| 3. Computation of radius of gyration (RG), hydrodynamic radius (RH) and ηsolvent (cP) at 293 K | ||||||||
| MW (kDa) | 29.155 | 57.586 | 87.705 | 115.337 | 29.155 | 57.586 | 87.705 | 115.337 |
| Assuming that the FP has a compact conformation | ||||||||
| Radius of molecule R (Å) | 15 | 20 | 20 | 20 | 15 | NA | NA | NA |
| Length of molecule L (Å) | 40 | 60 | 60 | 60 | 40 | NA | NA | NA |
| RG (Å) | 15.5 | 20 | 20 | 20 | 15.5 | NA | NA | NA |
| RH (Å) | 20 | 23 | 23 | 23 | 20 | NA | NA | NA |
| ηsolvent (Young) | 3.32 | 5.36 | 6.40 | 8.34 | 27.3 | NA | NA | NA |
| ηsolvent (He–Niemeyer) | 3.69 | 5.88 | 7.53 | 10.3 | 30.3 | NA | NA | NA |
| ηsolvent (Tyn–Gusek) | 4.57 | 7.18 | 9.86 | 14.1 | 37.5 | NA | NA | NA |
| ηsolvent (Stokes–Einstein) | 4.49 | 7.91 | 10.9 | 15.5 | 36.9 | NA | NA | NA |
| Assuming that the FP has an extended conformation | ||||||||
| Radius of molecule R (Å) | 15 | 15 | 35 | 35 | 15 | NA | NA | NA |
| Length of molecule L (Å) | 40 | 130 | 247 | 349 | 40 | NA | NA | NA |
| RG (Å) | 15.5 | 38 | 73 | 102 | 15.5 | NA | NA | NA |
| RH (Å) | 20 | 30 | 61 | 75 | 20 | NA | NA | NA |
| ηsolvent (Young) | 3.32 | 5.36 | 6.40 | 8.34 | 27.3 | NA | NA | NA |
| ηsolvent (He–Niemeyer) | 3.69 | 4.27 | 3.94 | 4.55 | 30.3 | NA | NA | NA |
| ηsolvent (Tyn–Gusek) | 4.57 | 3.78 | 2.70 | 2.76 | 37.5 | NA | NA | NA |
| ηsolvent (Stokes–Einstein) | 4.49 | 6.06 | 4.10 | 4.75 | 36.9 | NA | NA | NA |
BD, Brownian dynamics; CAP, chloramphenicol; Dsolvent, solvent‐dependent diffusion constant; ηsolvent, solvent‐dependent viscosity; NA, not appropriate; ND, not determined; RG, radius of gyration; RH, hydrodynamic radius; SFRAP, size of the experimental FRAP region in the BD model; T, temperature; Tmono, experimental FRAP mono‐exponential time constant.
Molecular weight (MW) was calculated directly from the protein sequences given in Appendix Table S1 (excluding the N‐terminal mitochondrial targeting sequence) using the pI/Mw tool (web.expasy.org/compute_pi). One (1) Dalton (Da) equals 1 g/mol.
Confocal laser scanning microscopy (CLSM) analysis: The data are presented as mean ± SEM. N indicates the number of mitochondria analyzed in at least two independent experiments. Mitochondrial length (Lmito) and mitochondrial diameter (Dmito) were determined from the FRAP images by quantifying the frame width at half‐maximal height (FWHM; equaling 2∙Rmito) of a Gaussian curve fitted to a 1 pixel wide intensity profile perpendicular to the short and long axis of the mitochondrial filament (see Appendix Fig S2F). Mitochondrial volume (Vmito) was calculated using a cylindrical approximation: Vmito = π ·(Rmito)2·Lmito. One (1) μm3 equals 10−15 liter, 0.001 picoliter (1 picoliter = 10−12 liter) and 1,000 attoliter (1 attoliter = 10−18 liter). Average Vmito values equaled 0.786 ± 0.0540(SD) μm3 = 786 attoliter (−CAP) and 0.810 μm3 = 810 attoliter (+CAP).
Electron microscopy (EM) analysis: The data are presented as mean ± SEM. N indicates the number of mitochondria analyzed in two independent experiments. Dmito and Rmito indicate the mitochondrial diameter and radius, respectively.
Fitting of the fluorescence recovery after photobleaching (FRAP) curve: y = y0 + Amono[1−EXP(−t/Tmono)]. N indicates the number of mitochondria analyzed in at least three independent experiments. The coefficient of determination (R2) is used as a measure of the goodness of fit (the closer R2 is to a value of one, the closer the fit is to the data points). The parameter errors reflect the standard error (SE) value from the fit, as reported by the fitting software. F0 indicates the fluorescence signal at the start of the fluorescence recovery (expressed as % of the prebleach value). F∞ indicates the fluorescence signal to which the fluorescence signal recovers given by: y0 + Amono (expressed as % of the prebleach value). The error in F∞ was calculated by summation of the errors in y0 and Amono. Fm indicates the mobile fraction given by: [(F∞−F0+)/(Fi−F0)], with Fi being the prebleach fluorescence equaling 100%.
Mathematical modeling: One (1) Angstrom (Å) equals 1·10−10 m. CBA, cristae‐blocked area (fraction of total transecting area). See Results for further details.