Open Access Research article

Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

Frederik W Lund1, Michael A Lomholt2, Lukasz M Solanko1, Robert Bittman3 and Daniel Wüstner1*

Author Affiliations

1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230, Denmark

2 MEMPHYS Center for Biomembrane Physics, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, DK-5230, Denmark

3 Department of Chemistry and Biochemistry, Queens College, The City University of New York, Flushing, NY, 11367, USA

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BMC Biophysics 2012, 5:20  doi:10.1186/2046-1682-5-20

Published: 18 October 2012

Additional files

Additional file 1:

Figure S1. Chemical structure of BODIPY-cholesterol (BChol) and dehydroergosterol (DHE). A, structure of BODIPY-cholesterol with the fluorophore (green, with light green underlay to highlight the fluorescent group) at carbon 24 of the sterol side chain. B, structure of DHE. Differences to cholesterol are indicated in green and red; i.e., an extra methyl group and double bond in the alkyl side chain and two additional double bonds in the steroid ring system. Only one additional double bond in the second ring distinguishes DHE from ergosterol (indicated in red). The three conjugated double bonds in the steroid ring (indicated in light blue) give DHE its slight fluorescence. Figure S2: Number and brightness analysis in HeLa cells labeled with BChol. Cells were repeatedly imaged on a 2P microscope with a pixel dwell time of 10 μsec and 100 frames in total. The apparent number (N) and brightness (B) of the BChol molecules were calculated from the intensity fluctuations per pixel positions, as described in the main text. A, the first 5 frames of the acquired sequence; B, N-map; C, B-map; D, intensity variance, (i.e., the second moment (2nd mt.) of the photon count distribution, as it enters in Eq. 6 in the main text. In addition, we calculated higher moments of the photon counting histogram using a plugin to Image J, kindly provided by Dr. Jay Unruh (Stowers Inst. for Microscopy, Kansas, USA). The 3rd, 4th and 5th moment were scaled identically using a FIRE LUT in the range of 0-950, and showed identical features. See text for further explanations. Figure S3: Photobleaching of BChol in CHO cells imaged with a wide field (WF) microscope (A) or a two-photon imaging system (B). In both cases 600 frames were acquired. Arrowheads in A indicate the BChol-labeled plasma membrane. The large central region in A is mostly slowly bleaching autofluorescence. C, quantification of BChol’s intensity for the WF sequence (light grey line, data and darkgrey line, monoexp. fit) and of the 2P sequence (black line). D, quantification of BChol’s intensity for another WF sequence with higher illumination intensity (grey symbols, data and grey line, fit); and the 2P sequence of Fig. 2 in the main text. Figure S4: Result of the reaction-diffusion simulation with diffusing particles shown in blue and binders shown in red. Figure S5: Analysis of the simulated data. Panel A shows the first frame of the simulated image stack. Panels B, C and D shows the diffusion map, the number of particles per pixel and the brightness of each pixel, respectively. By comparison with Figure 2 (in the main text) we can see that the simulation of Brownian diffusion with transient binding faithfully resembles the experimental data. The diffusion constant is lower in the areas with binding macromolecular structures, the majority of diffusing molecules are found in the area with binders and the brightness map shows a low degree of aggregation. Figure S6: Distribution of diffusing particles with and without exchange between the binders and the free particles with and without binding, A and D, respectively. B and E are maps of the apparent number of molecules (N), and C and F show maps of the apparent brightness (B). Figure S7: Simulation with increased mobility of the free particles. The binding and dissociation rate constants were kept at kon = 0.23 s-1 and koff = 0.5 s-1, while the diffusion coefficient of the particles (simulating sterol monomers was increased from 1 μm2/s to 300 μm2/s). Distribution of diffusing particles is shown in A, while B and C are maps of the apparent number of molecules (N), and the apparent brightness (B), respectively. Figure S8: Simulation with stronger binding: All settings were as described in Fig. S5 except for the unbinding rate constant, koff, which was reduced from 0.5 s-1 to 0.05 s-1. Here 5573 particles were bound to 100 binders compared to 278 particles in the previous experiment. Therefore, the bound particles (or vesicles) become more visible Distribution of diffusing particles is in A, while B and C are maps of the apparent number of molecules (N), and the apparent brightness (B), respectively. Figure S9: 2P time-lapse sequence showing shuttling of a small vesicle (in green) from a donor to an acceptor vesicle (from bottom to top of the image frames). The lower donor vesicle becomes first elongated, followed by formation and fission of a small vesicle which shuttles to and fuses with a stationary acceptor vesicle. Images in this example sequence were acquired every 4 s. The size of the frames is 1.73 x 2.59 μm. Figure S10: From the end-to-end distance of a trajectory one may estimate the degree of directed motion. That is, if a vesicle is subject to directed motion it is expected to traverse further away from the starting point than a vesicle which is not subject to directed motion. Panels A-C show the distribution of end-to-end distances in control cells (A) and in cells with either disrupted microtubules (B) or actin filaments (C). Also, shown in panel D is the mean end-to-end distance at each condition which further emphasize the decreased vesicle mobility upon disruption of the cytoskeletal filaments.

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