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Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells
© Lund et al.; licensee BioMed Central Ltd. 2012
Received: 22 May 2012
Accepted: 19 September 2012
Published: 18 October 2012
Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport.
We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t > ~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%.
The mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport.
Intracellular organelles contain very different amounts of cholesterol, but our knowledge about how these differences are established and maintained during continuous inter-compartment membrane traffic is very limited [1, 2]. The dynamics of these processes can, in principle, be adequately addressed only by imaging-based approaches. This, however, is limited by the poor behavior of most fluorescent cholesterol analogs (reviewed in [3–6]). Ultraviolet (UV)-sensitive wide field (UV-WF) and multiphoton microscopy of the intrinsically fluorescent sterol dehydroergosterol (DHE) as a close analog of cholesterol and ergosterol has provided new insight into cellular sterol trafficking . By this approach, it has been demonstrated that vesicular, ATP-dependent, and non-vesicular, ATP-independent, transport contribute to targeting of DHE from the plasma membrane to the endocytic recycling compartment (ERC) in various mammalian cells, although the relative contribution of each uptake mode differs between cell types [8, 9]. Dynamics of intracellular vesicles containing DHE has been assessed by particle tracking of time-lapse sequences recorded on an UV-WF set-up. Calculation of the mean square displacement (MSD) from the trajectories as well as temporal image correlation spectroscopy (TICS) of images acquired at a multiphoton microscope indicated that diffusion of sterol vesicles in the cytoplasm is hindered, probably by cytoskeletal structures [7, 10]. The poor fluorescence properties of DHE (UV emission, rapid bleaching, and low quantum yield), however, limited the availability of sufficient image data sets and thereby precluded an in-depth quantitative analysis of vesicular sterol transport.
A promising new cholesterol probe for some applications is BODIPY-tagged cholesterol (BChol), in which the borondipyrromethene fluorophore is situated in cholesterol’s aliphatic side chain [11, 12]. We recently compared membrane partitioning and intracellular transport of DHE with that of BChol . We found that DHE has a higher affinity for the liquid-ordered (lo) phase than BChol, but BChol still preferred this phase over the liquid-disordered (ld) phase (see Additional file 1: Figure S1 for the structure of both fluorescent sterols compared to cholesterol). We also showed that both sterols are targeted to the ERC, a major cellular sterol pool, with identical kinetics . Uptake of both sterols from the cell surface was strongly reduced in baby hamster kidney (BHK) cells overexpressing a dominant-negative clathrin heavy chain, and co-internalization of BChol with fluorescent transferrin (Tf), a marker for this uptake pathway, could be demonstrated . Both observations suggest that a large portion of plasma membrane sterol is internalized by clathrin-dependent endocytosis in these cells. BChol has also been used to study sterol trafficking in sphingolipid storage diseases  and to analyze lateral and rotational sterol diffusion in model membranes .
Two-photon (2P) excitation microscopy offers several advantages over the one-photon fluorescence microscopy approaches used so far to visualize BChol in living cells. The intrinsic sectioning capability of this technique combined with negligible bleaching propensity outside the focal region and deeper specimen penetration due to the long-wavelength-excitation enabled us to perform long-term tracking studies of vesicles containing BChol in live Chinese hamster ovarian (CHO) cells. In addition, we used raster image correlation spectroscopy (RICS) and TICS to map the diffusional mobility of BChol over entire cells. Single particle tracking applied to sterol vesicles was combined with mathematical modeling of the trajectories to decipher the diffusion modes under various conditions. We found evidence for anomalous subdiffusion of vesicles containing BChol on short time scales (t < ~5 s) with a transition to superdiffusion on longer time scales. We demonstrate that the superdiffusive mode is caused by directed transport along both microtubules and actin filaments. Confined diffusion of sterol vesicles might increase the likelihood for local non-vesicular sterol exchange by collision of the vesicles with surrounding organelle membranes.
Results and discussion
Comparison of diffusivity and availability of BChol in various cellular regions
Dynamics of vesicles containing BChol reveal anomalous diffusion characteristics
Anomalous subdiffusion is frequently observed for various cargo molecules in cells and is generally ascribed to the dynamics of the cytoskeleton with typical values of α ranging from α ~ 0.65-0.75, [26, 36–38]. Thus, the anomalous exponent we find for sterol-containing vesicles is slightly smaller than that reported for other vesicle cargo, but since the anomalous exponent was found to vary between different cell types, the value we find here is still within the expected range. The average transport velocity of endosomes and other vesicles along microtubules has been estimated to range from 0.25 μm/s to 2 μm/s [39–43]. Thus, the average velocity we find here for BChol-containing vesicles in CHO cells (i.e., 5.52 x 10-3 μm/s) is roughly a hundred times slower, indicating that directed transport plays a minor role in vesicular sterol trafficking in CHO cells. In references [44, 45], the velocity of vesicle transport was determined by selectively measuring the velocity of vesicles being actively transported. Here, on the other hand, we determine the average mobility of a large population of vesicles containing a small but significant subpopulation of vesicles being actively transported through the cytoplasm. Thus, the much slower average velocity we find here is likely due to a different experimental approach. Visual inspection of the data showed that few vesicles were almost entirely subject to directed transport, while the majority of vesicles containing BChol showed no directional transport over the observation time; see Figure 4E for examples of trajectories. Thus, it is likely that two differently mobile vesicle populations transport BChol in CHO cells. Analysis of the end-to-end distance of vesicle trajectories indicates that the population of vesicles with predominantly active transport is very small (Additional file 1: Figure S10). In fact, the majority of vesicle trajectories have an end-to-end distance of less than 1 μm, and out of the 210 vesicles tracked at 37°C in control cells, only eight vesicles had an end-to-end distance of more than 1 μm, up to ~3.2 μm; (see Additional file 1: Figure S10). We cannot rule out that these vesicles carry out special functions in intracellular sterol trafficking. Long-range active transport of endocytic vesicles primarily occurs along microtubules while actin filaments have been shown to support local short-distance vesicle movements [44, 45]. Furthermore, as discussed above, the filaments of the cytoskeleton should impact or even determine the diffusion modality of sterol vesicles (normal versus anomalous diffusion). Therefore, we proceeded to uncover the effects of disrupting either the microtubule or the actin filaments. Microtubule disruption was induced by treating the cells for 1 h with 33 μM nocodazole while actin was disrupted by incubation with 20 μM cytochalasin D for 1 h . Figure 5A shows the MSD after microtubule disruption (red, n = 132 vesicles from 15 cells) and actin disruption (blue, n = 111vesicles from 15 cells) compared to the MSD of vesicles in the control cells (black). It is evident that both treatments significantly reduced vesicle mobility. A fit of Eq. 1 to both MSDs showed that neither microtubule nor actin disruption altered the degree of anomaly significantly. In both cases, we found α = 0.65, while in control cells with an intact cytoskeleton the anomalous exponent was α = 0.62. Interestingly, the lowered MSD upon disruption of microtubules or actin filaments was caused by both a decrease in diffusion constant and by a decreased velocity. After microtubule disruption, the anomalous diffusion constant was reduced by 39% and the velocity by 52%. Similarly, the anomalous diffusion constant and the velocity were reduced by 40% and 33%, respectively, after disruption of the actin filaments; see Figure 5E, 5F. We infer that sterol-containing vesicles are transported along both microtubules and actin filaments although disruption of microtubules seems to have a slightly larger effect on active transport.
Significance of the results for regulation of intracellular cholesterol transport
It is well documented that different intracellular organelles contain very different amounts of cholesterol. However, much remains to be understood about how this spatiotemporal distribution is maintained. Using two-photon microscopy of the fluorescent cholesterol analog BChol, we found that diffusion of non-vesicular sterol in cells is very heterogeneous with diffusion constants ranging from 10-2 μm2/s close to the ERC to 1.3 μm2/s towards the plasma membrane. This non-vesicular sterol diffusion is likely interrupted by transient binding of BChol monomers to slowly moving vesicles (see Figures 1, 2 and Additional file 1: Figures S5 to S8). By particle tracking analysis we demonstrate that transport of vesicles containing BChol is governed by anomalous subdiffusion on a time scale less than ~5 s with a transition to superdiffusive motion consistent with directed transport on longer time scales. Both processes require an intact actin and microtubule network. The slow transport velocity in the superdiffusive mode indicates that active transport of vesicles containing sterols is not a major factor in vesicular sterol transport. Rather, most sterol vesicles move in small confined areas. This has likely two consequences. First, the predominantly subdiffusive motion of sterol vesicles in small confinement areas make inter-organelle vesicular sterol transport including vesicle budding, shuttling to a target organelle and fusion of the vesicle with the target organelle an inefficient transport mode. Budded vesicles will likely stay close to the donor membrane for prolonged time, as predicted for subddiffusive motion . Second, anomalous subdiffusion of sterol vesicles could increase the time for local collisional sterol transfer to adjacent organelles. In fact, exchange of cholesterol monomers between donor and acceptor liposomes has been shown to be enhanced by frequent vesicle collisions . We propose that confined diffusion of sterol vesicles in small subcellular domains increases the likelihood of local non-vesicular sterol exchange, either due to collisions of vesicles with adjacent membranous structures or by transport via sterol carrier proteins. This of course assumes that the sterol carrier proteins may pass through the barrier that confines the vesicle. The proposed mechanism could be an elegant way of coupling vesicular and non-vesicular sterol transport modes in mammalian cells.
Cell culture and labeling
Chinese Hamster Ovary (CHO) cells were grown in bicarbonate-buffered Ham’s F12 medium supplemented with 5% heat-inactivated fetal calf serum and antibiotics as previously described . Fetal calf serum and cell culture medium were purchased from Gibco BRL (Life Technologies, Paisley, Scotland), while all other chemicals except BChol were from Sigma Chemical (St. Louis, MO). Two to three days prior to experiments, cells were seeded on microscope slide dishes. Lipid probes were stored in ethanol at a concentration of 5 mM under nitrogen at – 80°C until use. BChol was synthesized and loaded on methyl-β-cyclodextrin as described previously, affording a solution containing BChol/cyclodextrin complexes (BChol-CD) . Cells were labeled with BChol-CD for 2 min at 37°C, washed with buffer medium containing 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 20 mM HEPES (pH 7.4) and chased for 25 min at 37°C prior to imaging. Microtubule disruption was induced by treating the cells for 1 h with 33 μM nocodazole while actin was disrupted by incubation with 20 μM cytochalasin D for 1 h .
Two-photon excitation microscopy
Fluorescence time lapse measurements of BChol were performed using a custom-built setup constructed around an Olympus IX70 microscope. The objective used was a 60x water immersion objective with a NA of 1.2. The excitation light source was a femtosecond Ti:Sa laser (Broadband Mai Tai XF W25 with a 10 W Millennia pump laser, 80 MHz pulse-frequency, tunable excitation range 710–980 nm, Spectra Physics, Mountain View, CA), and the excitation wavelength used was 930 nm. To collect BChol’s emission, a 540 ± 25 nm filter was used (BrightLine HC). The light was detected by a photomultiplier tube (Hamamatsu H7422P-40) operated in the photon counting mode. The data were acquired using simFCS software developed by the Laboratory for Fluorescence Dynamics, University of California, Irvine.
Image analysis was carried out using ImageJ (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/ij) or MatLab (MathWorks Inc., USA). For spatial registration of image stacks, “StackReg” developed by Dr. Thevenaz at the Biomedical Imaging group, EPFL, Lausanne, Switzerland was used . Prior to multiple particle tracking, the images were processed to enhance the signal to noise ratio by the PureDenoise algorithm [57, 58] for ImageJ and a 0.5 Gaussian filter applied to the image sequences. After removal of residual background, the image was processed with the spot enhancing Mexican hat filter implemented in the SpotTracker plugin for ImageJ by Daniel Sage .
Multiple particle tracking
where N is the number of frames, h is the time step between subsequent frames, and Δt is the time lag corresponding to n frames.
Model for combined subdiffusion and active transport
where CI is the upper 95% confidence interval and is the mean fitted value.
Temporal image correlation spectroscopy (TICS)
where angular brackets denote spatial and temporal averaging and τ is the timelag between subsequent images. TICS analysis was performed using simFCS analysis software developed by the Laboratory for Fluorescence Dynamics, University of California, Irvine.
Number & Brightness (N&B) analysis
N is directly proportional to the number of fluorescent particles, n, in a given pixel location.
Raster image correlation spectroscopy
where γ is a factor describing the geometry of the laser beam. In raster scanning mode, the correlation of the succeeding images is related on three different time scales. Along the horizontal direction the pixels are separated by the pixel dwell time (microseconds) while along the vertical direction the images are correlated by the line time (i.e., the time it takes to record the intensity of every pixel in a line plus the time it takes for the microscope to move to the next line). The line time is typically in milliseconds. Finally, the images are correlated by the time between two succeeding frames (seconds). Thus, RICS may be employed to measure the dynamics of particles with a wide range of diffusion coefficients. Diffusion maps and diffusion coefficients determined by RICS were calculated using simFCS software developed by the Laboratory for Fluorescence Dynamics, University of California, Irvine. [16, 17].
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