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

Abstract

BackgroundCholesterol 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.ResultsWe 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%.ConclusionsThe 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.

Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

“…To further confirm that our method can assess diffusion-limited FLIP data, we performed FLIP experiments using the fluorescent cholesterol probe, BODIPY-cholesterol (BChol) (Additional file 1: Figure S4). BChol diffuses by at least a factor 30 slower than eGFP due to frequent partition into organelle membranes in cells [37]. This allowed us to clearly discern a depletion-zone being accurately described by the StrExp function.…”

Imaging approaches for analysis of cholesterol distribution and dynamics in the plasma membrane

“…exist. For better statistical reliability due to higher overall molecular brightness and photostability such experiments were therefore repeated for BChol (TopFluorcholesterol; not shown but see [129]). We found again a homogeneous B-map and obtained B- Based on these results we wondered whether we can find experimental methods for directly assessing the probe orientation in the highly curved PM of mammalian cells.…”

“…RICS takes all of these time scales into consideration and, thus, can be used to study the diffusional properties of molecules moving too fast to be analyzed with TICS. For example RICS was used to determine the diffusion constant of EGFP in solution which was found to be 89 µm 2 /s compared to D = 90 ± 5 µm 2 /s as determined by single point FCS[162].We studied the mobility and distribution of BChol in Chinese Hamster Ovarian (CHO) cells using multiphoton excitation laser scanning microscopy[163]. Here, TICS analysis of cellular membrane areas including the peripheral PM showed diffusion constants ranging from ~1 -1.5 µm 2 /s while RICS analysis found diffusion constants of ~1 -1.2 µm 2 /s in similar areas of the cell.…”

“…Using N&B analysis on multiphoton image stacks of DHE(Fig. 5C-E)or BChol[129] therefore allowed us to determine whether the heterogeneous staining pattern of these sterols in the PM is caused by putative laterally segregated sterol domains. In such a case, we would have expected the brightness (B-map) to contain structural features with values largely exceeding one, or patches caused by the brighter sterol domains.…”

Analysis of Cholesterol Trafficking with Fluorescent Probes

“…In photon-counting the main source of noise in detection is the statistical uncertainty in the number of detected photons in a fixed time interval [100]. Computational progress in denoising routines allowed us to post-process individual frames of MP sequences of DHE stained cells achieving dramatic improvement in image quality [101,102]. After denoising MP microscopy images, we could follow the dynamics of individual vesicles containing DHE in living CHO cells with a time-resolution of 4 sec [101].…”