Search

Low-density lipoprotein (LDL) deposition, aggregation and retention in the endothelial sub-intima are critical initiating events during atherosclerosis. Macrophages digest aggregated LDL (agLDL) through a process called exophagy. High-density lipoprotein (HDL) plays an atheroprotective role, but studies attempting to exploit it therapeutically have been unsuccessful, highlighting gaps in our current understanding of HDL function. Here, we characterized the role of HDL during exophagy of agLDL. We find that atherosclerotic plaque macrophages contact agLDL and form an extracellular digestive compartment similar to that observed in vitro. During macrophage catabolism of agLDL in vitro, levels of free cholesterol in the agLDL are increased. HDL can extract free cholesterol directly from this agLDL and inhibit macrophage foam cell formation. Cholesterol-balanced hydroxypropyl-β-cyclodextrin similarly reduced macrophage cholesterol uptake and foam cell formation. Finally, we show that HDL can directly extract free cholesterol, but not cholesterol esters, from agLDL in the absence of cells. Together, these results suggest that the actions of HDL can directly extract free cholesterol from agLDL during catabolism, and provide a new context in which to view the complex relationship between HDL and atherosclerosis. [ABSTRACT FROM AUTHOR]

Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy is to use intrinsically fluorescent sterols, as dehydroergosterol (DHE), having minimal chemical alteration compared to cholesterol but giving low fluorescence signals in the UV region of the spectrum. Alternatively, one can use dye-tagged cholesterol analogs and in particular BODIPY-cholesterol (BChol), whose synthesis and initial characterization was pioneered by Robert Bittman. Here, we give a general overview of the properties and applications but also limitations of BODIPY-tagged cholesterol probes for analyzing intracellular cholesterol trafficking. We describe our own experiences and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we highlight the suitability of BChol for determining transport of lipoprotein-derived sterol using electron microscopy (EM) and show that this approach ideally complements fluorescence studies. [ABSTRACT FROM AUTHOR]

The endocytic pathway is a complex network of highly dynamic organelles, which has been traditionally studied by quantitative fluorescence microscopy. The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error-prone. We developed SpatTrack, an open source, platform-independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. SpatTrack performs 2D particle tracking, trajectory analysis and fitting of diffusion models to the calculated mean square displacement. It allows for spatial analysis of detected vesicle patterns including calculation of the radial distribution function and particle-based colocalization. Importantly, all analysis tools are supported by Monte Carlo simulations of synthetic images. This allows the user to assess the reliability of the analysis and to study alternative scenarios. We demonstrate the functionality of SpatTrack by performing a detailed imaging study of internalized fluorescence-tagged Niemann Pick C2 ( NPC2) protein in human disease fibroblasts. Using SpatTrack, we show that NPC2 rescued the cholesterol-storage phenotype from a subpopulation of late endosomes/lysosomes ( LE/ LYSs). This was paralleled by repositioning and active transport of NPC2-containing vesicles to the cell surface. The potential of SpatTrack for other applications in intracellular transport studies will be discussed. [ABSTRACT FROM AUTHOR]

Background: 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. Results: 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 D&#x03Bl; = 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%. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry (HDX-MS) has become a valuable tool in studies of protein dynamics and protein interactions. Isotopic exchange is typically initiated by diluting a protein solution into deuterated buffer at physiological conditions. For MS analysis, the exchange reaction is quenched by acidification and cooling and the labeled protein (or a digest thereof) is analyzed by mass spectrometry. An inevitable deuterium loss occurs during quench conditions (i.e., back-exchange). The primary structure of the peptide influences the back-exchange rate due to steric hindrance by bulky side groups and by inductive and charge effects. Here we show that the back-exchange in histidine repeats (His6) at HDX-MS quench conditions is complete within a few seconds using either acetic acid or formic acid in the quench solution, while aspartic acid repeats (Asp6) retain deuterons for several minutes using formic acid. We employ electron transfer dissociation to obtain residue-specific deuterium levels of the Asp repeat in K2D6IIKIIK using a hybrid linear ion trap-Orbitrap mass spectrometer. Our results show an unexpected uneven distribution of deuterium in the Asp repeat. The implication of the rapid back-exchange of His repeats for HDX-MS protein hydrogen exchange studies is discussed. We also discuss the implications of retained deuterons in the Asp repeat of K2D6IIKIIK when this peptide is used as a probe for the occurrence of hydrogen scrambling. [Copyright &y& Elsevier]

The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2's known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface. [ABSTRACT FROM AUTHOR]

Cholesterol is an important lipid component of the plasma membrane (PM) of mammalian cells, where it is involved in control of many physiological processes, such as endocytosis, cell migration, cell signalling and surface ruffling. In an attempt to explain these functions of cholesterol, several models have been put forward about cholesterol’s lateral and transbilayer organization in the PM. In this article, we review imaging techniques developed over the last two decades for assessing the distribution and dynamics of cholesterol in the PM of mammalian cells. Particular focus is on fluorescence techniques to study the lateral and inter-leaflet distribution of suitable cholesterol analogues in the PM of living cells. We describe also several methods for determining lateral cholesterol dynamics in the PM including fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT) and spot variation FCS coupled to stimulated emission depletion (STED) microscopy. For proper interpretation of such measurements, we provide some background in probe photophysics and diffusion phenomena occurring in cell membranes. In particular, we show the equivalence of the reaction-diffusion approach, as used in FRAP and FCS, and continuous time random walk (CTRW) models, as often invoked in SPT studies. We also discuss mass spectrometry (MS) based imaging of cholesterol in the PM of fixed cells and compare this method with fluorescence imaging of sterols. We conclude that evidence from many experimental techniques converges towards a model of a homogeneous distribution of cholesterol with largely free and unhindered diffusion in both leaflets of the PM. [ABSTRACT FROM AUTHOR]

Monocyte-derived cells use an extracellular, acidic, lytic compartment (a lysosomal synapse) for initial degradation of large objects or species bound to the extracellular matrix. Akin to osteoclast degradation of bone, extracellular catabolism is used by macrophages to degrade aggregates of low density lipoprotein (LDL) similar to those encountered during atherogenesis. However, unlike osteoclast catabolism, the lysosomal synapse is a highly dynamic and intricate structure. In this study, we use high resolution three dimensional imaging to visualize compartments formed by macrophages to catabolize aggregated LDL. We show that these compartments are topologically complex, have a convoluted structure and contain sub- regions that are acidified. These sub-regions are characterized by a close apposition of the macrophage plasma membrane and aggregates of LDL that are still connected to the extracellular space. Compartment formation is dependent on local actin polymerization. However, once formed, compartments are able to maintain a pH gradient when actin is depolymerized. These observations explain how compartments are able to maintain a proton gradient while remaining outside the boundaries of the plasma membrane. [ABSTRACT FROM AUTHOR]

Background: Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results: We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB’s). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments. Conclusions: We present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport. [ABSTRACT FROM AUTHOR]

Niemann-Pick disease type C2 is a lipid storage disorder in which mutations in the NPC2 protein cause accumulation of lipoprotein-derived cholesterol in late endosomes and lysosomes (LE/LYSs). Whether cholesterol delivered by other means to NPC2 deficient cells also accumulates in LE/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous diffusion in disease cells (D ∼ 4.6∙10 −4  μm 2 /sec; α∼0.76), a small pool of sterol could exchange rapidly with D ∼ 3 μm 2 /s between LE/LYSs, as shown by fluorescence recovery after photobleaching (FRAP). By quantitative lipid mass spectrometry we found that esterification of 13 C-labeled cholesterol but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM and LE/LYSs depends on a functional NPC2 protein. NPC2 likely acts inside LE/LYSs from where it increases non-vesicular sterol exchange with other organelles. [ABSTRACT FROM AUTHOR]