Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Incorporation of fluorophore–cholesterol conjugates into liposomal and mycobacterial membranes

Fluorescently-labeled steroids that emit intense blue light in nonpolar solvent (λ_em (CH₂Cl₂) ≈ 440 nm, Φ_F = 0.70) were prepared by treating cholesteryl chloroformate with 4-amino-1,8-naphthalimides. The lipid portion of the conjugates embeds into liposomal membrane bilayers in minutes, leaving the fluorophore exposed to the external aqueous environment. This causes a 40-nm red-shift in λ_em and significant quenching. DFT optimizations predict the conjugates to be about 30 Å long when fully extended, but rotation about the linker group can bring the compounds into an ‘L’-shape. Such a conformation would allow the cholesteryl anchor to remain parallel to the acyl chains of a membrane while the fluorescent group resides in the interfacial region, instead of extending beyond it. When incubated with Mycobacterium smegmatis mc2 155, a bacterial species known to use natural cholesterol, the labeled steroids support growth and can be found localized in the membrane fraction of the cells using HPLC. These findings demonstrate stable integration of fluorescent cholesterols into bacterial membranes in vivo, indicating that these compounds may be useful for evaluating cholesterol uptake in prokaryotic organisms.     

Protoplasts as tools in cell biology

Studies of plant protoplasts using both fluorescent dyes and electron dense probes have demonstrated endocytosis in plants. Ultrastructural work with soybean protoplasts using cationized ferritin (CF) revealed an endocytotic pathway from coated pits at the plasma membrane to coated vesicles, the partially coated reticulum, Golgi bodies, multivesicular bodies and finally the vacuole. Endocytosis may be responsible for membrane retrieval from the cell surface or degradation of elicitors or toxins during host-pathogen interactions. Immunofluorescence studies of dividing plant protoplasts have provided new information about the preprophase band (PPB) of microtubules and the shape of spindles. Studies of PPBs in soybean protoplast cultures permitted detailed examination of PPB development and an assessment of the usefulness of the PPB index for identifying morphogenic cultures. In multinucleate protoplasts the size and number of PPBs were apparently not controlled by nuclear number. Research with conifer protoplasts resulted in the discovery of new features of gymnosperm spindles.     

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.     

Fluorescent membrane probes’ behavior in lipid bilayers: insights from molecular dynamics simulations

Fluorescence spectroscopy and microscopy have been used as tools to study membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Two major points of concern arise regarding this matter, namely the incomplete understanding of the probe behavior inside the bilayer and the perturbation of the latter resulting from probe incorporation. To this effect, molecular dynamics (MD) simulations, by providing detailed atomic-scale information, represent a valuable way to characterize the location and dynamics of bilayer-inserted membrane probes, as well as the magnitude of perturbation they induce on the host lipid structure, and several important classes of reporter molecules have been studied in recent years. This article reviews the state of the art of MD simulations of bilayer-inserted fluorescent probes, focusing on the information that has been obtained from previous studies and hinting at future perspectives in this rapidly emerging field.     

Glycine transporter 1 associates with cholesterol-rich membrane raft microdomains

Membrane rafts, the highly-ordered, cholesterol-rich microdomains of the plasma membrane play important roles in cellular functions. In this study, GLYT1-CFP and GLYT2-CFP were constructed, followed by investigation of whether the tagged transporters associate with a fluorescence probe that labels membrane rafts (DilC16) by using Fluorescence Resonance Energy Transfer. A close association was observed between DiIC16 and GLYT1-CFP, but not for GLYT2-CFP. The glycine transport ability of GLYT1 is also highly dependent on the integrity of this area. Together, the results suggest that GLYT1 and membrane rafts are co-localized in the membrane, and that this influences the rate of glycine transport.     

Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of "lipid rafts" or "membrane rafts." Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-G(M1), which preferentially partitions into liquid-ordered phases. For both markers, we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, long-lived platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures, the glycosylphosphatidylinositol-anchored monomeric GFP homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.     

Molecular modelling of membrane sterols with the use of the GROMOS 96 force field

Membrane located sterols determine the structure and function of eucariotic cell membranes. Moreover, they are targets for important antifungal antibiotic amphotericin B. Knowledge about the geometry and dynamics of sterols in the environment of lipidic membranes is necessary to understand their functions. However, due to the dynamic character of the membrane, no experimental data about sterol behaviour on the molecular level is available. Hence molecular modelling simulations could be a source of useful information. The main goal of this paper is to prove the adequacy of the GROMOS 96 force field for molecular simulations of membrane sterols. We focused our attention on the reproduction of characteristic geometrical features observed in the crystal of cholesterol hemiethanolate by molecular dynamics simulations. The results presented clearly indicate that the GROMOS 96 force field can be a useful tool to simulate the highly lipophilic systems. Moreover, interactions responsible for the stability of such systems can also be recognised.     

Amphidinol 3 preferentially binds to cholesterol in disordered domains and disrupts membrane phase separation

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 μM, as in the case of methyl-β-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.     

The influence of saponins on cell membrane cholesterol

We studied the influence of structurally different saponins on the cholesterol content of cellular membranes. Therefore a cell culture model using ECV-304 urinary bladder carcinoma cells was developed. To measure the cholesterol content we used radiolabeled ³H-cholesterol which is chemically and physiologically identical to natural cholesterol. The cells were pre-incubated with ³H-cholesterol and after a medium change, they were treated with saponins to assess a saponin-induced cholesterol liberation from the cell membrane. In another experiment the cells were pre-incubated with saponins and after a medium change, they were treated with ³H-cholesterol to assess a saponin-induced inhibition of cholesterol uptake into the cell membrane. Furthermore, the membrane toxicity of all applied saponins was analyzed using extracellular LDH quantification and the general cytotoxicity was analyzed using a colorimetric MTT-assay and DNA quantification. Our results revealed a correlation between membrane toxicity and general cytotoxicity. We also compared the results from the experiments on the saponin-induced cholesterol liberation as well as the saponin-induced inhibition of cholesterol uptake with the membrane toxicity. A significant reduction in the cell membrane cholesterol content was noted for those saponins who showed membrane toxicity (IC₅₀ <60 μM). These potent membrane toxic saponins either liberated ³H-cholesterol from intact cell membranes or blocked the integration of supplemented ³H-cholesterol into the cell membrane. Saponins with little influence on the cell membrane (IC₅₀ >100 μM) insignificantly altered the cell membrane cholesterol content. The results suggested that the general cytotoxicity of saponins is mainly dependent on their membrane toxicity and that the membrane toxicity might be caused by the loss of cholesterol from the cell membrane.We also analyzed the influence of a significantly membrane toxic saponin on the cholesterol content of intracellular membranes such as those of endosomes and lysosomes. In these experiments ECV-304 cells were either incubated with ³H-cholesterol or with ³H-cholesterol and 5 μM saponin. After isolation of the endosomes/lysosomes their ³H-cholesterol content was measured. A significant influence of the saponins on the cholesterol content of endosomal/lysosomal membranes was not detected.     

Recent developments of genetically encoded optical sensors for cell biology

Optical sensors are powerful tools for live cell research as they permit to follow the location, concentration changes or activities of key cellular players such as lipids, ions and enzymes. Most of the current sensor probes are based on fluorescence which provides great spatial and temporal precision provided that high‐end microscopy is used and that the timescale of the event of interest fits the response time of the sensor. Many of the sensors developed in the past 20 years are genetically encoded. There is a diversity of designs leading to simple or sometimes complicated applications for the use in live cells. Genetically encoded sensors began to emerge after the discovery of fluorescent proteins, engineering of their improved optical properties and the manipulation of their structure through application of circular permutation. In this review, we will describe a variety of genetically encoded biosensor concepts, including those for intensiometric and ratiometric sensors based on single fluorescent proteins, Forster resonance energy transfer‐based sensors, sensors utilising bioluminescence, sensors using self‐labelling SNAP‐ and CLIP‐tags, and finally tetracysteine‐based sensors. We focus on the newer developments and discuss the current approaches and techniques for design and application. This will demonstrate the power of using optical sensors in cell biology and will help opening the field to more systematic applications in the future.     

The effect of thiol-active compounds and sterols on the membrane-associated hemolysin of Mycoplasma pulmonis

Abstract Previous studies had shown that Mycoplasma pulmonis contained a bovine serum albumin-dependent, membrane-associated hemolysin. Biochemical analyses were performed to further characterize this activity. The membrane-associated hemolytic activity could be activated by dithiothreitol and β-mercaptoethanol, and inactivated by oxidizing compounds, a sulfhydryl inhibitor and heat treatment. Cholesterol and other sterols were inhibitory in a stereo-specific manner, but they did not interfere with adherence of M. pulmonis to red blood cells. These results indicated that once attached, the M. pulmonis hemolysin recognized cholesterol in the opposing membrane leading to red cell lysis. Because of the unique location of this toxin and its sensitivity to cholesterol, the mycoplasma membrane hemolysins may belong to a unique class of bacterial toxins.     

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.     

Microscopy images as interactive tools in cell modeling and cell biology education

The advent of genomics, proteomics, and microarray technology has brought much excitement to science, both in teaching and in learning. The public is eager to know about the processes of life. In the present context of the explosive growth of scientific information, a major challenge of modern cell biology is to popularize basic concepts of structures and functions of living cells, to introduce people to the scientific method, to stimulate inquiry, and to analyze and synthesize concepts and paradigms. In this essay we present our experience in mixing science and education in Brazil. For two decades we have developed activities for the science education of teachers and undergraduate students, using microscopy images generated by our work as cell biologists. We describe open-air outreach education activities, games, cell modeling, and other practical and innovative activities presented in public squares and favelas. Especially in developing countries, science education is important, since it may lead to an improvement in quality of life while advancing understanding of traditional scientific ideas. We show that teaching and research can be mutually beneficial rather than competing pursuits in advancing these goals.     

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2-3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.     

The intriguing links between prominin-1 (CD133), cholesterol-based membrane microdomains, remodeling of apical plasma membrane protrusions, extracellular membrane particles, and (neuro)epithelial cell differentiation

Prominin-1 (CD133) is a cholesterol-interacting pentaspan membrane protein concentrated in plasma membrane protrusions. In epithelial cells, notably neuroepithelial stem cells, prominin-1 is found in microvilli, the primary cilium and the midbody. These three types of apical membrane protrusions are subject to remodeling during (neuro)epithelial cell differentiation. The protrusion-specific localization of prominin involves its association with a distinct cholesterol-based membrane microdomain. Moreover, the three prominin-1-containing plasma membrane protrusions are the origin of at least two major subpopulations of prominin-1-containing extracellular membrane particles. Intriguingly, the release of these particles has been implicated in (neuro)epithelial cell differentiation.     

Chemical biology tools for imaging-based analysis of organelle membranes and lipids

Membrane biology studies have revealed that in addition to providing structural support for compartment formation and membrane protein function, subcellular biomembranes are also critically involved in many biological events. To facilitate our understanding of the functions, biophysical properties and structural dynamics of organelle membranes, various exciting chemical biology tools have recently emerged. This short review aims to describe the latest molecular probes for organelle membrane studies. In particular, we will feature chemical strategies to visualize and quantitatively analyze the dynamic propeties of organelle membranes and lipids and discuss current limitations and potential future directions of this challenging research area.     

Secondary transport as an efficient membrane transport mechanism for plant secondary metabolites

Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, and phenolic compounds. Secondary metabolites have various functions including protection against pathogens and UV light in plants, and have been used as natural medicines for humans utilizing their diverse biological activities. Many of these natural compounds are accumulated in a particular compartment such as vacuoles, and some are even translocated from source cells to sink organs via long distance transport. Both primary and secondary transporters are involved in such compartmentation and translocation, and many transporter genes, especially genes belonging to the multidrug and toxin extrusion type transporter family, which consists of 56 members in Arabidopsis, have been identified as responsible for the membrane transport of secondary metabolites. Better understandings of these transporters as well as the biosynthetic genes of secondary metabolites will be important for metabolic engineering aiming to increase the production of commercially valuable secondary metabolites in plant cells.