Falk Schneider

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.

Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

The diffusion dynamics of lipids and GPI-anchored proteins is investigated using superresolution STED microscopy combined with single-molecule fluorescence correlation spectroscopy in the cellular membranes. The actin cytoskeleton is shown to play an essential role in the diffusion characteristics of molecules.

Reorganization of Lipid Diffusion by Myelin Basic Protein as Revealed by STED Nanoscopy

Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes.

Polarity-Sensitive Probes for Superresolution Stimulated Emission Depletion Microscopy

The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed. Polarity-sensitive dyes are powerful tools to characterize such lipid membrane order, employing, for example, confocal and two-photon microscopy. The investigation of potential nanodomains, however, requires the use of superresolution microscopy. Here, we test the performance of the polarity-sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA, and NR12S in superresolution stimulated emission depletion microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles, show the high potential of these dyes for probing nanoscale membrane heterogeneity.

Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy.

Abstract Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/−125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50  ±  15 nm and 120  ±  40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

Capturing resting T cells: the perils of PLL

Supported by a Royal Society University Research Fellowship (UF120277 to S.F.L.) and Research Professorship (RP150066 to D.K.); the EPSRC (EP/L027631/1 to A.P.,); the Wellcome Trust (098274/Z/12/Z to S.J.D., and WT101609MA to R.A.F.); PA Cephalosporin Fund (C.E.); the Wolfson Imaging Centre Oxford (funded by the Wolfson Foundation and Wellcome Trust; 104924/14/Z/14); the Micron Advanced BioImaging Unit (Wellcome Trust Strategic Award 091911); the Medical Research Council (MC_UU_12010/Unit Programmes G0902418 and MC_UU_12025); an MRC/BBSRC/EPSRC award (MR/K01577X/1); and a Marie Sklodowska-Curie Intra-European grant (707348 to I.U.).

Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging

The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.

Optimized processing and analysis of conventional confocal microscopy generated scanning FCS data

Highlights • FoCuS-scan is software for processing and analysis of large-scale scanning FCS data.• FoCuS-scan can correlate data acquired on conventional turn-key confocal systems.• We show the precision of diffusion measurements depends on experimental duration.• This article gives specific acquisition, analysis and interpretation details.

Nanoparticles can wrap epithelial cell membranes and relocate them across the epithelial cell layer

Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane’s disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.

Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion

Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.