Veronika Mueller

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon-rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution.

We report on a fiber laser-based stimulated emission-depletion microscope providing down to ∼20 nm resolution in raw data images as well as 15-19 nm diameter probing areas in fluorescence correlation spectroscopy. Stimulated emission depletion pulses of nanosecond duration and 775 nm wavelength are used to silence two fluorophores simultaneously, ensuring offset-free colocalization analysis. The versatility of this superresolution method is exemplified by revealing the octameric arrangement of Xenopus nuclear pore complexes and by quantifying the diffusion of labeled lipid molecules in artificial and living cell membranes.

Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment

Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion.

Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells.

The interaction of lipids and proteins plays an important role in plasma membrane bioactivity, and much can be learned from their diffusion characteristics. Here we present the combination of super-resolution STED microscopy with scanning fluorescence correlation spectroscopy (scanning STED-FCS, sSTED-FCS) to characterize the spatial and temporal heterogeneity of lipid interactions. sSTED-FCS reveals transient molecular interaction hotspots for a fluorescent sphingolipid analogue. The interaction sites are smaller than 80 nm in diameter and lipids are transiently trapped for several milliseconds in these areas. In comparison, newly developed fluorescent phospholipid and cholesterol analogues with improved phase-partitioning properties show more homogenous diffusion, independent of the preference for liquid-ordered or disordered membrane environments. Our results do not support the presence of nanodomains based on lipid-phase separation in the basal membrane of our cultured nonstimulated cells, and show that alternative interactions are responsible for the strong local trapping of our sphingolipid analogue.

STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue

We have developed a bright, photostable, and far-red emitting fluorescent phosphoglycerolipid analogue to probe diffusion characteristics of lipids in membranes. The lipid analogue consists of a saturated (C18) phosphoethanolamine and a hydrophilic far-red emitting fluorescent dye (KK114) that is tethered to the head group by a long polyethylenglycol linker. In contrast to reported far-red emitting fluorescent lipid analogues, this one partitions predominantly into liquid ordered domains of phase-separated ternary bilayers. We performed fluorescence correlation spectroscopy with a super-resolution STED microscope (STED-FCS) to measure the lateral diffusion of the new lipid analogue in the liquid ordered (Lo) and disordered (Ld) phase. On a mica support, we observed micrometer large phases and found that the lipid analogue diffuses freely on all tested spatial scales (40-250 nm) in both the Ld and Lo phase with diffusion coefficients of 1.8 microm2 s(-1) and 0.7 microm2 s(-1) respectively. This indicates that the tight molecular packing of the Lo phase mainly slows down the diffusion rather than causing anomalous sub-diffusion. The same ternary mixture deposited on acid-cleaned glass forms Lo nanodomains of < 40 nm to 300 nm in diameter as only revealed by STED microscopy, which demonstrates the severe influence of interactions with the substrate on the sizes of domains in membranes. When averaging over different positions, STEd-FCS measurements on such glass supported membranes displayed anomalous sub-diffusion. This anomaly can be attributed to a transient partitioning of the lipid analogue into the nano-domains, where diffusion is slowed down. Our results suggest that STED-FCS in combination with a Lo-partitioning fluorescent lipid analogue can directly probe the presence of Lo nano-domains, which in the future should allow the study of potential lipid rafts in live-cell membranes.

Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

Monitoring Clathrin-Mediated Endocytosis during Synaptic Activity

To visualize clathrin redistribution during endocytosis in hippocampal boutons, we used a fusion protein of clathrin light chain with enhanced green fluorescent protein. Both high potassium and electric field stimulation lead after a stimulus-dependent delay to a transient increase of fluorescence in synapses, but a slight and transient decrease in adjacent axonal segments. We conclude that the rise and fall of the signal in boutons, with decay kinetics remarkably similar to previous estimates of the endocytic time course, reflects coat assembly and disassembly. Thus, we could selectively measure clathrin-mediated endocytosis and separate its kinetics from other modes of membrane retrieval in CNS synapses. A long-lasting delay preceding the fluorescent transients shows that endocytosis during the first few seconds of continuing stimulation cannot be mediated by newly formed clathrin-coated pits. Therefore, a fast mode of endocytosis is either clathrin-independent or involves preassembled (easily retrievable) clathrin lattices at sites of endocytosis.

STED-FLCS: An Advanced Tool to Reveal Spatiotemporal Heterogeneity of Molecular Membrane Dynamics.

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.

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.

Molecular Interactions on the Plasma Membrane Studied by STED Fluorescence Microscopy

The direct and non-invasive observation of a whole range of cellular functionalities is impeded by the resolution limit of >200nm of a conventional far-field optical microscope. Prominent examples are molecular interactions on the plasma membrane such as the integration into lipid nanodomains (‘rafts’), which are considered to play a functional part in a whole range of membrane-associated processes. We report the detection of single diffusing lipid and protein molecules in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (STED) far-field nanoscopy or of fast single-molecule tracking. By combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (FCS) or by spatio-temporally following the movement of single lipids, we obtain new details of molecular membrane dynamics. For example, we reveal transient (∼ 10 ms) trapping of certain sphingolipids on the nanoscale in cholesterol-mediated molecular complexes. However, distinct differences show up between different lipids and molecules. Molecules such as certain transmembrane proteins show both trapping and a kind of hopping diffusion. The novel observations may highlight new details on lipid-protein interactions and their role in membrane bioactivity.