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New experimental techniques, especially in the context of observing molecular dynamics, reveal the plasma membrane to be heterogeneous and “scale rich,” from nanometers to microns and from microseconds to seconds. This is critical information, which shows that scale-dependent transport governs the molecular encounters that underlie cellular signaling. The data are rich and reaffirm the importance of the cortical cytoskeleton, protein aggregates, and lipidomic complexity on the statistics of molecular encounters. Moreover, the data demand simulation approaches with a particular set of features, hence the “manifesto.” Together with the experimental data, simulations that satisfy these requirements hold the promise of a deeper understanding of membrane spatiotemporal organization. Several experimental breakthroughs in measuring molecular membrane dynamics are reviewed, the constraints that they place on simulations are discussed, and the status of simulation approaches that aim to meet them are detailed.

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.

The high-affinity IgE receptor, FcεR1, is responsible for the sensitization of mast cells and basophils to IgE-targeted anti- gens. The current model of FcεR1 triggering rests on the principle of aggregation-driven phosphorylation, yet several recent observations have indicated that this is incomplete. Here we re-examine the minimal requirements for FcεR1 triggering and observe that it is induced without receptor aggregation by surface-associated antigen in a size-dependent manner.

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.

Lymphocyte T cells are responsible for cell-mediated adaptive immune responses, involving transient interactions of the T-cell receptor (TCR) with peptides presented by MHC proteins. A productive interaction triggers the T-cell signaling forming the immunological synapse. Initially, the TCR is phosphorylated by the Lck, a membrane-anchored tyrosine kinase, producing membrane microclusters. Understanding the T-cell pre-synaptic triggering implies comparing resting vs. activated states. Super-resolution imaging techniques (i.e. dSTORM and PALM) suggested protein pre-clustering into “nano-domains” in resting cells, contradicting the classical view of cluster formation upon activation. However, observations with cells touching non-activating surfaces probably don’t resemble a true resting state. Instead, avoiding contacts would allow better understanding the resting and early stages of activation. We studied live T-cells in suspension employing a hydrogel in a density gradient and used STED nanoscopy to unravel the plasma membrane distribution and dynamics of Lck in T-cells on glass and in suspension. T-cells suspended in the hydrogel do not triggered calcium, indicating absence of activation; while classically considered resting states triggered calcium in a similar fashion as when cells were deposited onto a surface functionalized by antibody (antiCD3 and antiCD28) coating. In contrast to surface-contacting, suspended T-cells showed mostly a uniform distribution of Lck. Nevertheless, Lck still displayed heterogeneity in mobility in the true resting state. We found several components of mobility rather than a single one. These different diffusion coefficients were remarkably slower when T-cells were in contact with any of the studied surfaces. Our results suggest that pre-clustering of signaling receptors and cell-surface proteins in resting T-cells needs reconsideration and that understanding the T cell activation requires a true resting state, which we can be obtained by means of hydrogels.

Nature of the cellular membrane heterogeneity has been a long-standing question in cell biology. One crucial measure for the membrane heterogeneity is the lipid packing. Measuring changes of lipid packing in the plasma membrane during cellular events has been an important part of elucidating the biological function of membrane physical chemistry. Here, we apply conventional and spectral imaging to figure out the nature of membrane heterogeneity and its role in immune signalling events.We observe bio-membranes sampling different lipid packing states that form coexisting domains (i.e., relatively ordered and disordered domains) with a variety of distinct compositions and functional characteristics. We first investigate the connection between membrane composition and lipid packing in synthetic liposomes and show that both the relatively ordered and disordered phase, and the difference between them, are tuned by lipid composition. We then give an example of live cells regulating the lipid packing phenotypes of their plasma membranes, accessing a variety of both ordered and disordered domains as a function of cellular activity. Finally, we find that in both synthetic and natural membranes, the tunable interdomain lipid packing disparity (i.e. the difference between coexisting domains) regulates component partitioning between domains and the functionality of membrane embedded lipidic receptors. By applying advanced image processing with spectral imaging, we show that even the marginal modulation of membrane heterogeneity is crucial for immune signalling events such as FCR and B-cell signalling.Membrane heterogeneity is an organizing principle for the membrane bio-activity by either concentrating certain molecules in transient domains or by physically adjusting the conformity of the molecules to favour or hinder their interactions in the membrane.

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Fluorescence fluctuation methods such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis (FIDA) have proven to be versatile tools for studying molecular interactions with single molecule sensitivity. Another well-known fluorescence technique is the measurement of the fluorescence lifetime. Here, we introduce a method that combines the benefits of both FIDA and fluorescence lifetime analysis. It is based on fitting the two-dimensional histogram of the number of photons detected in counting time intervals of given width and the sum of excitation to detection delay times of these photons. Referred to as fluorescence intensity and lifetime distribution analysis (FILDA), the technique distinguishes fluorescence species on the basis of both their specific molecular brightness and the lifetime of the excited state and is also able to determine absolute fluorophore concentrations. The combined information yielded by FILDA results in significantly increased accuracy compared to that of FIDA or fluorescence lifetime analysis alone. In this paper, the theory of FILDA is elaborated and applied to both simulated and experimental data. The outstanding power of this technique in resolving different species is shown by quantifying the binding of calmodulin to a peptide ligand, thus indicating the potential for application of FILDA to similar problems in the life sciences.

The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. The actin network undergoes constant reorganisation due to molecular turnover. Hence, cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents and the actin filament length-distribution, but quantitative data on these dynamics remains elusive. We combined single molecule speckle microscopy and photobleaching experiments with microscopic computer simulations to analyse how molecular binding dynamics of G-actin to filaments sets network turnover and consequently the mechanical properties of the cellular actin cortex in living cells. Using photobleaching experiments, we found that two filament families with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerisation resulting from addition of monomers to free barbed-ends and one with slow turnover dynamics with polymerisation resulting from formin-mediated filament growth. We show that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbedends. Additionally, we measured the molecular association rates and the distribution of travel-distances of actin monomers and formin dimers in speckle experiments and showed that this travel-distance distribution is consistent with the actin filament length-distribution found from photobleaching experiments and molecular simulations. Together, our results provide a quantitative characterisation of essential mechanisms underlying actin cortex homeostasis.

T-cell activation is widely thought to rely on cytoskeletal remodeling at all stages. Using super-resolution optical STED and lattice-light-sheet microscopy we show that during activation T cells sequentially rearrange their cortical actin using two networks of different-length F-actins creating three distinct structures. A cortical actin ring formed at the initial contact interface evolves into a rosette-shaped structure comprising a coarse central fiber network and flat lamellipodium as the cell spreads. The formation of actin “spikes” extending to the contact edge then marks a contraction phase leading to immunological synapse formation. We also show, however, that the earliest steps in T-cell signaling require remarkably little contact with activating surfaces and no cytoskeletal rearrangements. Blocking actin remodeling pharmacologically produces more rather than less early signaling. Our work shows that whereas a complex program of cytoskeletal reorganization is initiated by T-cell activation, early signaling occurs entirely independently of cytoskeletal remodeling.

In this study we introduce the combination of two-color global fluorescence correlation spectroscopy (2CG-FCS) and Förster resonance energy transfer (FRET) as a very powerful combination for monitoring biochemical reactions on the basis of single molecule events. 2CG-FCS, which is a new variation emerging from the family of fluorescence correlation spectroscopy, globally analyzes the simultaneously recorded auto- and cross-correlation data from two photon detectors monitoring the fluorescence emission of different colors. Overcoming the limitations inherent in mere auto- and cross-correlation analysis, 2CG-FCS is sensitive in resolving and quantifying fluorescent species that differ in their diffusion characteristics and/or their molecular brightness either in one or both detection channels. It is able to account for effects that have often been considered as sources of severe artifacts in two-color and FRET measurements, the most prominent artifacts comprising photobleaching, cross talk, or concentration variations in sample preparation. Because of its very high statistical accuracy, the combination of FRET and 2CG-FCS is suited for high-throughput applications such as drug screening. Employing beam scanning during data acquisition even further enhances this capability and allows measurement times of

Currently two of the most important - as well as controversial - proposed membrane-organizing principles are the “lipid rafts” hypothesis, proposed by K. Simons, and the “picket-fence” model, proposed by A. Kusumi. To date, both hypotheses encounter obstacles for full acceptance due to technical limitations on the available superresolution and single molecule techniques and limitations on the correspondent probes utilized by such techniques applied to living cells.Trying to elucidate the lipid hop diffusion dilemma, which would be the underlying principle for the “picket-fence” model, we planned a series of STED-FCS experiments to probe the diffusion of phospholipids on the membrane of different cell types. The superresolution of STED microscopy allows FCS experiments to probe areas comparable in size to the compartments created by the actin cytoskeleton, arguably one of the major structures responsible for lipid and protein segregation in the cell membrane.Using STED-FCS, we were able to detect phospholipid hop diffusion in two of the cell lines studied (NRK and IA32) and free diffusion was observed for the other cell lines under consideration (Ptk2, Vero, Hela). Different treatments to deplete the actin cytoskeleton on IA32 and NRK cells resulted in the extinction or hindrance of phospholipid hop diffusion. The same result was observed in a mutant cell line Arp 2/3 depleted originated from IA32, evidencing the major role of actin cytoskeleton on the compartmentalization of lipids. Simulations connecting membrane content and actin cytoskeleton density provided insight into the reason for apparent different diffusion modes in different cells.Whereas lipid hop diffusion could still not be confirmed by SPT experiments using fluorescent tags as markers due to technical limitations, it was now for the first time evidenced by STED-FCS, which is the time-domain counterpart of that technique.

The eukaryotic cell membrane is connected to a dense actin rich cortex. We present FCS and STED experiments showing that dense membrane bound actin networks have severe influence on lipid phase separation. A minimal actin cortex was bound to a supported lipid bilayer via biotinylated lipid streptavidin complexes (pinning sites). In general, actin binding to ternary membranes prevented macroscopic liquid-ordered and liquid-disordered domain formation, even at low temperature. Instead, depending on the type of pinning lipid, an actin correlated multi-domain pattern was observed. FCS measurements revealed hindered diffusion of lipids in the presence of an actin network. To explain our experimental findings, a new simulation model is proposed, in which the membrane composition, the membrane curvature, and the actin pinning sites are all coupled. Our results reveal a mechanism how cells may prevent macroscopic demixing of their membrane components, while at the same time regulate the local membrane composition. DOI: http://dx.doi.org/10.7554/eLife.01671.001, eLife digest All cells are surrounded by a lipid membrane that protects the cell, controls the movement of molecules into and out of the cell, and passes messages about environmental conditions to the cell. This membrane is made of two layers of molecules called lipids, with various proteins embedded in it. There are many different types of lipid molecules that together help to keep the membrane flexible. Moreover, lipid molecules of particular types can also come together to form ‘rafts’ that help the membrane to carry out its various roles. Given the complexity of the cell membrane, cell biologists often use simpler model membranes and computer simulations to explore how the different types of lipid molecules are organized within the membrane. According to the ‘picket fence’ model the cell membrane is divided into small compartments as a result of its interaction with the dense network of actin fibers that acts as a skeleton inside the cell. Recent computer simulations have predicted that these interactions can influence the distribution of lipids and proteins within the membrane. In particular, they can prevent the drastic re-arrangement of lipids into regions of high and low viscosity at low temperature. This temperature dependent re-arrangement of the membrane is known as lipid phase separation. Honigmann et al. have now used computer simulations and two advanced techniques—super-resolution optical STED microscopy and fluorescence correlation spectroscopy—to explore the properties of a model membrane in the presence of a dense network of actin fibers in fine detail. The results show that, in agreement with the simulation predictions of the ‘picket fence’ model, the actin fibers bound to the membrane prevent lipid phase separation happening at low temperatures. Moreover, the actin fibers also help to organize the distribution of lipids and proteins within the membrane at physiological temperatures. Honigmann et al. also suggest that the actin fibers cause the membrane to curve in a way that can reinforce the influence of the ‘picket fence’. The results show that the ‘raft’ and ‘picket fence’ models are connected, and that a cell can control the properties of its membrane by controlling the interactions between the membrane and the actin fibers that make up the skeleton of the cell. DOI: http://dx.doi.org/10.7554/eLife.01671.002

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.

Cholesterol-assisted lipid interactions such as the integration into lipid nanodomains (‘rafts’) are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of >200nm of a conventional far-field optical microscope. We report the detection of single diffusing lipid molecules in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (STED) far-field nanoscopy. Combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (FCS) or other single-molecule techniques, we obtain new details of molecular membrane dynamics. For example, unlike phosphoglycerolipids, sphingolipids or ‘raft’-associated proteins are transiently (∼ 10 ms) trapped on the nanoscale in cholesterol-mediated molecular complexes.

The plasma membrane seems to be dynamically structured in regions of different composition and function. However, the spatial and temporal scale of the respective membrane structure is not directly accessible by the diffraction limited resolution of conventional far-field optical microscopes. We report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (STED) far-field nanoscopy. By combining a (tunable) resolution of down to 30 nm with tools such as fluorescence correlation spectroscopy (FCS), we obtain new details of molecular membrane dynamics. Sphingolipids are transiently (∼ 10 ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while glycero-phospho-lipids diffuse freely. The results are compared to STED experiments on model membranes, which highlight that the nanoscale trapping in cells is not correlated with liquid order partitioning in model systems. The novel observations shine new light on the distribution and interaction of lipids and proteins in the plasma membrane with respect to the lipid raft hypothesis.

Prominent problems in biology cannot be solved due to the limited resolution of conventional optical microscopy. For example, a whole range of membrane-associated processes are considered to be mediated through cholesterol-assisted interactions such as the formation of lipid nanodomains or ‘rafts’. The direct and non-invasive observation of lipid or membrane protein dynamics in living cells, which are believed to occur on small spatial scales, is impeded by the resolution limit of >200nm of a conventional far-field fluorescence microscope or by the limited time resolution of single-particle tracking. We combine single-molecule based techniques such as fluorescence correlation spectroscopy (FCS) with stimulated emission depletion (STED) far-field microscopy to accesses a superior spatial and temporal resolution for observing the diffusion characteristics of molecules in the plasma membrane of living cells. Tuning the detection area between 250 nm and 30 nm in diameter, we directly reveal marked differences between different lipid or molecular classes. For example, sphingolipids or ‘raft’-associated proteins are transiently trapped on the nanoscale in cholesterol-mediated molecular complexes. The presented direct detection of molecular dynamics in nanoscale areas of tunable size constitutes a powerful approach to study the dynamics of biomolecules in living cells.

Membrane nanodomains based on phase-segregation of lipids have emerged as a key organizing principle of the plasma membrane. They have been shown to play important roles in signal transduction and membrane trafficking. We have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-NTA) head groups for selective targeting of His-tagged proteins into liquid ordered or liquid disordered lipid phases. The stable, non-covalent interaction of His-tagged proteins to the tris-NTA moiety can be employed not only for efficient specific tethering of spectroscopic probes, but also for versatile manipulation of membrane nanodomains. In giant unilamellar vesicles strong partitioning of tris-NTA lipids into different lipid phases was observed. For a saturated tris-NTA lipid, at least 10-fold preference for the liquid ordered phase was found. In contrast, an unsaturated NTA lipid shows a comparable preference for the liquid disordered phase. Simular partitioning of the tris-NTA lipids was observed in solid-supported membranes on mica. Partitioning into submicroscopic membrane domains formed in solid-supported membranes was confirmed by superresolution imaging techniques (FPALM, STED). Single molecule tracking of His-tagged proteins tethered to solid-supported phase-separating membranes revealed clear differences in the diffusion behavior of the different NTA-lipids. By using vesicles as a carrier, multivalent NTA lipids were efficiently incorporated into the plasma membrane of live cells. After formation of giant plasma membrane vesicles (GPMV), efficient partitioning of the lipid probes into the respective membrane phases was confirmed. We have employed these probes for exploring lipid diffusion, morphology and spatiotemporal dynamics of membrane nanodomains in vitro and live cells by single molecule tracking and STED FCS.