Stefan Wieser

Tracking single molecules in the live cell plasma membrane—Do’s and Don’t’s

In recent years, the development of fast and highly sensitive microscopy has changed the way of thinking of cell biologists: it became more and more important to study the structural origin for cellular function, and industry turned its attention to the improvement of the required instruments. Optical microscopy has now reached a milestone in sensitivity by resolving the signal of a single, fluorescence-labeled biomolecule within a living cell. First steps towards these pioneering studies were set by methods developed in the late eighties for tracking single biomolecules labeled with fluorescent latex spheres or gold-particles. Meanwhile, a time-resolution of milliseconds for imaging weakly fluorescent cellular structures like small organelles, vesicles, or even single molecules is state-of-the-art. The advances in the fields of microscopy brought new cell biological questions into reach. The investigation of a single fluorescent molecule-or simultaneously of an ensemble of individual molecules-provides principally new information, which is generally hidden in ensemble-averaged signals of molecules. In this paper we describe strategies how to make use of single molecule trajectories for deducing information about nanoscopic structures in a live cell context. In particular, we focus our discussion on elucidating the plasma membrane organization by single molecule tracking. A diffusing membrane constituent--e.g. a protein or a lipid--experiences a manifold of interactions on its path: the most rapid interactions represent the driving force for free diffusion; stronger or correlated interactions can be frequently observed as subdiffusive behavior. Correct interpretation of the data has the potential to shine light on this enigmatic organelle, where membrane rafts, protein microdomains, fences and pickets still frolic through the text-book sketches. We summarize available analytical models and point out potential pitfalls, which may result in quantitative or three even qualitative misinterpretations.

Cortical contractility triggers a stochastic switch to fast amoeboid cell motility.

Summary 3D amoeboid cell migration is central to many developmental and disease-related processes such as cancer metastasis. Here, we identify a unique prototypic amoeboid cell migration mode in early zebrafish embryos, termed stable-bleb migration. Stable-bleb cells display an invariant polarized balloon-like shape with exceptional migration speed and persistence. Progenitor cells can be reversibly transformed into stable-bleb cells irrespective of their primary fate and motile characteristics by increasing myosin II activity through biochemical or mechanical stimuli. Using a combination of theory and experiments, we show that, in stable-bleb cells, cortical contractility fluctuations trigger a stochastic switch into amoeboid motility, and a positive feedback between cortical flows and gradients in contractility maintains stable-bleb cell polarization. We further show that rearward cortical flows drive stable-bleb cell migration in various adhesive and non-adhesive environments, unraveling a highly versatile amoeboid migration phenotype.

Actin Flows Mediate a Universal Coupling between Cell Speed and Cell Persistence

Cell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.

Different Types of Cell-to-Cell Connections Mediated by Nanotubular Structures

Communication between cells is crucial for proper functioning of multicellular organisms. The recently discovered membranous tubes, named tunneling nanotubes, that directly bridge neighboring cells may offer a very specific and effective way of intercellular communication. Our experiments on RT4 and T24 urothelial cell lines show that nanotubes that bridge neighboring cells can be divided into two types. The nanotubes of type I are shorter and more dynamic than those of type II, and they contain actin filaments. They are formed when cells explore their surroundings to make contact with another cell. The nanotubes of type II are longer and more stable than type I, and they have cytokeratin filaments. They are formed when two already connected cells start to move apart. On the nanotubes of both types, small vesicles were found as an integral part of the nanotubes (that is, dilatations of the nanotubes). The dilatations of type II nanotubes do not move along the nanotubes, whereas the nanotubes of type I frequently have dilatations (gondolas) that move along the nanotubes in both directions. A possible model of formation and mechanical stability of nanotubes that bridge two neighboring cells is discussed.

Versatile Analysis of Single-Molecule Tracking Data by Comprehensive Testing against Monte Carlo Simulations

We propose here an approach for the analysis of single-molecule trajectories which is based on a comprehensive comparison of an experimental data set with multiple Monte Carlo simulations of the diffusion process. It allows quantitative data analysis, particularly whenever analytical treatment of a model is infeasible. Simulations are performed on a discrete parameter space and compared with the experimental results by a nonparametric statistical test. The method provides a matrix of p-values that assess the probability for having observed the experimental data at each setting of the model parameters. We show the testing approach for three typical situations observed in the cellular plasma membrane: i), free Brownian motion of the tracer, ii), hop diffusion of the tracer in a periodic meshwork of squares, and iii), transient binding of the tracer to slowly diffusing structures. By plotting the p-value as a function of the model parameters, one can easily identify the most consistent parameter settings but also recover mutual dependencies and ambiguities which are difficult to determine by standard fitting routines. Finally, we used the test to reanalyze previous data obtained on the diffusion of the glycosylphosphatidylinositol-protein CD59 in the plasma membrane of the human T24 cell line.

Spot variation fluorescence correlation spectroscopy allows for superresolution chronoscopy of confinement times in membranes.

Resolving the dynamical interplay of proteins and lipids in the live-cell plasma membrane represents a central goal in current cell biology. Superresolution concepts have introduced a means of capturing spatial heterogeneity at a nanoscopic length scale. Similar concepts for detecting dynamical transitions (superresolution chronoscopy) are still lacking. Here, we show that recently introduced spot-variation fluorescence correlation spectroscopy allows for sensing transient confinement times of membrane constituents at dramatically improved resolution. Using standard diffraction-limited optics, spot-variation fluorescence correlation spectroscopy captures signatures of single retardation events far below the transit time of the tracer through the focal spot. We provide an analytical description of special cases of transient binding of a tracer to pointlike traps, or association of a tracer with nanodomains. The influence of trap mobility and the underlying binding kinetics are quantified. Experimental approaches are suggested that allow for gaining quantitative mechanistic insights into the interaction processes of membrane constituents.

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

Single molecule diffusion analysis on cellular nanotubules: Implications on plasma membrane structure below the diffraction limit

Cells are frequently interconnected by tunneling nanotubules, recently discovered cylindrical structures which facilitate material exchange. We employed here single molecule fluorescence microscopy to study the diffusion of the glycosylphosphatidylinositol-protein CD59 in the plasma membrane of tunneling nanotubules in living cells at subwavelength resolution. Our study provides the nanotubule radius in vivo, yielding a surprisingly narrow size distribution (7nm standard deviation) around a mean value of 65nm. Moreover, by separating longitudinal and transverse mobilities, we find isotropic diffusion behavior.

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides—in particular a CAMP with Lysine–Leucine–Lysine repeats (termed KLK)—affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents.

Lpe10p modulates the activity of the Mrs2p-based yeast mitochondrial Mg2+channel

Saccharomyces cerevisiae Lpe10p is a homologue of the Mg2+‐channel‐forming protein Mrs2p in the inner mitochondrial membrane. Deletion of MRS2, LPE10 or both results in a petite phenotype, which exhibits a respiratory growth defect on nonfermentable carbon sources. Only coexpression of MRS2 and LPE10 leads to full complementation of the mrs2Δ/lpe10Δ double disruption, indicating that these two proteins cannot substitute for each other. Here, we show that deletion of LPE10 results in a loss of rapid Mg2+ influx into mitochondria, as has been reported for MRS2 deletion. Additionally, we found a considerable loss of the mitochondrial membrane potential (ΔΨ) in the absence of Lpe10p, which was not detected in mrs2Δ cells. Addition of the K+/H+‐exchanger nigericin, which artificially increases ΔΨ, led to restoration of Mg2+ influx into mitochondria in lpe10Δ cells, but not in mrs2Δ/lpe10Δ cells. Mutational analysis of Lpe10p and domain swaps between Mrs2p and Lpe10p suggested that the maintenance of ΔΨ and that of Mg2+ influx are functionally separated. Cross‐linking and Blue native PAGE experiments indicated interaction of Lpe10p with the Mrs2p‐containing channel complex. Using the patch clamp technique, we showed that Lpe10p was not able to mediate high‐capacity Mg2+ influx into mitochondrial inner membrane vesicles without the presence of Mrs2p. Instead, coexpression of Lpe10p and Mrs2p yielded a unique, reduced conductance in comparison to that of Mrs2p channels. In summary, the data presented show that the interplay of Lpe10p and Mrs2p is of central significance for the transport of Mg2+ into mitochondria of S. cerevisiae.