Verena Ruprecht

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.

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-GM1, 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.

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.

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.

Two-color single molecule tracking combined with photobleaching for the detection of rare molecular interactions in fluid biomembranes

Experimentalists are increasingly confronted with the demand to single out rare interaction events under a vast excess of non-interacting molecules. We recently presented an approach how to virtually dilute fluorescently labeled membrane constituents by combining photobleaching and single molecule microscopy, termed “Thinning Out Clusters while Conserving the Stoichiometry of Labeling” (TOCCSL; Moertelmaier et al., Appl. Phys. Lett., 2005, 87, 263903). Using this approach, single molecule microscopy can be performed even at arbitrarily high surface densities of fluorescent probe molecules. Here, we extended this method for two color microscopy. We provide a detailed statistical description of false positives and false negatives. In particular, we quantified the increase in sensitivity by tracking the colocalized objects over successive images. Proof of principle experiments were performed by measuring the interaction between Alexa647-labeled Cholera Toxin B (CTX-B-Alexa647) and Bodipy-labeled GM1 (Bodipy-GM1) diffusing in a fluid supported lipid bilayer. We directly observed single Cholera Toxin B molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. Each colocalized spot could be further analyzed with respect to its diffusion constant, yielding a clear anticorrelation between occupancy and mobility. We finally demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified.

UV laser ablation to measure cell and tissue-generated forces in the zebrafish embryo in vivo and ex vivo.

Mechanically coupled cells can generate forces driving cell and tissue morphogenesis during development. Visualization and measuring of these forces is of major importance to better understand the complexity of the biomechanic processes that shape cells and tissues. Here, we describe how UV laser ablation can be utilized to quantitatively assess mechanical tension in different tissues of the developing zebrafish and in cultures of primary germ layer progenitor cells ex vivo.

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.

What Can We Learn from Single Molecule Trajectories

Diffusing membrane constituents are constantly exposed to a variety of forces that influence their stochastic path. Single molecule experiments allow for resolving trajectories at extremely high spatial and temporal accuracy, thereby offering insights into en route interactions of the tracer. In this review we discuss approaches to derive information about the underlying processes, based on single molecule tracking experiments. In particular, we focus on a new versatile way to analyze single molecule diffusion in the absence of a full analytical treatment. The method is based on comprehensive comparison of an experimental data set against the hypothetical outcome of multiple experiments performed on the computer. Since Monte Carlo simulations can be easily and rapidly performed even on state-of-the-art PCs, our method provides a simple way for testing various - even complicated - diffusion models. We describe the new method in detail, and show the applicability on two specific examples: firstly, kinetic rate constants can be derived for the transient interaction of mobile membrane proteins; secondly, residence time and corral size can be extracted for confined diffusion.

Chapter Two – Measuring Colocalization by Dual Color Single Molecule Imaging: Thresholds, Error Rates, and Sensitivity

Dual color single molecule tracking is a state-of-the-art fluorescence microscopy technique to study molecular associations. Featuring high spatial and temporal resolution, the method allows for catching associations with extremely high reliability. In this review, we describe robust data analysis approaches to identify and characterize molecular associations from dual color fluorescence images and show how colocalization can be statistically discriminated from accidental colocalizations. We discriminate the case of static analysis where colocalizations are revealed from individual dual color fluorescence images, and the case of dynamic analysis where colocalizations are tracked in time.Abstract Dual color single molecule tracking is a state-of-the-art fluorescence microscopy technique to study molecular associations. Featuring high spatial and temporal resolution, the method allows for catching associations with extremely high reliability. In this review, we describe robust data analysis approaches to identify and characterize molecular associations from dual color fluorescence images and show how colocalization can be statistically discriminated from accidental colocalizations. We discriminate the case of static analysis where colocalizations are revealed from individual dual color fluorescence images, and the case of dynamic analysis where colocalizations are tracked in time.

Measuring Colocalization by Dual Color Single Molecule Imaging: Thresholds, Error Rates, and Sensitivity

Dual color single molecule tracking is a state-of-the-art fluorescence microscopy technique to study molecular associations. Featuring high spatial and temporal resolution, the method allows for catching associations with extremely high reliability. In this review, we describe robust data analysis approaches to identify and characterize molecular associations from dual color fluorescence images and show how colocalization can be statistically discriminated from accidental colocalizations. We discriminate the case of static analysis where colocalizations are revealed from individual dual color fluorescence images, and the case of dynamic analysis where colocalizations are tracked in time.Abstract Dual color single molecule tracking is a state-of-the-art fluorescence microscopy technique to study molecular associations. Featuring high spatial and temporal resolution, the method allows for catching associations with extremely high reliability. In this review, we describe robust data analysis approaches to identify and characterize molecular associations from dual color fluorescence images and show how colocalization can be statistically discriminated from accidental colocalizations. We discriminate the case of static analysis where colocalizations are revealed from individual dual color fluorescence images, and the case of dynamic analysis where colocalizations are tracked in time.