TY - JOUR AB - Nonadherent polarized cells have been observed to have a pearlike, elongated shape. Using a minimal model that describes the cell cortex as a thin layer of contractile active gel, we show that the anisotropy of active stresses, controlled by cortical viscosity and filament ordering, can account for this morphology. The predicted shapes can be determined from the flow pattern only; they prove to be independent of the mechanism at the origin of the cortical flow, and are only weakly sensitive to the cytoplasmic rheology. In the case of actin flows resulting from a contractile instability, we propose a phase diagram of three-dimensional cell shapes that encompasses nonpolarized spherical, elongated, as well as oblate shapes, all of which have been observed in experiment. AU - Callan Jones, Andrew AU - Ruprecht, Verena AU - Wieser, Stefan AU - Heisenberg, Carl-Philipp J AU - Voituriez, Raphaël ID - 1239 IS - 2 JF - Physical Review Letters TI - Cortical flow-driven shapes of nonadherent cells VL - 116 ER - TY - JOUR AU - Callan Jones, Andrew AU - Ruprecht, Verena AU - Wieser, Stefan AU - Heisenberg, Carl-Philipp J AU - Voituriez, Raphaël ID - 1275 IS - 13 JF - Physical Review Letters TI - Callan-Jones et al. Reply VL - 117 ER - TY - JOUR AB - During metazoan development, the temporal pattern of morphogen signaling is critical for organizing cell fates in space and time. Yet, tools for temporally controlling morphogen signaling within the embryo are still scarce. Here, we developed a photoactivatable Nodal receptor to determine how the temporal pattern of Nodal signaling affects cell fate specification during zebrafish gastrulation. By using this receptor to manipulate the duration of Nodal signaling in vivo by light, we show that extended Nodal signaling within the organizer promotes prechordal plate specification and suppresses endoderm differentiation. Endoderm differentiation is suppressed by extended Nodal signaling inducing expression of the transcriptional repressor goosecoid (gsc) in prechordal plate progenitors, which in turn restrains Nodal signaling from upregulating the endoderm differentiation gene sox17 within these cells. Thus, optogenetic manipulation of Nodal signaling identifies a critical role of Nodal signaling duration for organizer cell fate specification during gastrulation. AU - Sako, Keisuke AU - Pradhan, Saurabh AU - Barone, Vanessa AU - Inglés Prieto, Álvaro AU - Mueller, Patrick AU - Ruprecht, Verena AU - Capek, Daniel AU - Galande, Sanjeev AU - Janovjak, Harald L AU - Heisenberg, Carl-Philipp J ID - 1100 IS - 3 JF - Cell Reports TI - Optogenetic control of nodal signaling reveals a temporal pattern of nodal signaling regulating cell fate specification during gastrulation VL - 16 ER - TY - JOUR AB - 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. AU - Maiuri, Paolo AU - Rupprecht, Jean AU - Wieser, Stefan AU - Ruprecht, Verena AU - Bénichou, Olivier AU - Carpi, Nicolas AU - Coppey, Mathieu AU - De Beco, Simon AU - Gov, Nir AU - Heisenberg, Carl-Philipp J AU - Lage Crespo, Carolina AU - Lautenschlaeger, Franziska AU - Le Berre, Maël AU - Lennon Duménil, Ana AU - Raab, Matthew AU - Thiam, Hawa AU - Piel, Matthieu AU - Sixt, Michael K AU - Voituriez, Raphaël ID - 1553 IS - 2 JF - Cell TI - Actin flows mediate a universal coupling between cell speed and cell persistence VL - 161 ER - TY - JOUR AB - 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. AU - Ruprecht, Verena AU - Wieser, Stefan AU - Callan Jones, Andrew AU - Smutny, Michael AU - Morita, Hitoshi AU - Sako, Keisuke AU - Barone, Vanessa AU - Ritsch Marte, Monika AU - Sixt, Michael K AU - Voituriez, Raphaël AU - Heisenberg, Carl-Philipp J ID - 1537 IS - 4 JF - Cell TI - Cortical contractility triggers a stochastic switch to fast amoeboid cell motility VL - 160 ER - TY - JOUR AB - In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic 'roadmap' that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity. AU - Lamprecht, Constanze AU - Plochberger, Birgit AU - Ruprecht, Verena AU - Wieser, Stefan AU - Rankl, Christian AU - Heister, Elena AU - Unterauer, Barbara AU - Brameshuber, Mario AU - Danzberger, Jürgen AU - Lukanov, Petar AU - Flahaut, Emmanuel AU - Schütz, Gerhard AU - Hinterdorfer, Peter AU - Ebner, Andreas ID - 1925 IS - 12 JF - Nanotechnology TI - A single-molecule approach to explore binding uptake and transport of cancer cell targeting nanotubes VL - 25 ER - TY - CHAP AB - 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. AU - Smutny, Michael AU - Behrndt, Martin AU - Campinho, Pedro AU - Ruprecht, Verena AU - Heisenberg, Carl-Philipp J ED - Nelson, Celeste ID - 6178 SN - 1064-3745 T2 - Tissue Morphogenesis TI - UV laser ablation to measure cell and tissue-generated forces in the zebrafish embryo in vivo and ex vivo VL - 1189 ER - TY - JOUR AB - 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. AU - Weghuber, Julian AU - Aichinger, Michael C. AU - Brameshuber, Mario AU - Stefan Wieser AU - Verena Ruprecht AU - Plochberger, Birgit AU - Madl, Josef AU - Horner, Andreas AU - Reipert, Siegfried AU - Lohner, Karl AU - Henics, Tamas AU - Schuetz, Gerhard J ID - 3286 IS - 10 JF - Biochimica et Biophysica Acta (BBA) - Biomembranes TI - Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells VL - 1808 ER - TY - JOUR AB - 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. AU - Ruprecht, Verena AU - Axmann, Markus AU - Wieser, Stefan AU - Schuetz, Gerhard ID - 3287 IS - 8 JF - Current Protein & Peptide Science TI - What can we learn from single molecule trajectories? VL - 12 ER - TY - JOUR AB - 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. AU - Ruprecht, Verena AU - Wieser, Stefan AU - Marguet, Didier AU - Schuetz, Gerhard ID - 3285 IS - 11 JF - Biophysical Journal TI - Spot variation fluorescence correlation spectroscopy allows for superresolution chronoscopy of confinement times in membranes VL - 100 ER -