Biomedical subjects

Gaudenz Danuser

Publications and source records attributed to Gaudenz Danuser.

13 recordsLinked to original sources

PubMed2007

Differential transmission of actin motion within focal adhesions.

Cell migration requires the transmission of motion generated in the actin cytoskeleton to the extracellular environment through a complex assembly of proteins in focal adhesions. We developed correlational fluorescent speckle microscopy to measure the coupling of focal-adhesion proteins to actin filaments. Different classes of focal-adhesion structural and regulatory molecules exhibited varying degrees of correlated motions with actin filaments, indicating hierarchical transmission of actin motion through focal adhesions. Interactions between vinculin, talin, and actin filaments appear to constitute a slippage interface between the cytoskeleton and integrins, generating a molecular clutch that is regulated during the morphodynamic transitions of cell migration.

Actin Cytoskeleton↗

PubMed2006

Linking data to models: data regression.

Mathematical models are an essential tool in systems biology, linking the behaviour of a system to the interactions between its components. Parameters in empirical mathematical models must be determined using experimental data, a process called regression. Because experimental data are noisy and incomplete, diagnostics that test the structural identifiability and validity of models and the significance and determinability of their parameters are needed to ensure that the proposed models are supported by the available data.

Animals↗

PubMed2006

Comparative autoregressive moving average analysis of kinetochore microtubule dynamics in yeast.

To elucidate the regulation of kinetochore microtubules (kMTs) by kinetochore proteins in Saccharomyces cerevisiae, we need tools to characterize and compare stochastic kMT dynamics. Here we show that autoregressive moving average (ARMA) models, combined with a statistical framework for testing the significance of differences between ARMA model parameters, provide a sensitive method for identifying the subtle changes in kMT dynamics associated with kinetochore protein mutations. Applying ARMA analysis to G1 kMT dynamics, we found that 1), kMT dynamics in the kinetochore protein mutants okp1-5 and kip3Delta are different from those in wild-type, demonstrating the regulation of kMTs by kinetochore proteins; 2), the kinase Ipl1p regulates kMT dynamics also in G1; and 3), the mutant dam1-1 exhibits three different phenotypes, indicating the central role of Dam1p in maintaining the attachment of kMTs and regulating their dynamics. We also confirmed that kMT dynamics vary with temperature, and are most likely differentially regulated at 37 degrees C. Therefore, when elucidating the role of a protein in kMT regulation using a temperature-sensitive mutant, dynamics in the mutant at its nonpermissive temperature must be compared to those in wild-type at the same temperature, not to those in the mutant at its permissive temperature.

Algorithms↗

PubMed2006

Kinesin 5-independent poleward flux of kinetochore microtubules in PtK1 cells.

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from approximately 0.7 to approximately 0.5 microm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar "pulling-in" mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.

Animals↗

PubMed2006

Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.

Fluorescent speckle microscopy (FSM) is a technology used to analyze the dynamics of macromolecular assemblies in vivo and in vitro. Speckle formation by random association of fluorophores with a macromolecular structure was originally discovered for microtubules. Since then FSM has been expanded to study other cytoskeleton and cytoskeleton-binding proteins. Specialized software has been developed to convert the stochastic speckle image signal into spatiotemporal maps of polymer transport and turnover in living cells. These maps serve as a unique quantitative readout of the dynamic steady state of the cytoskeleton and its responses to molecular and genetic interventions, allowing a systematic study of the mechanisms of cytoskeleton regulation and its effect on cell function. Here, we explain the principles of FSM imaging and signal analysis, outline the biological questions and corresponding methodological advances that have led to the current state of FSM, and give a glimpse of new FSM modalities under development.

Animals↗

PubMed2005

Integrin-dependent actomyosin contraction regulates epithelial cell scattering.

The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions. We show that HGF does not trigger any detectable decrease in E-cadherin function, but increases integrin-mediated adhesion. Time-lapse imaging suggests that tension on cell-cell junctions may disrupt cell-cell adhesion. Varying the density and type of extracellular matrix proteins shows that scattering correlates with stronger integrin adhesion and increased phosphorylation of the myosin regulatory light chain. To directly test the role of integrin-dependent traction forces, substrate compliance was varied. Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates. We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.

Actomyosin↗

PubMed2005

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

Actin Depolymerizing Factors↗

PubMed2005

Tracking retrograde flow in keratocytes: news from the front.

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1-3 microm/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization.

Actins↗

PubMed2004

Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy.

We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion. However, how they are orchestrated to produce directed cell movements is poorly understood. Our approach to data analysis allows us to examine their interdependence. Our maps confirm the previously described organization of flow into a lamellipodium and a lamellum, both exhibiting retrograde flow; and a convergence zone, where lamellum retrograde flow meets with slow anterograde flow of cortical F-actin at the ventral side of the cell body. The turnover maps show the well known actin polymerization at the leading edge, but also indicate that approximately 90% of the polymer disassembles at the lamellipodium-lamellum junction. Strong depolymerization is also found in the convergence zone, where meshwork contraction is prominent. To determine whether contraction and depolymerization are coupled events, we have treated cells with calyculin A, which is known to promote myosin activity. Stimulated contraction was accompanied by accelerated retrograde flow and increased depolymerization throughout the lamellum, whereas disassembly at the lamellipodium-lamellum junction remained unaffected. There appear to be two distinct depolymerization mechanisms, of which one depends directly on meshwork contraction.

Actins↗

PubMed2003

FRET or no FRET: a quantitative comparison.

Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. In biological applications, this technique has become popular to qualitatively map protein-protein interactions, and in biophysical projects it is used as a quantitative measure for distances between a single donor and acceptor molecule. Numerous approaches can be found in the literature to quantify and map FRET, but the measures they provide are often difficult to interpret. We propose here a quantitative comparison of these methods by using a surface FRET system with controlled amounts of donor and acceptor fluorophores and controlled distances between them. We support the system with a Monte Carlo simulation of FRET, which provides reference values for the FRET efficiency under various experimental conditions. We validate a representative set of FRET efficiencies and indices calculated from the different methods with different experimental settings. Finally, we test their sensitivity and draw conclusions for the preparation of FRET experiments in more complex and less-controlled systems.

Algorithms↗

PubMed2003

Microcontact printing of novel co-polymers in combination with proteins for cell-biological applications.

Microcontact printing (microcP) is a cost effective and simple method to create chemically micropatterned surfaces for cell biological applications. We have combined the technique with the spontaneous molecular assembly of a polycationic PEG-grafted copolymer, poly-L-lysine-g-poly(ethylene glycol) (PLL-g-PEG). PLL-g-PEG with omega-functionalized PEG chains was print-transferred onto tissue culture polystyrene (TCPS) or glass substrates, resulting in patterns with a lateral resolution down to 1 microm. Subsequently, dipping in an aqueous solution of non-functionalized PLL-g-PEG was used to backfill the non-printed regions of the surface, rendering them highly protein and thus cell resistant. In a second approach, proteins were stamped and a PLL-g-PEG backfill was applied for passivation of the bare surface regions. Printing of peptide(RGD)-functionalized PLL-g-PEG or proteins combined with a subsequent PLL-g-PEG backfill can be applied to a wide variety of substrate materials with negatively charged surfaces such as TCPS, glass and many metal oxides. We have tested the printed surfaces with human foreskin fibroblasts for cell adhesion and long-term performance and with fish epidermal keratocytes for cell motility and short-time behaviour. Both cell types reacted selectively to the surface micropatterns. Fibroblasts adhered to the printed (adhesive) regions only, where they remained attached up to at least 1 week and were even able to proliferate. Keratocyte spreading and motility were also directed by the geometry of the underlying patterns. The results prove that microcP in conjunction with the use of PLL-g-PEG and its derivatives provides a simple and robust alternative to previously reported micropatterning methods for future cell biological and biotechnological applications.

Animals↗

PubMed2002

New directions for fluorescent speckle microscopy.

Fluorescent Speckle Microscopy (FSM) is a technology for analyzing cytoskeleton dynamics, giving novel insight into their roles in living cells. New applications of FSM, together with the development of computer-based FSM image analysis, will make FSM the first microscopy-based method to deliver quantitative kinetic readouts at high spatial and temporal resolution for a wide variety of macromolecular systems. Here, we review the most recent applications and developments and give a glimpse of future directions and potentials of FSM.

Actins↗

PubMed2002

Single muscle fiber contraction is dictated by inter-sarcomere dynamics.

This paper presents first results from a study where we developed a generic framework for analysing inter-sarcomere dynamics. Our objective is to find an accurate description of a muscle as a linear motor composed of parallel and series coupled subunits. The quality of theoretical models can be tested through their ability to predict experimental observations. With the current method we have found rigorous mathematical explanations for mechanisms such as sarcomere popping, extra tension and homogenization. These phenomena have been observed for many years in single fibers experiments, yet have never been completely understood in terms of a mechanical model. Now they can be explained on a theoretical basis. Interestingly, rather simplistic descriptions of each of the various molecular components in the sarcomere (actin-myosin cross-bridges, titin and contributions from passive elastic components) are sufficient to predict these behaviors. The complexity of a real muscle fiber is addressed through rigorous coupling of the single component models in a system of differential equations. We examine the properties of the differential equations, based on a down-stripped model, which permits the derivation of analytical solutions. They suggest that the contraction characteristics of inter-connected sarcomeres are essentially dictated by the initial distribution of the sarcomeres on the force-length curve and their starting velocities. The complete model is applied to show the complexity of inter-sarcomere dynamics of activated fibers in stretch-release experiments with either external force or length control. Seemingly contradictory and unexpected observations from single fiber experiments, which have hitherto been discussed with the argument of uncontrollable biological variability, turn out to be a consistent set of possible fiber responses. They result from a convolution of multiple relatively simple rules each of them defining a certain characteristics of the single sarcomere.

Animals↗