Load fluctuations drive actin network growth

The growth of actin filament networks is a fundamental biological process that drives a variety of cellular and intracellular motions. During motility, eukaryotic cells and intracellular pathogens are propelled by actin networks organized by nucleation-promoting factors, which trigger the formation of nascent filaments off the side of existing filaments in the network. A Brownian ratchet (BR) mechanism has been proposed to couple actin polymerization to cellular movements, whereby thermal motions are rectified by the addition of actin monomers at the end of growing filaments. Here, by following actin--propelled microspheres using three--dimensional laser tracking, we find that beads adhered to the growing network move via an object--fluctuating BR. Velocity varies with the amplitude of thermal fluctuation and inversely with viscosity as predicted for a BR. In addition, motion is saltatory with a broad distribution of step sizes that is correlated in time. These data point to a model in which thermal fluctuations of the microsphere or entire actin network, and not individual filaments, govern motility. This conclusion is supported by Monte Carlo simulations of an adhesion--based BR and suggests an important role for membrane tension in the control of actin--based cellular protrusions.Comment: To be published in PNA

In Silico Reconstitution of Actin-Based Symmetry Breaking and Motility

Computational modeling and experimentation in a model system for actin-based force generation explain how actin networks initiate and maintain directional movement

Rheological Characterization of the Bundling Transition in F-Actin Solutions Induced by Methylcellulose

In many in vitro experiments Brownian motion hampers quantitative data analysis. Therefore, additives are widely used to increase the solvent viscosity. For this purpose, methylcellulose (MC) has been proven highly effective as already small concentrations can significantly slow down diffusive processes. Beside this advantage, it has already been reported that high MC concentrations can alter the microstructure of polymer solutions such as filamentous actin. However, it remains to be shown to what extent the mechanical properties of a composite actin/MC gel depend on the MC concentration. In particular, significant alterations might occur even if the microstructure seems unaffected. Indeed, we find that the viscoelastic response of entangled F-actin solutions depends sensitively on the amount of MC added. At concentrations higher than 0.2% (w/v) MC, actin filaments are reorganized into bundles which drastically changes the viscoelastic response. At small MC concentrations the impact of MC is more subtle: the two constituents, actin and MC, contribute in an additive way to the mechanical response of the composite material. As a consequence, the effect of methylcellulose on actin solutions has to be considered very carefully when MC is used in biochemical experiments

An Experimental and Computational Study of the Effect of ActA Polarity on the Speed of Listeria monocytogenes Actin-based Motility

Listeria monocytogenes is a pathogenic bacterium that moves within infected cells and spreads directly between cells by harnessing the cell's dendritic actin machinery. This motility is dependent on expression of a single bacterial surface protein, ActA, a constitutively active Arp2,3 activator, and has been widely studied as a biochemical and biophysical model system for actin-based motility. Dendritic actin network dynamics are important for cell processes including eukaryotic cell motility, cytokinesis, and endocytosis. Here we experimentally altered the degree of ActA polarity on a population of bacteria and made use of an ActA-RFP fusion to determine the relationship between ActA distribution and speed of bacterial motion. We found a positive linear relationship for both ActA intensity and polarity with speed. We explored the underlying mechanisms of this dependence with two distinctly different quantitative models: a detailed agent-based model in which each actin filament and branched network is explicitly simulated, and a three-state continuum model that describes a simplified relationship between bacterial speed and barbed-end actin populations. In silico bacterial motility required a cooperative restraining mechanism to reconstitute our observed speed-polarity relationship, suggesting that kinetic friction between actin filaments and the bacterial surface, a restraining force previously neglected in motility models, is important in determining the effect of ActA polarity on bacterial motility. The continuum model was less restrictive, requiring only a filament number-dependent restraining mechanism to reproduce our experimental observations. However, seemingly rational assumptions in the continuum model, e.g. an average propulsive force per filament, were invalidated by further analysis with the agent-based model. We found that the average contribution to motility from side-interacting filaments was actually a function of the ActA distribution. This ActA-dependence would be difficult to intuit but emerges naturally from the nanoscale interactions in the agent-based representation

Quantitative description of temperature induced self-aggregation thermograms determined by differential scanning calorimetry

A novel thermodynamic approach for the description of differential scanning calorimetry (DSC) experiments on self-aggregating systems is derived and presented. The method is based on a mass action model where temperature dependence of aggregation numbers is considered. The validity of the model was confirmed by describing the aggregation behavior of poly(ethylene oxide)-poly(propylene oxide) block copolymers, which are well-known to exhibit a strong temperature dependence. The quantitative description of the thermograms could be performed without any discrepancy between calorimetric and van 't Hoff enthalpies, and moreover, the aggregation numbers obtained from the best fit of the DSC experiments are in good agreement with those obtained by light scattering experiments corroborating the assumptions done in the derivation of the new model

Interplay of Magnetic Interactions and Active Movements in the Formation of Magnetosome Chains

Magnetotactic bacteria assemble chains of magnetosomes, organelles that contain magnetic nano-crystals. A number of genetic factors involved in the controlled biomineralization of these crystals and the assembly of magnetosome chains have been identified in recent years, but how the specific biological regulation is coordinated with general physical processes such as diffusion and magnetic interactions remains unresolved. Here, these questions are addressed by simulations of different scenarios for magnetosome chain formation, in which various physical processes and interactions are either switched on or off. The simulation results indicate that purely physical processes of magnetosome diffusion, guided by their magnetic interactions, are not sufficient for the robust chain formation observed experimentally and suggest that biologically encoded active movements of magnetosomes may be required. Not surprisingly, the chain pattern is most resembling experimental results when both magnetic interactions and active movement are coordinated. We estimate that the force such active transport has to generate is compatible with forces generated by the polymerization or depolymerization of cytoskeletal filaments. The simulations suggest that the pleiotropic phenotypes of mamK deletion strains may be due to a defect in active motility of magnetosomes and that crystal formation in magneteosome vesicles is coupled to the activation of their active motility in M. gryphiswaldense, but not in M. magneticum

A Novel Pseudopodial Component of the Dendritic Cell Anti-Fungal Response: The Fungipod

Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the “fungipod”, formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 µm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion