ABSTRACT
The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.
Subject(s)
Actins/physiology , Cell Movement/physiology , Models, Biological , Animals , Biomechanical Phenomena/physiology , Computer Simulation , Fluorescence , Kinetics , Magnetics , Polymers , Rabbits , ThermodynamicsABSTRACT
The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point.
Subject(s)
Actin Cytoskeleton/chemistry , Stress, Mechanical , Biophysical Phenomena , Elasticity , Entropy , Gelsolin/chemistry , Kinetics , Magnetics , Models, Biological , PolymerizationABSTRACT
Oil and water can only be mixed by dispersing droplets of one fluid in the other. When two droplets approach one another, the thin film that separates them invariably becomes unstable, causing the droplets to coalesce. The only known way to avoid this instability is through addition of a third component, typically a surfactant, which stabilizes the thin film at its equilibrium thickness. We report the observation that a thin fluid film of oil separating two water droplets can lead to an adhesive interaction between the droplets. Moreover, this interaction prevents their coalescence over timescales of several weeks, without the use of any surfactant or solvent.
ABSTRACT
Recently a new di-gold(I) organometallic complex [1,3-(Ph(3)PAu)(2)-C(6)H(4)] (KF0101) has been synthesised and found to exhibit cytotoxic activity in vitro. Subsequently it has been demonstrated that KF0101 shows little or no cross-resistance against a number of the cisplatin resistant ovarian cancer cell lines in vitro suggesting a different mode of action for the drug. In this study, syncrotron radiation infrared microspectroscopy (SR-IRMS) has been used on drug treated single A2780 cells in order to determine if this different mode of action can be identified spectroscopically. The aim of the study was to establish: (i) if single cell SR-IRMS could be used to give insight into the cellular response on treatment with different cytotoxic agents relative to non-treated cells (control) and (ii) that if the cytotoxic drugs elicit a different biochemical response these responses could be distinguished from each other. The most striking features obtained after Principal Components Analysis (PCA) of Resonant Mie Scattering (RMieS) corrected single cell spectra of drug treated ovarian A2780 cells are: (i) The spectra obtained for the control are quite heterogeneous and several hundred spectra are required to adequately define the nature of the control; (ii) after drug treatment at the IC50 level for 24 h with cisplatin, KF0101, methotrexate, paclitaxel or 5-fluorouracil the cell spectra, as represented on a PCA scores plot, generally concentrate in certain well defined areas of the control, there are however a small number of spectra that fall outside of the area defined by the control; and (iii) a differentiation between cell spectra obtained on treatment with different drugs is observed which fits well with different in vitro cell culture behaviour and a flow cytometry cell cycle analysis of the control and drug treated cells. Inspection of the loading plots shows that PC1 is essentially the same for all plots and reflects changes in cell biochemistry related to the cell cycle. PC2, however, on comparison of the control versus cisplatin or cisplatin versus KF0101 is indicative of differences induced by drug treatment and has been termed as cell cycle-plus behaviour. These data are shown to be consistent with that obtained using bench-top IRMS by averaging a number of single cell spectra and carrying out a PCA, but SR-IRMS offers more insight into how the drug is affecting the cell population. More importantly, this approach enables the influence of the cell cycle on both the control and drug treated samples to be taken into consideration when evaluating the drug-cell interaction.