Membrane rigidity regulates E. coli proliferation rates.

Combining single cell experiments, population dynamics and theoretical methods of membrane mechanics, we put forward that the rate of cell proliferation in E. coli colonies can be regulated by modifiers of the mechanical properties of the bacterial membrane. Bacterial proliferation was modelled as mediated by cell division through a membrane constriction divisome based on FtsZ, a mechanically competent protein at elastic interaction against membrane rigidity. Using membrane fluctuation spectroscopy in the single cells, we revealed either membrane stiffening when considering hydrophobic long chain fatty substances, or membrane softening if short-chained hydrophilic molecules are used. Membrane stiffeners caused hindered growth under normal division in the microbial cultures, as expected for membrane rigidification. Membrane softeners, however, altered regular cell division causing persistent microbes that abnormally grow as long filamentous cells proliferating apparently faster. We invoke the concept of effective growth rate under the assumption of a heterogeneous population structure composed by distinguishable individuals with different FtsZ-content leading the possible forms of cell proliferation, from regular division in two normal daughters to continuous growing filamentation and budding. The results settle altogether into a master plot that captures a universal scaling between membrane rigidity and the divisional instability mediated by FtsZ at the onset of membrane constriction.

A gradient-based, GPU-accelerated, high-precision contour-segmentation algorithm with application to cell membrane fluctuation spectroscopy.

We present a novel intensity-gradient based algorithm specifically designed for nanometer-segmentation of cell membrane contours obtained with high-resolution optical microscopy combined with high-velocity digital imaging. The algorithm relies on the image oversampling performance and computational power of graphical processing units (GPUs). Both, synthetic and experimental data are used to quantify the sub-pixel precision of the algorithm, whose analytic performance results comparatively higher than in previous methods. Results from the synthetic data indicate that the spatial precision of the presented algorithm is only limited by the signal-to-noise ratio (SNR) of the contour image. We emphasize on the application of the new algorithm to membrane fluctuations (flickering) in eukaryotic cells, bacteria and giant vesicle models. The method shows promising applicability in several fields of cellular biology and medical imaging for nanometer-precise boundary-determination and mechanical fingerprinting of cellular membranes in optical microscopy images. Our implementation of this high-precision flicker spectroscopy contour tracking algorithm (HiPFSTA) is provided as open-source at www.github.com/michaelmell/hipfsta.

Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase.

ATP synthase is a rotating membrane protein that synthesizes ATP through proton-pumping activity across the membrane. To unveil the mechanical impact of this molecular active pump on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large nonequilibrium deformations at discrete hot spots in lipid vesicles and thus inducing an overall membrane softening. The enhanced nonequilibrium fluctuations are compatible with an accumulation of active proteins at highly curved membrane sites through a curvature-protein coupling mechanism that supports the emergence of collective effects of rotating ATP synthases in lipid membranes.

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells.

Erythrocytes are flexible cells specialized in the systemic transport of oxygen in vertebrates. This physiological function is connected to their outstanding ability to deform in passing through narrow capillaries. In recent years, there has been an influx of experimental evidence of enhanced cell-shape fluctuations related to metabolically driven activity of the erythroid membrane skeleton. However, no direct observation of the active cytoskeleton forces has yet been reported to our knowledge. Here, we show experimental evidence of the presence of temporally correlated forces superposed over the thermal fluctuations of the erythrocyte membrane. These forces are ATP-dependent and drive enhanced flickering motions in human erythrocytes. Theoretical analyses provide support for a direct force exerted on the membrane by the cytoskeleton nodes as pulses of well-defined average duration. In addition, such metabolically regulated active forces cause global membrane softening, a mechanical attribute related to the functional erythroid deformability.

Fluctuation dynamics of bilayer vesicles with intermonolayer sliding: experiment and theory.

The presence of coupled modes of membrane motion in closed shells is extensively predicted by theory. The bilayer structure inherent to lipid vesicles is suitable to support hybrid modes of curvature motion coupling membrane bending with the local reorganization of the bilayer material through relaxation of the dilatational stresses. Previous experiments evidenced the existence of such hybrid modes facilitating membrane bending at high curvatures in lipid vesicles [Rodríguez-García, R., Arriaga, L.R., Mell, M., Moleiro, L.H., López-Montero, I., Monroy, F., 2009. Phys. Rev. Lett. 102, 128201.]. For lipid bilayers that are able to undergo intermonolayer sliding, the experimental fluctuation spectra are found compatible with a bimodal schema. The usual tension/bending fluctuations couple with the hybrid modes in a mechanical interplay, which becomes progressively efficient with increasing vesicle radius, to saturate at infinity radius into the behavior expected for a flat membrane. Grounded on the theory of closed shells, we propose an approximated expression of the bimodal spectrum, which predicts the observed dependencies on the vesicle radius. The dynamical features obtained from the autocorrelation functions of the vesicle fluctuations are found in quantitative agreement with the proposed theory.

Bending stiffness of biological membranes: what can be measured by neutron spin echo?

Large vesicles obtained by the extrusion method represent adequate membrane models to probe membrane dynamics with neutron radiation. Particularly, the shape fluctuations around the spherical average topology can be recorded by neutron spin echo (NSE). In this paper we report on the applicable theories describing the scattering contributions from bending-dominated shape fluctuations in diluted vesicle dispersions, with a focus on the relative relevance of the master translational mode with respect to the internal fluctuations. Different vesicle systems, including bilayer and non-bilayer membranes, have been scrutinized. We describe the practical ranges where the exact theory of bending fluctuations is applicable to obtain the values of the bending modulus from experiments, and we discuss about the possible internal modes that could be alternatively contributing to shape fluctuations.