Quantitative investigation of negative membrane curvature sensing and generation by I-BARs in filopodia of living cells.

Membrane curvature has recently been recognized as an active regulator of cellular function, with several protein families identified as sensors and generators of membrane curvature. Amongst them, the inverse Bin/Amphiphysin/Rvs (I-BAR) domain family has been implicated in the sensing and generation of membrane structures with negative membrane curvature e.g. filopodia or dendritic spines. However, to date, quantitative biophysical investigations of I-BAR domains have mostly taken place in reconstitution. Here, we use fluorescence microscopy to quantitatively investigate membrane curvature sensing and generation by I-BARs in filopodia of living cells. As a model system, we selected two prototypic members of the I-BAR family, the insulin receptor substrate p53 and missing-in-metastasis. Our data demonstrated how I-BARs sense negative membrane curvature in the complex environment of live cells by revealing a dependence on membrane curvature for both their binding affinity to membranes and their saturation density. The non-monotonic dependence of protein sorting with negative membrane curvature allowed us to apply previously developed thermodynamic models to provide estimates of the effective intrinsic curvature and bending rigidity of the two I-BARs bound at the plasma membrane. Our results agree with studies performed on the insulin receptor substrate p53 in reconstitution. To quantitate membrane curvature generation by I-BARs we measured how their overexpression reduces the peak and the width of the size distribution of filopodia, resulting in filopodia populations with smaller and more uniform diameters. Our findings provide a quantitative biophysical insight in the ability of I-BARs to sense and generate negative membrane curvature in the crowded environment of living cells.

Contact angle at the leading edge controls cell protrusion rate.

Plasma membrane tension and the pressure generated by actin polymerization are two antagonistic forces believed to define the protrusion rate at the leading edge of migrating cells [1-5]. Quantitatively, resistance to actin protrusion is a product of membrane tension and mean local curvature (Laplace's law); thus, it depends on the local geometry of the membrane interface. However, the role of the geometry of the leading edge in protrusion control has not been yet investigated. Here, we manipulate both the cell shape and substrate topography in the model system of persistently migrating fish epidermal keratocytes. We find that the protrusion rate does not correlate with membrane tension, but, instead, strongly correlates with cell roundness, and that the leading edge of the cell exhibits pinning on substrate ridges-a phenomenon characteristic of spreading of liquid drops. These results indicate that the leading edge could be considered a triple interface between the substrate, membrane, and extracellular medium and that the contact angle between the membrane and the substrate determines the load on actin polymerization and, therefore, the protrusion rate. Our findings thus illuminate a novel relationship between the 3D shape of the cell and its dynamics, which may have implications for cell migration in 3D environments.

Accounting for inertia effects to access the high-frequency microrheology of viscoelastic fluids.

We study the Brownian motion of microbeads immersed in water and in a viscoelastic wormlike micelles solution by optical trapping interferometry and diffusing wave spectroscopy. Through the mean-square displacement obtained from both techniques, we deduce the mechanical properties of the fluids at high frequencies by explicitly accounting for inertia effects of the particle and the surrounding fluid at short time scales. For wormlike micelle solutions, we recover the 3/4 scaling exponent for the loss modulus over two decades in frequency as predicted by the theory for semiflexible polymers.

Hydrodynamic and subdiffusive motion of tracers in a viscoelastic medium.

We investigate the diffusive motion of micron-sized spherical tracers in a viscoelastic actin filament network over the time span of 8 orders of magnitude using optical-tweezers single-particle tracking. The hydrodynamic interactions of a tracer with the surrounding fluid are shown to dominate at microsecond time scales, while subdiffusive scaling due to viscoelastic properties of the medium emerges at millisecond time scales. The transition between these two regimes is analyzed in the frame of a minimal phenomenological model which combines the Basset force and the generalized Stokes force. The resulting Langevin equation accounts for various dynamical features of the thermal motion of endogenous or exogenous tracers in viscoelastic media such as inertial and hydrodynamic effects at short times, subdiffusive scaling at intermediate times, and eventual optical trapping at long times. Simple analytical formulas for the mean-square displacement and velocity autocorrelation function are derived.