Nanometric thermal fluctuations of weakly confined biomembranes measured with microsecond time-resolution

Abstract

We probe the bending fluctuations of bio-membranes using highly deflated giant unilamellar vesicles (GUVs) bound to a substrate by a weak potential arising from generic interactions. The substrate is either homogeneous, with GUVs bound only by the weak potential, or is chemically functionalized with a micro-pattern of very strong specific binders. In both cases, the weakly adhered membrane is seen to be confined at a well-defined distance above the surface while it continues to fluctuate strongly. We quantify the fluctuations of the weakly confined membrane at the substrate proximal surface as well as of the free membrane at the distal surface of the same GUV. This strategy enables us to probe in detail the damping of fluctuations in the presence of the substrate, and to independently measure the membrane tension and the strength of the generic interaction potential. Measurements were done using two complementary techniques -dynamic optical displacement spectroscopy (DODS, resolution: 20 nm, 10 ms), and dual wavelength reflection interference contrast microscopy (DW-RICM, resolution: 4 nm, 50 ms). After accounting for the spatio-temporal resolution of the techniques, an excellent agreement between the two measurements was obtained. For both weakly confined systems we explore in detail the link between fluctuations on the one hand and membrane tension and the interaction potential on the other hand.

The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering

“…Many studies have shown that the plasma membrane of cells, including lymphocytes, display thermally induced stochastic displacements or fluctuations with an amplitude of several tens of nanometers at frequencies of 0.1–1 s (67, 68, 77, 78). We therefore speculate that mechanical forces can be generated, albeit inefficiently, by stochastic membrane fluctuations (sometimes the opposing T cell and APC membranes at sites of TCR engagement are fluctuating in the same direction and sometimes they move in opposite directions; forces should only be generated when they move in opposite directions).…”

Statistical Mechanics of an Elastically Pinned Membrane: Equilibrium Dynamics and Power Spectrum

“…In the presence of an impermeable wall, damping coefficients are modified [51,52], which in the case of protein mediated adhesion, typically has an effect only on the amplitude of the first few membrane modes [49]. Furthermore, if the membrane is surrounded by two different fluids with viscosities η 1 and η 2 , the viscosity η in the damping coefficients is replaced by the arithmetic mean η = (η 1 + η 2 )/2 [15].…”

“…In order to compare with experiments, it is necessary to account for the finite temporal and spatial resolutions of the set-up [15,54]. Averaging the true membrane profile u(r, t) over a spatial domain A and a time interval τ , gives rise the so-called apparent membrane profile u A τ (r, t)…”

“…While occurring on time scales between 10 −9 and 10 −5 s [7][8][9][10], the fluctuations are overdamped by the surrounding fluid [11][12][13][14]. Nonetheless, mean fluctuation amplitudes of up to 100 nm have been observed experimentally [15]. In a vicinity of a substrate, these fluctuations are known to contribute to an effective potential which prevents the membrane from non-specifically adhering to the underlying scaffold [1,3,10,[16][17][18][19][20][21].…”

“…In a vicinity of a substrate, these fluctuations are known to contribute to an effective potential which prevents the membrane from non-specifically adhering to the underlying scaffold [1,3,10,[16][17][18][19][20][21]. The scaffold in turn affects the hydrodynamic damping of the membrane reflected in changes of the time dependent correlation function and the associated power spectrum, the so-called power spectral density (PSD) [15,[22][23][24]. Moreover, in the cellular environment, active processes couple with the membrane fluctuations [23][24][25][26][27][28][29][30][31][32][33], resulting in the violation of the fluctuationdissipation theorem in the activated state of the cell [34][35][36].…”