Effects of cholesterol on nano-mechanical properties of the living cell plasma membrane

Abstract

In this study, we investigated the effects of membrane cholesterol content on the mechanical properties of cell membranes by using optical tweezers. We pulled membrane tethers from human embryonic kidney cells using single and multi-speed protocols, and obtained time-resolved tether forces. We quantified various mechanical characteristics including the tether equilibrium force, bending modulus, effective membrane viscosity, and plasma membrane-cytoskeleton adhesion energy, and correlated them to the membrane cholesterol level. Decreases in cholesterol concentration were associated with increases in the tether equilibrium force, tether stiffness, and adhesion energy. Tether diameter and effective viscosity increased with increasing cholesterol levels. Disruption of cytoskeletal F-actin significantly changed the tether diameters in both non-cholesterol and cholesterol-manipulated cells, while the effective membrane viscosity was unaffected by F-actin disruption. The findings are relevant to inner ear function where cochlear amplification is altered by changes in membrane cholesterol content.

Fig. 1

Typical temporal plasma membrane tether force plot for a HEK cell in response to a single-speed pulling protocol. Force plot shows tether formation (TF), tether elongation (TE), and tether force relaxation (REL) regions. Tether is formed and elongated at a constant pulling rate of force, 1μm/s to 20μm. F_max-maximum tether force, F_eq-equilibrium tether F₁-tether force at the end of elongation.

Fig. 2

Plasma membrane tether equilibrium forces versus plasma membrane cholesterol concentration for HEK cells with intact and disrupted F-actin.

Fig. 3

Bright-field photomicrographs of HEK cell–membrane tether–bead assembly for cells with intact F-actin (a, b, c) and disrupted F-actin (d, e, f). The (b, e) images represent non-cholesterol manipulated cells. The (a, d) images represent the photomicrographs under cholesterol depletion (5.7 pmol/μg protein), and (c, f) represent the cholesterol enrichment condition (17.3 pmol/μg protein).

Fig. 4

Plasma membrane-cytoskeleton adhesion energy per unit area versus membrane cholesterol concentration for HEK cells with intact and disrupted F-actin cells. Inset shows plasma membrane tether diameter estimates for HEK cells with intact and disrupted F-actin versus membrane cholesterol concentration in pMol/μg protein.

Fig. 5

Typical temporal plasma membrane tether force plot for a HEK cell in response to multi-speed pulling protocol. This plot is for a HEK cell incubated in 5 mM MβCD-cholesterol for 30 minutes in order to elevate the membrane cholesterol content. The initial negative value of the force before the beginning of tether pulling results from the cell pushing the bead in a direction opposite to that of the stage movement. Inset shows single exponential curve fit to the 3^rd segment of membrane tether force profile. R²=0.95 for this pulling rate, and >0.89 for other pulling rates (data not shown).

Fig. 6

(A) Steady-state membrane tether forces linearly fit to their corresponding pulling rates for the membrane force plot shown in Fig. 5. (B) Membrane tether effective viscosities versus membrane cholesterol concentrations for HEK cells with intact and disrupted F-actin.

Fig. 7

Two-stage plasma membrane tether force relaxation, the first rapid force relaxation followed by a longer one. The force plot is fit to a single and bi-exponential force decays.

Fig. 8

(A) Static membrane tether forces as a function of tether length for control HEK cells and cells under cholesterol depletion and cholesterol enrichment conditions. The numbers on each force plot indicate the corresponding membrane cholesterol concentration (pMol/μg protein). (B) Plasma membrane tether stiffness (k) in HEK cells estimated using static forces shown in (A) versus membrane cholesterol concentration. (C) Plasma membrane tether stiffness in HEK cells estimated using force relaxation-corrected method (krc) versus membrane cholesterol concentrations.