Membrane elastic properties and cell function

Abstract

Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.

Figure 1. Tether extraction from microglial cell.

(A) Image of the extracted tether in bright field with Image J shadow north processing filter applied. (B) Zoom of the white rectangle in A. Scale bar for A is 10 µm and for B is 5 µm. (C) A typical tether extraction force curve, indicating the maximum force F_m and the approach to F ₀, the steady-state tether force.

Figure 2. Tether extraction and radius measurements for neurons.

(A–C) Images of the cortical (CX) neurons cytoskeleton stained for F-actin with phaloidin-FITC, in green (A), β-tubulin III, in red (B) and the merge of both images (C). (D–F) Images of the ganglionic eminence (GE) neurons cytoskeleton stained for F-actin with phaloidin-FITC, in green (D), β-tubulin III, in red (E) and the merge of both images (F). (C) and (F) also display numbers locating the 3 different regions from which tethers were extracted, (1) cell body; (2) neurite; (3) growth cone. (G) Mean values of the tether force, , extracted from both Neuron CX and Neuron GE in regions 1, 2 and 3. (H) SEM image of a typical tether extracted from Neuron GE. Scale bar is 1 µm. (I) Mean values of the tether radius, R, extracted from both Neuron CX and Neuron GE in regions 1, 2 and 3. Standard errors were used as error bars in (G) and (H). At least 20 different experiments were performed for each situation in (G) and (H). (J)–(L) Images of tethers extracted from Neuron GE stained for F-actin with phaloidin-FITC, in green (J), β-tubulin III, in red (K) and the merge of both images (L). Scale bar is 5 µm.

Figure 3. Tether extraction and radius measurements for microglial cells.

(A–C) Images of the control microglia cytoskeleton stained for F-actin with phaloidin-FITC, in green (A), stained with F480, in red (B) and both images merged (C). (D–F) Images of the microglia+LPS cytoskeleton stained for F-actin with phaloidin-FITC, in green (C), F4/80, in red (D) and both images merged (F). Scale bars for A–F are all 10 µm. (G) Mean values of the tether force, , extracted from microglial cells. (H) SEM image of a typical tether extracted from microglia+LPS. Scale bar is 1 µm. (I) Image of tether extracted from control microglia stained for F-actin with phaloidin-FITC, in green. Scale bar is 5 µm. (J) Mean values of the tether radius, R, extracted from both microglial cell conditions. Standard errors were used as error bars in (G) and (J). At least 20 different experiments were performed for each situation in (G) and (J) (*** means p<0.0001 in t-test statistics).

Figure 4. Tether extraction and radius measurements for macrophage cells.

(A–C) Images of the control macrophage cytoskeleton stained for F-actin with phaloidin-FITC, in green (A), stained with CD68, in red (B) and both images merged (C). (D–F) Images of the macrophage+LPS cytoskeleton stained for F-actin with phaloidin-FITC, in green (C), CD68, in red (D) and both images merged (F). Scale bars for A-F are all 10 µm. (G) Mean values of the tether force, , extracted from macrophage cells in both conditions. (H) SEM image of a typical tether extracted from macrophage+LPS cells. Scale bar is 1 µm. (I) Mean values of the tether radius, R, extracted from macrophage cells in both conditions. Standard errors were used as error bars in (G) and (I). At least 20 different experiments were performed for each situation in (G) and (I). (*** means p<0.0001 in t-test statistics).

Figure 5. Tether extraction and radius measurements for astrocytes and glioblastoma cells.

(A-B) Images of the astrocyte cytoskeleton stained for F-actin with phaloidin-FITC, in green (A) and GFAP, in red (B). (C-D) Images of glioblastomas U-87 MG and GBM95 cytoskeleton, respectively stained for F-actin with phaloidin-FITC, in green. Scale bars for A-D are all 10 µm. (E) Mean values of the tether force, , extracted from astrocytes and glioblastomas cells. (F) Mean values of the tether radius,R, extracted from astrocytes and glioblastomas cells. Standard errors were used as error bars in (E) and (F). At least 20 different experiments were performed for each situation in (E) and (F). (G-I) Images of tethers extracted from astrocytes, stained for F-actin with phaloidin-FITC, in green (G) and GFAP, in red (H). (G) and (H) merged in (I). Scale bars for G-I are all 5 µm.

Figure 6. Plasma membrane vesicles growth dynamics.

(A) Time 0min, U-87 MG cells immediately after treating with the PMV solution. (B) Time 10 min, appearance of small PMVs, indicated with white arrows. (C) Time 20 min, PMVs growth. (D) Time 30 min, some PMVs reach their maximum size. Scale bar is 50 µm.

Figure 7. Membrane tether extraction from PMV.

(A) Selection of images of the tether extraction experiment from a PMV surface. (1) Initial moment, bead is being pressed against the PMV with the optical trap, (2) moment when the force reaches the maximum value and, (3) membrane tether from PMV already formed. Scale bar is 10 µm. (B) Force curve of a tether extraction from PMV. 1, 2, and 3 represent points in the plot from the frames in (A).

Figure 8. F_m and F₀ values obtained from PMV.

(A) Mean values of F_m found for PMV in each of the cell types studied. (B) Mean values of F₀ found for PMV in each of the cell types studied. Standard errors were used as error bars. At least 20 different experiments were performed for each situation. Colors in the plots represent the different cell types used, as indicated in the legend.

Figure 9. Patch radius measured from the bead/membrane contact in PMV.

(A–B) representative image (A) and zoom (B) indicating how R_p is obtained. Scale bar for A is 10 µm and for B is 5 µm. (C) Plot of the mean values of R_p for each of the cell types used. Standard errors were used as error bars. At least 20 different experiments were performed for each situation. Colors in the plots represent the different cell types used, as indicated by the legend.