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 Fm and the approach to F0, the steady-state tether force.
Table 1.
Surface tension and bending modulus values.
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.
Fm and F0 values obtained from PMV.
(A) Mean values of Fm 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 Rp is obtained. Scale bar for A is 10 µm and for B is 5 µm. (C) Plot of the mean values of Rp 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.