T-lymphocyte passive deformation is controlled by unfolding of membrane surface reservoirs

Abstract

T-lymphocytes in the human body routinely undergo large deformations, both passively, when going through narrow capillaries, and actively, when transmigrating across endothelial cells or squeezing through tissue. We investigate physical factors that enable and limit such deformations and explore how passive and active deformations may differ. Employing micropipette aspiration to mimic squeezing through narrow capillaries, we find that T-lymphocytes maintain a constant volume while they increase their apparent membrane surface area upon aspiration. Human resting T-lymphocytes, T-lymphoblasts, and the leukemic Jurkat T-cells all exhibit membrane rupture above a critical membrane area expansion that is independent of either micropipette size or aspiration pressure. The unfolded membrane matches the excess membrane contained in microvilli and membrane folds, as determined using scanning electron microscopy. In contrast, during transendothelial migration, a form of active deformation, we find that the membrane surface exceeds by a factor of two the amount of membrane stored in microvilli and folds. These results suggest that internal membrane reservoirs need to be recruited, possibly through exocytosis, for large active deformations to occur.

FIGURE 1:

T-lymphocyte volume is conserved during micropipette aspiration. (A) Diagram of a micropipette aspiration experiment. We impose an aspiration pressure P₀ + ΔP (ΔP assumes only negative values) and measure the initial cell diameter, D₀, the final cell diameter, D_f, the micropipette diameter, D_p, and the final entry length, L. (B) Plot of the final cell volume, V_f (see Supplemental Figure S1 for details on volume measurement), as a function of initial cell volume, V₀. Data points fall on the dotted line of slope 1 (V_f = V₀), showing that cell volume is conserved.

FIGURE 2:

The apparent stiffness of a resting T-lymphocyte increases with apparent membrane surface area. (A) Example of a resting T-lymphocyte being aspirated into a micropipette while its apparent stiffness is measured using profile microindentation. Scale bar, 10 μm. (B) Plot of the aspiration pressure, ΔP, used to hold the resting T-lymphocytes during profile microindentation as a function of time. Each resting T-lymphocyte is indented ∼50 times, once every 30 s. Black, aspiration pressure is −15 Pa; green, −50 Pa; red, −100 Pa. (C) Histogram of the indentation scaling exponent β found during the profile microindentations. We fit the force–indentation curve using the relation F = α δ^β, with F the force, δ the indentation, and α and β two fitting parameters. (D) Plot of the apparent stiffness, E, as a function of the normalized apparent membrane surface area, A/A₀, where A₀ is the initial membrane surface area and A is the membrane surface area at the time when E is measured. Bars represent SD. Five cells and 201 microindentations. The number of cells decreases for large values of A/A₀. The three last points to the right correspond to only 5, 7, and 2 microindentations, respectively, compared with >30 on average for the six points to the left. Dotted line represents the best fit using the phenomenological relation E = E₀ for A/A₀ < 1 + ε and E = E₀ + k[A/A₀ − (1 + ε)] for A/A₀ > 1 + ε. (E) Plot of E (left axis, dotted line) and A/A₀ (right axis, continuous line) averaged over five cells, as a function of time.

FIGURE 3:

T-lymphocyte membrane ruptures at a well-defined entry length L* during micropipette aspiration. (A–C) Example of membrane rupture triggered using micropipette aspiration for (A) a resting T-lymphocyte, (B) a Jurkat cell, and (C) a lymphoblast. Scale bar, 10 μm. The time is indicated in the top right-hand corner, with t = 0 s chosen as the time at which the aspiration pressure goes from −20 Pa to ΔP. A background of bright-field light is kept to visualize the cell throughout the experiment. On membrane rupture, propidium iodide enters the cell and binds to DNA, emitting a bright fluorescent signal (red arrows). (D) Plot of the entry length at rupture, L*, vs. micropipette diameter, D_p, for three cell types: resting T-lymphocytes (14 cells), Jurkat cells (27 cells), and lymphoblasts (14 cells). Bars represent SD.

FIGURE 4:

Effect of the aspiration pressure on T-lymphocyte membrane rupture. (A) Plot of the entry length at rupture, L*, as a function of the absolute value of the aspiration pressure, ΔP, for resting T-lymphocytes (white circles) and Jurkat cells (black squares). (B) Plot of the duration of micropipette aspiration time, T, to rupture as a function of the absolute value of ΔP for resting T-lymphocytes (white circles) and Jurkat cells (black squares). Bars indicate SD. For resting T-lymphocytes, micropipette diameters between 2.0 and 2.8 μm were included (14 ruptured cells). For Jurkat cells, micropipette diameters between 5.8 and 6.2 μm were included (10 ruptured cells).

FIGURE 5:

T-lymphocyte membrane rupture occurs at a critical increase in apparent membrane surface area, A*/A₀. (A–C) “Phase diagram” of cell state after micropipette aspiration, depending on the micropipette diameter, D_p, and the membrane expansion, defined as the final apparent membrane surface area, A_f, divided by the initial apparent membrane surface area, A₀. The phase diagrams are given for various cell types and conditions: (A) resting T-lymphocytes, (B) Jurkat cells, and (C) lymphoblasts. A red cross indicates a cell whose membrane ruptured, a black circle a cell that stayed trapped after 5 min of aspiration, and a green diamond a cell that was entirely aspirated inside the micropipette. The red filling indicates that in this zone, cell membranes are expected to rupture. The gray filling indicates that in this zone, a cell is expected to stay trapped inside the micropipette without rupturing or being entirely aspirated. The cutoff on the vertical axis between the rad and gray zones is chosen as the mean increase in apparent membrane surface area. The green filling indicates that in this zone, cells are expected to be entirely aspirated, based on geometrical considerations and volume conservation (Supplemental Movie S6). (D) Boxplot of the normalized apparent membrane surface area at rupture, A*/A₀, for the cell types and conditions in A–C.

FIGURE 6:

Maximum membrane surface area increase during micropipette aspiration is of the same order of magnitude as the excess membrane contained in microvilli and membrane folds. (A–C) Scanning electron microscopy images of a lymphoblast (A, B) and a Jurkat cell (C). Scale bars, 2 μm (A), 400 nm (B), 5 μm (C). (D) Histogram of lymphoblast microvilli length (432 measurements, 10 cells). The length is 284 ± 140 nm (mean ± SD). (E) Histogram of lymphoblast microvilli diameter (48 measurements, one cell, as we observed that the diameter was well conserved across cells). The diameter is 62 ± 13 nm (mean ± SD). (F) Plot of the maximum increase in apparent membrane surface area during micropipette aspiration experiments (defined as the apparent membrane surface area at rupture, A*, minus the initial apparent membrane surface area, A₀) as a function of the estimated excess membrane contained in microvilli and membrane folds, A_mv, for resting T-lymphocytes (blue), Jurkat cells (red), and lymphoblasts (black). Squares represent the mean; bars represent the SD. Dotted line represents the A* − A₀ = A_mv (slope equal to 1) line.

FIGURE 7:

Evolution of lymphoblast membrane surface area during transendothelial migration and cell spreading. (A) Time lapse of a lymphoblast transmigrating between human aortic endothelial cells. Scale bar, 20 μm. Images were taken every 15 s. The projected surface area S_proj is represented in yellow before (leftmost image) and after (rightmost image) transendothelial migration. The pore diameter, D_pore, is estimated by taking the image in which the lymphoblast width is identical above and below the pore (yellow arrows). (B) D_pore during transendothelial migration as a function of the lymphoblast’s projected diameter before transendothelial migration (computed using D₀ = S_proj/2π, and D₀ is an equivalent diameter for a sphere whose projected area is S_proj). (C) Histogram of the duration of transmigration. The mean duration is 3 ± 2 min (mean ± SD). (D) Scanning electron microscopy images of lymphoblasts spreading on a substrate coated with anti-CD3 plus anti-CD28 activating antibodies. Scale bars, 100 μm, 20 μm, 10 μm (left to right). Yellow arrows indicate spread cells. (E) Boxplots of the apparent membrane surface area of T-lymphocytes under both passive (white-filled box) and active (blue-filled box) deformations. The bottom and top of the box indicate the 25th and 75th percentiles, respectively. Red plus signs indicate outliers. From left to right, resting T-lymphocytes initially (column 1, A₀, n = 14) and at rupture (column 2, A*, n = 14) aspirated using a micropipette, lymphoblasts at rest (column 3, A₀, n = 14) and at rupture (column 4, A*, n = 14) aspirated using a micropipette, lymphoblasts spread on anti-CD3 plus anti-CD28 monoclonal antibodies (column 5, aCD3, n = 17), and lymphoblasts after transendothelial migration (column 6, TEM, n = 15). ***p < 0.001. n.s., p > 0.05.