The secretion-coupled endocytosis correlates with membrane tension changes in RBL 2H3 cells

Abstract

Stimulated secretion in endocrine cells and neuronal synapses causes a rise in endocytosis rates to recover the added membrane. The endocytic process involves the mechanical deformation of the membrane to produce an invagination. Studies of osmotic swelling effects on endocytosis indicate that the increased surface tension is tightly correlated to a significant decrease of endocytosis. When rat basophilic leukemia (RBL) cells are stimulated to secrete, there is a dramatic drop in the membrane tension and only small changes in membrane bending stiffness. Neither the shape change that normally accompanies secretion nor the binding of ligand without secretion causes a drop in tension. Further, tension decreases within 6 s, preceding shape change and measurable changes in endocytosis. After secretion stops, tension recovers. On the basis of these results we suggest that the physical parameter of membrane tension is a major regulator of endocytic rate in RBL cells. Low tensions would stimulate endocytosis and high tensions would stall the endocytic machinery.

Figure 1

(A) Time course of RBL cell volume change during cell swelling. An RBL cell was transferred from normal medium to hypotonic buffer (50% water/50% medium) by a micropipet. The cell volume (measured from the cell diameter) increased very fast (within ∼2 min) and then stayed constant. (B) Photomicrograph of a membrane tension measurement from a RBL cell using a tether. In the diagram, T is the in-plane membrane tension. The tether force (F) can be calculated from the displacement of the bead in the laser trap (Δr) and the calibration of the trap. (C) Tether force is plotted versus time and shows elongation and stationary periods. A tether was formed from a control RBL cell. The tether force reached ∼39 pN during the elongation period, but it dropped to ∼21 pN when held at a constant length. (D) Time course of tether force during the RBL cell swelling. A tether was formed and kept at a constant length (before ∼40 s), and the hypotonic buffer (50% water/ 50% medium) was introduced to the cell through the flow chamber (during the time ∼44– 53 s). The static tether force increased very fast before it reached the limit of the trap strength and the bead escaped from the trap. This time course of tether force is consistent with the time course of cell volume change (A) in that the time of most rapid swelling corresponds to the time of highest tether force. (E) Average static tether force of swollen RBL cells and after recovery from swollen to isotonicity. The static tether force of swollen cells (31.59 ± 0.63 pN, n = 15) is significantly larger than the control cells (22.31 ± 1.41 pN, n = 17). The tether force was measured between 5 and 15 min after cells were put into hypotonic buffer. After the swollen cells were transferred to normal isotonic medium, the tether force decreased to 20.78 ± 0.63 pN (n = 11) slightly below the control value. The error bars are SEM. (F) RBL cell endocytosis rate measured by Lucifer yellow uptake with flow cytometry. The average fluorescence intensity of 10⁵ cells was measured as described in materials and methods. When the cells were incubated with hypotonic buffer, the endocytosis rate was inhibited.

Figure 3

(A) Tether force during tether formation after stimulation of secretion with DNP-BSA (1 μg/ml). The tether force during the elongation was decreased (∼20 pN) compared to control and it decreased further to ∼10 pN when held at a constant length (see Fig. 1 C, legend). (B) The average static tether force for the RBL cells during stimulated secretion. The tether force decreased in cells stimulated to secrete by the addition of 1 and 2 μg/ml DNP-BSA compared to the control cells. There is no substantial difference between the tether forces of nonsecreting cells, when secretion was blocked by DNP-lysine, and the control cells. The error bars are SD.

Figure 2

RBL cell endocytosis rate is increased upon stimulation of secretion and can be inhibited by removing extracellular Ca²⁺ with EGTA. Endocytosis was measured by FACS analysis for 10⁵ cells each sample after Lucifer yellow incubation for control cells or cells treated with 1 μg/ml DNP-BSA or with 1μg/ml DNP-BSA and 10 mM EGTA.

Figure 6

(A) The cell side scattering (SSC) and forward scattering (FSC) measured by FACS. All cells were incubated with Lucifer yellow dye for 20 min; (a) control, (b) cells with 1 μg/ml DNP-BSA, (c) cells with 10 mM EGTA and 1 μg/ml DNP-BSA. Cells in c have significant increase of SSC which indicates that more dramatic membrane ruffling occurs. (B) Membrane tether force was not affected by cell shape changes caused by the treatment with both EGTA and DNP-BSA. The DNP-BSA concentration is 1 μg/ml and EGTA is 10 mM. The error bars are SD.

Figure 6

(A) The cell side scattering (SSC) and forward scattering (FSC) measured by FACS. All cells were incubated with Lucifer yellow dye for 20 min; (a) control, (b) cells with 1 μg/ml DNP-BSA, (c) cells with 10 mM EGTA and 1 μg/ml DNP-BSA. Cells in c have significant increase of SSC which indicates that more dramatic membrane ruffling occurs. (B) Membrane tether force was not affected by cell shape changes caused by the treatment with both EGTA and DNP-BSA. The DNP-BSA concentration is 1 μg/ml and EGTA is 10 mM. The error bars are SD.

Figure 4

Time course of the decrease in tether force during secretion. Extension of the tether started at the time of ∼8.5 s and ceased at ∼12 s. DNP-BSA (1 μg/ml) was added by flow at the time of ∼13 s. The stationary tether force started to decrease at ∼19 s, and this meant that the tether force dramatically decreased within 6 s after the addition of the antigen. In four repeats of this experiment, the average tether static force decreased from ∼24 pN to 12.43 ± 1.56 (n = 4) pN within 10 s after the addition of DNP-BSA.

Figure 5

(A) Photomicrograph showing the method of measuring the relative tether thickness from the DIC image. The intensity of the contrast is related to the tether thickness (Schnapp et al., 1988). The relative tether contrast was determined through integration of changes in intensity along orthogonal scans across the DIC image of the tether. (B) The comparison of tether force with the relative tether diameter. For the resting cells (Control) and secreting cells (1 μg/ml DNP-BSA) the tether force decreased by ∼50%, whereas the relative tether diameter increased by ∼35%.