Figure 4. Detection of dynamic macrophage membrane protrusions and their role in binding phagocytic targets. (A and B) Suspended RAW macrophages stably expressing a GPI-linked GFP were treated with actin-perturbing agents or vehicle only (DMSO) and allowed to sediment by gravity onto uncoated glass coverslips, while monitoring fluorescence by TIR-FM. Panel A illustrates the behavior of two control cells shortly after making contact with the coverslip surface. Arrowheads illustrate contact points not present in previous image, arrows point to sites where contacts visible in previous frame were lost; bar, 7 µm. In B, the contact area revealed by TIR-FM in experiments like A is plotted over time for control and drug-treated cells. Data are integrated mean areas ± SEM obtained by analyzing at least 10 cells from three independent experiments for each condition. (C–E) Scanning electron micrographs of RAW cells exposed briefly (40 s) to IgG-opsonized latex beads. The presence of membrane protrusions making contact with the phagocytic target is evident in control (C and D) but not in latrunculin-treated (E) cells; bars: 1 µm (C); 2 µm (D and E). (F and G) Use of laser tweezers to detect capture of particles placed in the vicinity of macrophages. IgG-opsonized sheep red blood cells (sRBCs) were trapped by an optical tweezer and placed in proximity to control (T = 0, top) or drug-treated cells (T = 0, bottom). The position of the optical trap is indicated by the square brackets. After 60 s (T = 60) the trap was relocated to a position away from the cell, as indicated by the brackets. Note that the sRBCs are captured by protrusions on the control cells (arrow), displacing them from the trap, but not by the jasplakinolide-treated cells; bar, 8 µm. (G) Quantitative analysis of target capture measured from optical trap experiments like that in F. The percentage of phagocytic targets that remained in the optical trap is presented as a function of time. Data are means of a total of 30 trapped sRBCs analyzed for each condition tested in three independent experiments.
Figure 4.

Detection of dynamic macrophage membrane protrusions and their role in binding phagocytic targets. (A and B) Suspended RAW macrophages stably expressing a GPI-linked GFP were treated with actin-perturbing agents or vehicle only (DMSO) and allowed to sediment by gravity onto uncoated glass coverslips, while monitoring fluorescence by TIR-FM. Panel A illustrates the behavior of two control cells shortly after making contact with the coverslip surface. Arrowheads illustrate contact points not present in previous image, arrows point to sites where contacts visible in previous frame were lost; bar, 7 µm. In B, the contact area revealed by TIR-FM in experiments like A is plotted over time for control and drug-treated cells. Data are integrated mean areas ± SEM obtained by analyzing at least 10 cells from three independent experiments for each condition. (C–E) Scanning electron micrographs of RAW cells exposed briefly (40 s) to IgG-opsonized latex beads. The presence of membrane protrusions making contact with the phagocytic target is evident in control (C and D) but not in latrunculin-treated (E) cells; bars: 1 µm (C); 2 µm (D and E). (F and G) Use of laser tweezers to detect capture of particles placed in the vicinity of macrophages. IgG-opsonized sheep red blood cells (sRBCs) were trapped by an optical tweezer and placed in proximity to control (T = 0, top) or drug-treated cells (T = 0, bottom). The position of the optical trap is indicated by the square brackets. After 60 s (T = 60) the trap was relocated to a position away from the cell, as indicated by the brackets. Note that the sRBCs are captured by protrusions on the control cells (arrow), displacing them from the trap, but not by the jasplakinolide-treated cells; bar, 8 µm. (G) Quantitative analysis of target capture measured from optical trap experiments like that in F. The percentage of phagocytic targets that remained in the optical trap is presented as a function of time. Data are means of a total of 30 trapped sRBCs analyzed for each condition tested in three independent experiments.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal