Figure 9.

Optical tweezer (OT) as a tool to analyze PML nuclear body assembly. (A) Schematic depiction of erythrocyte-mediated force application (EMFA) based on OTs. Polyethylenimine coated erythrocytes attach unspecifically to the surfaces of the adherent target cells. Erythrocytes serve as very efficient “force transmitting devices” for axial force application on cells. The cell layer is moved into the region of the desired position in such a way that the laser focus (yellow ellipse) locates slightly below the erythrocyte. Immediately after switching on the laser the erythrocyte is pulled toward the focus due to the gradient force of the laser light, which causes a brief physical force onto the cell. The experimental setup used here consists of a continuous wave (cw) diode pumped Nd-YAG-laser (Spectra Physics) emitting at 1064 nm. The laser beam is coupled into an inverted confocal laser scanning microscope (LSM 510, Carl Zeiss Jena) and was focused via a high numerical aperture objective (100×, 1.30 NA) into the object plane. (B) U2OS cells expressing EGFP-tagged PML-IV were subjected to EMFA as shown in (A). In phase contrast (PhC) imaging, the position of the nucleus relative to erythrocytes can be monitored during the course of the experiment (upper panels). The behavior of PML nuclear bodies was monitored by confocal sectioning (middle panels, images show maximum intensity projections). The nuclear region of force application is shown as a magnified view in the bottom panels. Arrowheads indicate de novo formation of PML NBs. Scale bar, 5 µm.