Figure 3. Role of the Cytoskeleton in Movement of Nonlocalized mRNA

(A) Retention of the LacZ-hGH mRNA after triton extraction. Cos cells transfected with pGRE-Z-24-GH were extracted with 0.1% triton for 1 min at room temperature (right) or were fixed directly (left), and they were hybridized in situ with an MS2 probe. Unextracted cells show a stronger cytoplasmic staining (6.5×). (68 × 68 µm).

(B–D) Simultaneous localization of microtubules and mRNA movements in live cells. Cos cells were transiently cotransfected with pMS2-YFP, pCFP-tubulin, and pGRE-Z-24-hGH and were imaged live in the CFP and YFP wavelengths. Images were captured in the CFP channel, immediately followed by a movie in the YFP channel (3 images per second for 15 s) and, again, a CFP image. This reduced the artifacts due to cytoskeletal remodeling during recording of mRNA movements. (B) Left: CFP image; right: maximum intensity image projection of YFP movie. The scale bar represents 10 µm. (C) Magnification of merged images of microtubules (red) with YFP (green). The scale bar represents 2 µm. Arrows point to the center of mass of a “directed” particle at t = 0 and t = 1.67 s. The distance between the center of mass at these time points is 1.3 µm (particle speed = 780 nm/s). (D) Magnification of merged images of microtubules (red) with YFP (green). The scale bar represents 2 µm. Arrows point to the center of mass of two “static” particles that colocalize with microtubules.