Article Figures & Data
Figures
- FIG. 1.
Western blotting with specific antibodies to GFP of PC12 Tet-On (control) and stable transfected PC12/FMR7 cells with (+) and without (−) DOX induction. PC12/FMR7 cells show the presence of a band of approximately 110 kDa after DOX induction with the anti-GFP antibody, which is the expected size for the full-length FMRP-GFP fusion protein (80 plus 27 kDa). In contrast, PC12/FMR7 cells without DOX induction are devoid of the 110-kDa band, representing the absence of nonspecific induction (leakage). In PC12 Tet-On cells no detection of the 110-kDa band was observed. Numbers at left show molecular masses in kilodaltons.
- FIG. 2.
Light microscopic appearance of movement of FMRP-GFP granules into neurites of PC12/FMR7 cells after DOX induction. The FMRP-GFP fusion protein was visualized either directly by immunofluorescence of the GFP molecules (A to C) or by an indirect immunoperoxidase technique with specific antibodies to GFP (D to F). Without DOX induction (A and D; time zero) no reaction product could be visualized. After 30 min of DOX induction FMRP-GFP fusion protein was present solely in the cell body (B and E). Exposure of the cells to DOX for 2 h revealed a strong labeling in the cell body and a punctate labeling pattern in the neurites (C and F, arrows). Especially in the bright-field images from the immunoperoxidase-incubated cells, swellings along the neurite with high concentrations of granules could be observed (F, arrows). Panels G and H illustrate the endogenous FMRP expression in untransfected PC12 cells and primary mouse hippocampal neurons, respectively. PC12/FMR7 cells were cultured for 30 min in the presence of DOX (I); subsequently cells were cultured for another 2 h in medium containing DOX with (J) and without (K) the presence of cycloheximide. In the presence of cycloheximide the FMRP-GFP fusion protein is still able to reach the neurites (K, arrows). Magnifications: A to C, ×720; D to F, ×960; G, ×1,200; H, ×960; I and K, ×1,440; J, ×800.
- FIG. 3.
(A) Figure corresponding to time-lapse series 1 (available online); individual FMRP-GFP labeled granules were observed by time-lapse microscopy. The time-lapse series shows the movement of green fluorescent granules in PC12/FMR7 cells after DOX induction (2 h) in a time-lapse study of frames every 5 s for 500 s. Note the movement of fluorescent granules between the swelling along the neurite (square 2) and the growth cone (rectangle 1). (B) Quantification of the emission intensities over time (500 s). The overall emission intensity in the cell body (square 3) remains the same over time, whereas the emission intensity within the swelling (square 2) follows a cycle every 3 min. The emission profile of the growth cone shows a gradual increase of intensity during the recorded time (rectangle 1).
- FIG. 4.
Figure corresponding to time-lapse series 2 (available online); individual time-lapse frames were more highly integrated than those of time-lapse series 1 (every second for 300 s). We have focused on the movement of granules between the swelling along the neurite and the growth cone already described for time-lapse series 1. A selection of consecutive time-lapse frames illustrates the bidirectional movement (arrows) of an individual granule. The bidirectional movement of this particular granule switched later in the time-lapse series to a distal unidirectional movement followed by appearance in the growth cone.
- FIG.5.
PC12/FMR7 cells (after 24 h of DOX treatment) double labeled on the one hand for FMRP-GFP (A, D, G, and J) and on the other hand for total RNA (B), ribosomal subunits (P0) (E), kinesin heavy chain (H), and FXR1P (K). For each individual colocalization experiment we have included an image that merges both separate images (green and red). Colocalization results in a yellow color (C, F, I, and L). Photographs show part of a neurite and growth cone with two populations of granules (larger and smaller). Both types of granules show colocalization. Magnifications: A to I, ×2,880; J to L, ×1,600.
- FIG.6.
Shown are fluorescent images of PC12/FMR7 cells after 30 min of DOX induction followed by further treatment in (i) control medium (with DOX) for another 2 h (A, B, G, and H), (ii) medium supplemented with cytochalasin D for another 2 h (D and E), and (iii) medium supplemented with nocodazole for another 2 h (J and K). Colocalization experiments were performed with phalloidin conjugated with Texas red to visualize F-actin (B and E) and antibodies to tyrosine-tubulin to visualize microtubules (H and K). Treatment with cytochalasin D had no effect on the localization of FMRP-GFP granules in the neurites (compare green fluorescence of panels A and D), while the F-actin labeling in the neurites was completely abolished compared to that in untreated cells (compare red fluorescence of panels B and E). Treatment with nocodazole abolished the microtubules (compare red fluorescence of panels H and K; arrows in panel K point to a neurite with only very faint staining for tubulin), and FMRP-GFP granules were not transported into the neurites (compare green fluorescence of panels G and J, arrows). The two individual images (red and green) of each experiment were merged (C, F, I, and L) to visualize colocalization (yellow). FMRP-GFP granules in the neurites were not colocalized with either F-actin or tubulin. Magnifications: A to I, ×2,400; J to L, ×1,360.
