Article Figures & Data
Figures
- Fig. 1.
Emission intensity profiles of RNA, DNA, proteins, and lipids incubated with SYTO 14 and scanned at an absorbance of 500 nm. Peak emission intensity for RNA was sixfold greater than that for either proteins or lipids. It differed from proteins, lipids, and DNA with a significance of p < 0.001. The emission intensity for DNA differed from proteins and lipids with a significance of p < 0.05.
- Fig. 2.
Cortical neurons colabeled with SYTO 14 (a) and Mitotracker (b).Arrowhead indicates mitochondria that colocalize with both markers. Small arrows indicate granules only observed with SYTO 14 labeling. Scale bar, 20 μm.
- Fig. 3.
Cortical neurons labeled with SYTO 14 before (a) and after (b) RNase treatment.Arrows indicate SYTO 14-labeled granules. Scale bar, 10 μm.
- Fig. 4.
Cortical neurons labeled with SYTO 14 and translational components. a, SYTO 14-labeled granules (small arrows) and mitochondria (large arrow) staining in neurites. b, Poly(A+) mRNA-containing granules detected in neurites (small arrows) and no signal at location of mitochondria (large arrow). Scale bar, 5 μm. c, SYTO 14-labeled granules in neurites. d, β-Actin mRNA-containing granules in neurites. Examples of colocalization are indicated with arrows. Scale bar, 5 μm.e, SYTO 14-labeled granule staining in neurites.f, Ribosomal 60S subunit labeling in neurites. Examples of colocalization are indicated with arrows.Arrowhead in e points to start of axon-like neurite. Scale bar, 10 μm. g, SYTO 14-labeled granule staining in neurites. h, EF1α labeling in neurites. Examples of colocalization are indicated witharrows. Phase contrast showed that indicated granules were located on neurite branches (data not shown). Scale bar, 5 μm.
- Fig. 5.
Time-lapse analysis of SYTO 14-labeled small granule movement in neurites. a–f, Images after granule (small arrow) and mitochondria (large arrow) at times 0, 1, 2, 3, 4, 5 min. a′, Image taken of mitochondria (large arrow) just before time-lapse. f′, Image taken of mitochondria just after time-lapse. Scale bar, 10 μm.
- Fig. 6.
Distribution of SYTO 14-labeled granules in 40 μm segments in the neurite shafts of 4-d-old cortical neuronal cultures. Treatment with cytochalasin-D does not significantly alter the distribution of RNA granules, whereas treatment with colchicine decreases the number by 62% (p < 0.001).
- Fig. 7.
A, Cortical neurons labeled with SYTO 14, photoconverted in the presence of DAB, and viewed with an electron microscope. Arrows point to electron-dense DAB reaction product that corresponds to the precise location of the SYTO 14 dye. The labeled structures appear as clusters of ribosomes (arrow) and organelles that seem to be mitochondria (arrowhead). Sections are 90 μm thick. Scale bar, 500 nm. B, Electron micrograph of a single RNA granule. The site shown here precisely corresponds to a SYTO 14-labeled granule in a culture of cortical neurons. Scale bar, 100 nm.
Tables
Filler Mitochondria Granules Background Resorufin 90.2 ± 1.7 65.4 ± 0.7 22.3 ± 0.4 Cy5 89.7 ± 1.6 31.5 ± 0.5 31.3 ± 0.6 Average signal intensities of SYTO 14-labeled mitochondria (n = 100) and nonmitochondria granules (n = 100) detected with a Resorufin filter and the corresponding signal intensities in the identical regions caused by Mitotracker labeling detected with the Cy5 filter. Background intensities were generated by examining the average pixel intensity in random 1 × 1 μm regions in the neurite shaft that did not contain any detectable mitochondria or nonmitochondria granules (n = 100). Signal intensities of mitochondria and nonmitochondria granules are significantly above background detected with the Resorufin filter (p < 0.001). Mitochondria intensities are significantly above background with the cy5 filter (p < 0.001), whereas the signal from granules is not significantly above background.
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