Gagnon JA , Kreiling JA , Powrie EA , Wood TR , Mowry KL .

GM071049 NIGMS NIH HHS , T32-GM07601 NIGMS NIH HHS , P40 OD010997 NIH HHS , R01 GM071049 NIGMS NIH HHS , T32 GM007601 NIGMS NIH HHS

	Figure 2. Dynein is associated with Vg1 RNA.(A) Oocyte lysates were immunoprecipitated with nonspecific mouse IgG or mouse anti-dynein intermediate chain (DIC), and bound Vg1 and EF1α RNAs were detected by RT-PCR. (B) Oocyte lysates were immunoprecipitated as in (A) and blotted after SDS-PAGE with antibodies against DIC, Vg1RBP/Vera (Vera), Staufen (Stau), or ribosomal protein S6 (rS6). (C) Oocyte lysates were immunoprecipitated as in (B) and blotted with antibodies for DIC, the p150Glued subunit of dynactin (p150Glued), dynein heavy chain (DHC), dynein light intermediate chain (DLIC), and dynein light chain (DLC). (D) Control oocytes (−) or oocytes expressing p150Glued CC1 (+) were lysed and immunoprecipitated as in (A); bound Vg1 and EF1α RNAs were detected by RT-PCR. (E) Lysates from control (−) or p150Glued CC1 expressing (+) oocytes were immunoprecipitated as in (B) and blotted with antibodies for DIC, p150Glued, Vera, Stau, and rS6.
	Figure 3. Dynein and kinesin mediate distinct steps in vegetal RNA transport.(A–D) Fluorescently labeled VLE RNA was microinjected into oocytes expressing (A) kinesin-1 rigor mutant (K1r), (B) p150Glued CC1 (CC1), (C) both K1r and CC1, or (D) no exogenous protein. Representative oocytes are shown with the vegetal pole towards the bottom. Scale bars, 50 µm. (E) Quantification of in vivo interference results for oocytes expressing no exogenous protein (control, n = 167), kinesin-1 rigor (K1r, n = 159), CC1 domain of p150Glued (CC1, n = 209), and both K1r and CC1 (n = 208). Black bars indicate normal localization, gray denotes cup accumulation, and white bars indicate accumulation of RNA in the lower vegetal cytoplasm. Error bars indicate standard deviation.
	Figure 4. Live imaging of RNA localization reveals RNA transport dynamics.(A) Diagram of VLE RNA (VLE-MS2) and nonlocalizing β-globin RNA (βG-MS2) tagged with multimerized MS2 binding sites, which recruit MS2 coat protein fused to mCherry (mCh-MCP). (B) Oocytes expressing mCh-MCP and injected with βG-MS2 RNA exhibit uniform cytoplasmic fluorescence. (C) Oocytes expressing mCh-MCP and injected with VLE-MS2 RNA exhibit strong vegetal fluorescence (red). (B–C) Images of live oocytes are shown, with vegetal poles towards the bottom; scale bars, 20 µm. (D) Diagram of oocyte showing regions used for analysis: cup region immediately adjacent to the nucleus on the vegetal side (Region 1), the upper vegetal cytoplasm (Region 2), the lower vegetal cytoplasm (Region 3), and the animal hemisphere (Region 4). The 5 µm circular regions for FRAP are indicated in red and are spatially defined in Materials and Methods. (E) Calculated half times of recovery and diffusion coefficients from FRAP analysis. βG-MS2 RNA mobility was measured in Regions 2–4 and VLE-MS2 RNA mobility was measured in Region 4. After nocodazole treatment, VLE-MS2 RNA mobility was measured in Regions 2 and 3. ± indicates standard error of the mean. (F) Averaged halftimes (t½) of recovery for indicated regions in control oocytes (black bars) or oocytes with dynein function disrupted by expression of CC1 (white bars). Control (Region 1, n = 10; Region 3, n = 10), Disrupted dynein (Region 1, n = 21; Region 3, n = 22). Error bars show standard error of the mean. Half times of recovery (t1/2) and diffusion coefficients were calculated as described in Materials and Methods. The p values were generated using a two-tailed unpaired Student's t test; ** p = 0.0054, * p = 0.299.
	Figure 5. Distinct regions of RNA transport directionality.Oocytes expressing PA-mCh-MCP were microinjected with VLE-MS2 RNA. (A) Prior to activation of PA-mCh-MCP in live oocytes (t = 0), minimal fluorescence is observed. The activation point is shown by the small white dot and the oocyte nucleus is outlined in white. Scale bar, 20 µm. (A′) By 7 s after activation of PA-mCh-MCP, robust fluorescence (red) is evident at and around the activation point (white dot). (A″–A′″) By 240–480 s after activation, PA-mCh-MCP tethered to RNA can be visualized asymmetrically around the activation point. (A′″) The four collection quadrants are indicated by white circles surrounding the activation point: V and A indicate the collection quadrants on the vegetal and animal sides of the activation point, respectively. L and R indicate the collection quadrants on the left and right sides. (B–C) After activation of PA-mCh-MCP in (B) the upper vegetal cytoplasm (Region 2) or (C) the lower vegetal cytoplasm (Region 3), corrected fluorescence intensities in the V (black) and A (grey) quadrants were plotted over time. (D, E) The ratios of V∶A (red) and L∶R (blue) intensities for oocytes activated in (D) the upper vegetal cytoplasm (Region 2) and (E) the lower vegetal cytoplasm (Region 3) were plotted over time.
	Figure 6. Analysis of net transport after photoactivation.Oocytes expressing PA-mCh-MCP were injected with VLE-MS2 RNA and photoactivated as in Figure 5. (A) Collection windows were defined at 5 µm (brown) and 15 µm (green) from the site of photoactivation (gray) in all four directions: Animal (A), Vegetal (V), Left (L), and Right (R). (B) Data from an oocyte activated in the lower vegetal cytoplasm and collected on the vegetal side of the activation point are shown. Fluorescence intensity collected in the defined windows is plotted over time, along with a moving average trendline (period = 10). The timepoints of intensity maxima (MAX) for the 5 µm (t1) and 15 µm (t2) collection windows are indicated by dashed boxes. (C) The percentages of VLE-MS2 RNA moving in the Vegetal, Animal, Left, and Right directions were calculated by measuring change in fluorescence intensity in collection windows 5 µm and 15 µm away from the activation point (see Materials and Methods for details). Standard deviation is indicated by ±, n = 5. RNA movement in the animal direction could not be detected (not detected, n.d.) in Region 2 because intensity maxima in the collection windows were simultaneous, suggesting no transport in this direction within Region 2. (D) Transport rates were calculated for the motile fraction of VLE-MS2 RNA in Regions 2 and 3 by identifying time points (t1, t2) of peak fluorescence intensity in collection windows 5 µm and 15 µm away from the activation point (see Materials and Methods for details). The calculated rates of net RNA transport are shown, with error (±) given as standard deviation, n = 5.
	Figure 7. Model for vegetal RNA localization.The vegetal cytoplasm is depicted, with the vegetal cortex at the bottom. The oocyte nucleus is shown in gray and the perinuclear cup is indicated in gold. (A) The oocyte microtubules are shown in black with orientation indicated by plus and minus. The proposed arrangement of microtubules is based on the appearance of a subpopulation of microtubule plus-ends at the vegetal cortex following breakdown of the mitochondrial cloud [12], which has been proposed to contain a microtubule organizing center [52]. (B) Vg1 mRNA enriched at the perinuclear cup is first transported by the dynein molecular motor in the upper vegetal cytoplasm in an initial highly directional step toward the vegetal cortex (blue). Microtubules are shown in grey. (C) Repeated cycles of bidirectional transport dependent on kinesin molecular motors occur in the lower vegetal cytoplasm (purple), until Vg1 mRNA exits the transport cycle by becoming anchored at the vegetal cortex (red).
	Figure 1. Dynein is required for vegetal RNA localization.(A–C) Fluorescently labeled VLE RNA was microinjected into oocytes expressing (A) p150Glued CC1 domain, (B) p50-dynamitin, or (C) no exogenous protein. (D) Quantification of in vivo interference results (control [control, n = 69], p150Glued CC1 [CC1, n = 97], and p50-dynamitin [Dyna., n = 98]). Black bars indicate normal localization; gray denotes cup accumulation. Error bars indicate standard deviation. (E) Oocytes microinjected with fluorescently labeled VLE RNA were probed with anti-dynein. Shown is a confocal section with dynein in green (E), VLE RNA in red (E′), and co-localization in yellow (E″). (F) Zoomed view of (E″) showing the vegetal cytoplasm. (A–F) Representative confocal images of fixed oocytes are shown, with the vegetal pole towards the bottom. Scale bars, 50 µm.

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