Messitt TJ , Gagnon JA , Kreiling JA , Pratt CA , Yoon YJ , Mowry KL .

GM071049 NIGMS NIH HHS , R01 GM071049-13 NIGMS NIH HHS , T32-GM07601 NIGMS NIH HHS , R01 GM071049-16 NIGMS NIH HHS , T32 GM007601 NIGMS NIH HHS , R01 GM071049 NIGMS NIH HHS

	Figure 1. Kinesin-1 Interacts with VLE RNA(A and B) Immunofluorescence was carried out using SUK4 antibodies on st. III oocytes injected with fluorescently labeled VLE RNA. Shown is a confocal section with kinesin-1 in green ([A]; αSUK4) and VLE RNA in red (A′); the overlap of VLE RNA and SUK4 staining (A′) is shown in white. (B) Higher-magnification view of VLE RNA and KHC colocalization (white) in the vegetal oocyte cytoplasm. For (A) and (B), scale bars = 50 μm.(C) S10 lysates from oocytes injected with VLE or XβM RNA (lanes 1 and 4 show 10% of total) were immunoprecipitated with mouse IgG (lanes 2 and 5) or αSUK4 (lanes 3 and 6). Bound RNA was detected by RT-PCR using primers for VLE (lanes 1–3) or XβM (lanes 4–6).(D) S10 lysates from uninjected oocytes were immunoprecipitated with mouse IgG (lanes 2 and 5) or αSUK4 (lanes 3 and 6). Bound RNA was detected by RT-PCR using primers for Vg1 (lanes 1–3) or EF1α (lanes 4–6). Lanes 1 and 4 show 20% of total input RNA.
	Figure 2. Kinesin-1 Has a Role in Vegetal RNA Transport(A–C) Confocal images of representative oocytes injected with fluorescently labeled VLE RNA following injection with (A) IgG (control), (B) αSUK2 (non-function-blocking KHC antibody), or (C) αSUK4 (function-blocking αKHC) antibodies. Scale bars = 50 μm.(D) Quantification of antibody interference results. Oocytes were analyzed for VLE RNA localization by confocal microscopy, with percent of oocytes exhibiting localization scored relative to the IgG control, which was set to 100%.
	Figure 3. Kinesin-1 Rigor Mutation Disrupts Vegetal RNA Transport(A–E) Alexa-546-labeled VLE RNA was injected into oocytes expressing (A) no exogenous protein (control, n = 311), (B) kinesin-1 heavy chain (XKHC, n = 253), (C) kinesin-1 rigor mutant (XKHC-T92I, n = 260), (D) Eg5 (XEg5, n = 116), or (E) Eg5 rigor mutant (XEg5-T105N, n = 123), all injected at 500 nM. Representative confocal images of the vegetal hemisphere cytoplasm are shown; scale bars = 50 μm.(F) Comparison of the percentage of oocytes (gray bars, ± SD) exhibiting vegetal localization scored relative to the average for control oocytes (A), which was set to 100%.
	Figure 4. The Kinesin-1 Rigor Mutation Has an Intermediate Phenotype that May Provide Insight into Kinesin-1 Function(A and B) Oocytes were injected with either (A) XKHC-FLAG or (B) XKHC-T92I-FLAG at 125 nM. After 16 hr expression, fluorescently labeled VLE RNA was injected, and localization was assayed after 8 hr by confocal microscopy. Representative images are shown for the “normal” phenotype (A) and “intermediate” phenotype (B), with the vegetal hemisphere toward the bottom.(C and D) Oocytes were injected with either (C) XKHC-encoding or (D) XKHC-T92I-encoding RNAs at 500 nM, followed by injection of fluorescently labeled VLE RNA. Oocytes were harvested at 2, 4, 6, and 8 hr, and localization was assayed as above. Representative images of the vegetal cytoplasm are shown.(E and F) Oocytes were injected with (E) XKlp3b-encoding or (F) XKlp3b-T103I-encoding RNAs at 125 nM, and assayed for VLE localization as in (A) and (B).(G) Oocytes (from [A], [B], [E], and [F]) were scored for localization by confocal microscopy, and the percentage that exhibited the intermediate rigor phenotype is indicated, along with the number of oocytes (n) assayed.For (A)–(F), scale bars = 50 μm.
	Figure 6. Markers of Microtubule Polarity Reveal a Vegetal Population of Plus Ends(A and B) Immunofluorescence was performed on st. III oocytes using (A) EB1 antibodies or (B) no primary antibody. (C) Depiction of vegetal views shown in (A), (B), (D), (E), and (I). The optical section, 10–25 μm from the vegetal pole, is depicted as a gray grid relative to the oocyte animal-vegetal axis. (D–F) Immunofluorescence was performed on st. III oocytes using both EB1 and α-tubulin antibodies. (D) An optical section shows EB1 in green (D) and α-tubulin in red (D′). (E) Colocalization of EB1 (green) and microtubules (red), viewed at high magnification (scale bar = 5 μm). Arrowheads point to examples of microtubule ends colocalized with EB1. (F) An optical section, with the vegetal pole oriented toward the bottom, shows α-tubulin in red (F) and EB1 in green (F′). Arrowhead points to a microtubule end colocalized with EB1; scale bar = 2 μm. (G) Immunofluorescence was performed on st. III oocytes using γ-tubulin antibodies. A cross-section through the vegetal cytoplasm is shown, with the vegetal pole at the bottom. (H) Immunofluorescence was performed on Alexa 546 VLE-injected oocytes using EB1 antibodies. An optical cross-section of the vegetal cytoplasm is shown, with the vegetal pole at the bottom. EB1 is shown in green (H), VLE RNA is shown in red (H′), and the overlap of VLE RNA and EB1 is visualized in white (H′). (I) Vegetal view (as in [C]) of EB1 and VLE colocalization in a st. III oocyte. EB1 is shown in green (I), VLE RNA is shown in red (I′), and VLE RNA and EB1 overlap is shown in white (I′). (J) An optical cross-section of the animal hemisphere from (H) is shown, with VLE RNA as red (J) and EB1 as green (J′). Scale bars represent 50 μm (A, B, D, and G–J), 5 μm (E), or 2 μm (F).

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