Supplemental Material

This article contains the following supporting material:

• Movie S01 - GFP-Exu particles move in a linear fashion within nurse cells
  GFP-Exu transport in nurse cells of a wild-type egg chamber (Figure 1A). Red lines highlight linear movements. Acquisition was 1fps for 3 min. Playback is 15fps. Scale bar=10 μm.
• Movie S02 - GFP-Staufen particles move in a linear fashion within nurse cells
  GFP-Staufen transport in nurse cells of a wild-type egg chamber (Figure 1A). Red lines highlight linear movements. Acquisition was 1fps for 3 min. Playback is 15fps. Scale bar=10 μm.
• Movie S03 - GFP-Exu particles move in a linear fashion within nurse cells
  A projection-movie of GFP-Exu particles as they move in a linear fashion within nurse cells of an egg chamber (Figure 3). Note the lack of transport within the oocyte at this rapid rate of acquisition (1fps for 3 min). The projection is of every third frame. The movie loops twice. Scale bar=20 μm.
• Movie S04 - Comparison of cytoplasmic streaming within the oocyte
  The left-hand egg chamber is expressing UASp-ΔGl, which retains cytoplasmic streaming. The right-hand egg chamber is null for the kinesin heavy chain, which has no cytoplasmic streaming. Acquisition was 1f/30s. Playback is 10fps.
• Movie S05 - RNP disassembly
  As GFP-Exu RNPs are transported through ring canals into the oocyte, they appear to be disassembled (Figure 5). Due to the depth of these Z-series projections (10 μm), we believe these RNP are disassembled, and not simply moving out of the plane of focus. Scale bar=10 μm.
• Movie S06 - bcd mRNA injected to the oocyte of a wild-type egg chamber
  Fluorescently labeled bcd mRNA injected to the oocyte of a wild-type egg chamber accumulates to the cortex (Figure 6A). Acquisition was 1f/30s for 10min. Playback is 10fps.
• Movie S07 - bcd mRNA injected to the oocyte of a ΔGl mutant egg chamber
  Fluorescently labeled bcd mRNA injected to the oocyte of a ΔGl mutant egg chamber fails to accumulates to the cortex (Figure 6A), which suggests dynein is required for the cortical accumulation. Acquisition was 1f/30s for 10min. Playback is 10fps.
• Movie S08 - bcd mRNA injected to the oocyte of a kinesin null egg chamber
  Fluorescently labeled bcd mRNA injected to the oocyte of a kinesin null egg chamber accumulates to the cortex (Figure 6A), which indicates cytoplasmic streaming does not account for the cortical accumulation. Acquisition was 1f/30s for 10min. Playback is 10fps.
• Movie S09 - bcd particles move in a linear fashion within the oocyte
  A rapid (1fps) acquisition sequence of fluorescently labeled bcd mRNA injected into the oocyte of a wild-type egg chamber (Figure 6B). Note the linear movements of bcd particles and the random directions of transport. Playback is 15fps. Scale bar=10 μm.
• Movie S10 - Comparison of GFP-Exu RNP transport through ring canals into the oocyte.
  The left-hand egg chamber is wild-type for dynein function, the right-hand egg chamber expresses the UASp-ΔGl. In the ΔGl egg chamber, there is less transport through the ring canals compared to the wild-type egg chamber, suggesting that dynein is required for transport through ring canals into the oocyte. Red asterisk indicate ring canals. Acquisition was 1f/30s. Playback is 10fps.
• Figure S1 - GFP-Exu is present in RNA-containing particles
  A) Sucrose gradients of GFP-Exu ovary extracts treated with RNase or RNase inhibitor. In the presence of inhibitor, GFP-Exu migrates as a large particle near the bottom of the gradient. After RNase treatment, GFP-Exu migrates as a much smaller particle, ~7S. When RNA remains intact, the majority of GFP-Exu is in a large complex. Standards were run on a parallel gradient. E, extract.
  B) GFP-Exu (green) is recruited to and colocalizes with injected bcd mRNA (red) particles within the oocyte (early). GFP-Exu and bcd mRNA colocalize at the cortex within 20 minutes after injection (late). GFP-Exu and bcd mRNA are shown separately for comparison. Scale bar=10 μm.
• Figure S2 - Characterization of ΔGlued
  A) Western blot of a pull-down experiment reveals the predicted interaction between dynein intermediate chain and Glued as previously reported. Bacterially expressed GST-dynein intermediate chain (GST-74) associates with the truncated product of ΔGlued. Full-length Glued normally runs as a 150/135 kD doublet.
  B) The dominant Glued¹ mutation and expression of the ΔGlued construct in the nervous system produce the same tail-flip phenotype.
  C) Hatching rates from females expressing varying amounts of ΔGlued. High levels of expression result in sterility. WT=wild-type, ΔGl=UASp-ΔGl, nos=nanos-GAL4
  D) High expression levels of ΔGlued achieved by two copies of the transgene and two copies of the nanos-GAL4 driver result in a small oocyte with extremely low levels of dynein accumulation as determined by immunofluorescence compared to wild-type.
  
• Figure S3 - Localization of GFP-Exu and GFP-Staufen in the presence of different motor mutations.
  A) Proper localization and maintenance of GFP-Exu to the anterior margin is dependent on both dynein and kinesin I function. The posterior accumulation of GFP-Staufen does not require dynein function, however it does require kinesin I function. Dhc-, Dhc6-6/Dhc6-12; Khc-, Khc²⁷.Scale bar=20 μm.
  B) In late stage 9 and 10a oocytes, GFP-Exu localizes to the posterior of the oocyte in wild-type egg chambers. This posterior accumulation is disrupted in egg chambers mutant for dynein function.
  
• Figure S4 - Cytoskeleton distribution in wild-type and mutant egg chambers.
  Microtubule distribution appears unaffected in egg chambers where dynein or kinesin I function is disrupted (tubulin panel). Actin distribution is normal in the dynein mutants; in contrast, the kinesin I mutant egg chambers show aberrant actin distribution in the oocyte. The inset is a three-fold magnification of the disrupted actin distribution within the Khc²⁷ mutant oocyte. Scale bar=20 μm.
• Supplementary Information