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									Supplementary Information

Figure Legends

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m.



Figure S2: Characterization of Glued

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 Glued construct in the nervous

system produce the same tail-flip phenotype.

C) Hatching rates from females expressing varying amounts of Glued. High levels of

expression result in sterility. WT=wild-type, Gl=UASp-Gl, nos=nanos-GAL4

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D) High expression levels of Glued 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m.

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m.




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Materials and Methods:

Construction and characterization of the UASp-Gl plasmid

To generate the UASp-Gl construct, a DNA fragment encoding 826 amino acids of the

N-terminus of the Glued protein was amplified with pfu polymerase (Stratagene) from the

plasmid phs-tGl (Fan and Ready, 1997) using two primers: 5'-

GAATTCATGTTTGTGCGACCCACGCAG-3' and 5'-

GGCAGAGTGTGCTCTCGTGC-3'. This fragment excludes scrambled sequences of the

second chromosome present in the 5' end of both the original Glued cDNA clone C39

(Swaroop et al., 1987) and the plasmid phs-tGl. The scrambled sequence corresponds to

base pairs 675 through 1009 of the transcript of CG7085 mapped to the second

chromosome. The PCR fragment was subcloned into a pUASp vector (Rorth, 1998).

Transgenic flies were obtained by standard P-element mediated germline transformation

(Karess and Rubin, 1984).

       When expressed in the nervous system, the UASp-ΔGl product generates the

same tail flipping phenotype (Figure S2B) and the same rough eye phenotype (data not

shown) that were previously described for the original Glued1 mutation (Schroer et al.,

1988; Echeverri et al., 1996; Waterman-Storer et al., 1997; Martin et al., 1999; Valetti et

al., 1999; Ling et al., 2004). Mutations in the dynein heavy chain (McGrail et al., 1995;

Gepner et al., 1996), and the dynein intermediate chain subunit (Boylan and Hays, 2002),

produce similar phenotypes. High levels of UASp-Gl expression during early oogenesis

block oocyte growth (Figure S2D) and results in female sterility (Figure S2C).




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Biochemical analyses

The method of sucrose gradient fractionation was based on that published by Wilhelm et

al, 2000. Extracts from hand-dissected ovaries were treated with RNasin (Promega) or

RNaseA (Sigma). 700ug total protein from each treatment were sedimented through 5 ml

gradients of 10-40% sucrose, 5 hours at 200,000 x g, and collected into 20 fractions.

Sedimentation standards were run in parallel gradients. Equal volume from each fraction

was analyzed by SDS-PAGE and western blotting using standard methods. For pull-down

experiments, a full-length dynein intermediate chain cDNA (Boylan et al., 2000) was

subcloned into the PGEX-1 vector (New England Biolabs) and expressed as a GST

fusion protein using standard procedures. As a control, the vector was used to express

GST only. Bacterially expressed proteins were bound to Glutathione-Sepharose 4B

(Amersham), then incubated with ovary extracts from flies expressing Glued, or wild-

type controls. Pellets were washed 3 times and the bound proteins were eluted into

sample buffer for SDS-PAGE. Chemiluminescence reagents were from Tropix-Applied

Biosystems. Anti-GFP (Clontech) was diluted 1:1000. Rabbit polyclonal anti-Glued was

diluted 1:6000 (Waterman-Storer and Holzbaur, 1996).




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Movie Legend:

Movie S1: 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m.



Movie S2: 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m.



Movie S3: 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m.



Movie S4: 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.




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Movie S5: 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m), we

believe these RNP are disassembled, and not simply moving out of the plane of focus.

Scale bar=10m.



Movie S6: 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 S7: bcd mRNA injected to the oocyte of a Gl mutant egg chamber

Fluorescently labeled bcd mRNA injected to the oocyte of a Gl 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 S8: 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.




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Movie S9: 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m.



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-Gl. In the Gl 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.




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