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First published online 26 February 2008
doi: 10.1242/jcs.019091

Journal of Cell Science 121, 887-894 (2008)
Published by The Company of Biologists 2008
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The dynamics of fluorescently labeled endogenous gurken mRNA in Drosophila

Angela M. Jaramillo1,2, Timothy T. Weil2, Joseph Goodhouse2, Elizabeth R. Gavis2 and Trudi Schupbach1,2,*

1 Howard Hughes Medical Institute, Princeton University, NJ 08544, USA
2 Department of Molecular Biology, Princeton University, NJ 08544, USA


Figure 1
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  		Fig. 1. Live imaging of endogenous grk mRNA. (A-C) grk*GFP expressed in the younger stages of mid-oogenesis. (A) Stage 7 shows posterior accumulation. (B) In early stage 8, there is often a lateral-cortical lining of grk*GFP on the side of the oocyte nucleus (asterisk). (C) During late 8, there is no longer a posterior lining of grk*GFP but instead the beginnings of an anterior ring with significant accumulation at the ventral anterior. (D,E) grk*RFP expressed in older stages of mid-oogenesis. From stages 9 to 10, there is a prominent dorsal-anterior cap and a strong reduction of grk*RFP at the ventral-anterior corner. (F) Control line expressing MCP-RFP alone. MCP-GFP and MCP-RFP have a nuclear localization sequence, and, as a consequence, when no grk mRNA with stem loops is present, the protein will enter the follicle cell nuclei (arrowhead) and nurse cell nuclei (arrow). Bars, 50 µm.

 

Figure 2
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  		Fig. 2. Actin is not necessary for anchorage of grk mRNA. (A-C) Dissected live grk*RFP egg chambers treated with DMSO, the solvent used to dissolve the actin-destabilizing drugs. grk*RFP is localized properly from young to late stages. (D-F) Egg chambers were treated with the actin-destabilizing drugs cytochalasin D and latrunculin A for 45-60 minutes. There is no obvious disruption of grk*RFP.

 

Figure 3
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  		Fig. 3. grk*RFP appears to remain unaltered after treatment with microtubule-destabilizing drugs. (A-C) Dissected live grk*RFP egg chambers treated with ethanol show properly maintained grk*RFP. (D-F) Egg chambers were treated with the microtubule-destabilizing drugs colchicine and colcemid for 45-60 minutes. grk*RFP does not appear to be disrupted in either young or older egg chambers.

 

Figure 4
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  		Fig. 4. grk mRNA is a dynamic molecule. FRAP experiments were performed on grk*GFP egg chambers at stages 6-9. (A-C) Images of grk*GFP egg chambers at stages 6-9, with a small box representing the area that is bleached during a FRAP experiment. (D,E) Line graphs representing the average relative fluorescence of grk*GFP during several FRAP experiments. (D) During stage 6, a defined area of fluorescent grk*GFP at the oocyte posterior was bleached and about 70% was recovered. (E) grk*GFP localized at the dorsal-anterior cap during late stage 9 was bleached and showed reduced recovery. (F) Bar graph representing the percentage of fluorescence recovery from stages 6 to late stage 9. There is a steady decrease in the amount of recovery as the oocyte progresses through development. (G) Bar graph representing the half-time of recovery (t1/2) for stages 6 to late 9. Although recovery steadily decreased, the rate did not, suggesting there could be a similar mechanism occurring during stages 6 to early 9. A minimum of five FRAP experiments were performed for each data point. Bars represent the standard deviation; e, early; l, late.

 

Figure 5
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  		Fig. 5. grk mRNA recovery does not specifically involve depletion of nearby localized sources. (A,B) Images of stage 7 (A) and late stage 8 (B) egg chambers from flies expressing grk*GFP illustrating the region bleached during the FRAP experiment (black box), plus arrows pointing to the unbleached areas of grk*GFP monitored for fluorescence loss. (C,D) Line graphs representing average relative fluorescence of grk*GFP and two unbleached neighboring pools of grk*GFP during FRAP experiments. In panel C, the two posterior pools that surrounded the bleached region of grk*GFP during stage 7 show no dramatic decrease in fluorescence intensity. The pool on the right of the bleached region is represented by the dark gray line, whereas the pool on the left is the pale gray line. (D) Likewise, the surrounding sources of localized grk*GFP found at the dorsal-anterior cap during late stage 8 also displayed no measurable decrease while recovery occurred (right, dark gray; left, pale gray). This suggests that recovery is not due to nearby localized sources of grk*GFP.

 

Figure 6
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  		Fig. 6. Mislocalized grk mRNA at the anterior is dynamic. FRAP experiments were performed on K10; grk*GFP and grk*GFP; sqd1 egg chambers. Images of grk*GFP (A), K10; grk*GFP (B,D) and grk*GFP; sqd1 (C) egg chambers during mid-to-late stage 9. (B-D) Mislocalized grk*GFP forms an anterior ring at the edge of the oocyte. A defined area of fluorescent grk mRNA at the dorsal (arrowhead) and ventral-anterior (arrow) sides was bleached during FRAP experiments. (E) Previously we noted that grk*GFP localized at the dorsal-anterior cap during mid-to-late stage 9 had very reduced recovery (~25% recovery) during FRAP experiments. By contrast, grk*GFP found both at the dorsal and ventral-anterior sides of K10 and sqd1 mutants was more dynamic (~45%). (F) Bar graph representing the half-time of recovery (t1/2) for grk*GFP during mid-to-late stage 9 from controls and mutant lines. Recovery rates are much faster in the mutants, similar to the younger stages in the control line (Fig. 4). A minimum of five FRAP experiments were taken for each data point. Error bars represent standard deviations.

 

Figure 7
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  		Fig. 7. Reducing dynein activity leads to reduced grk mRNA recovery. FRAP experiments were performed on grk*GFP; {alpha}tubGal4/+;UAS-Dmn/+ and grk*GFP; dhc6-10/6-12 egg chambers. Images of grk*GFP (A,B), grk*GFP; {alpha}tubGal4/+;UAS-Dmn/+ egg (C,D) and grk*GFP; dhc6-10/6-12 (E,F) egg chambers during stages 6-7 (A,C,E) and late 8 to early 9 (B,D,F). (A,B) Properly localized grk*GFP lines the posterior cortex of young egg chambers, whereas grk*GFP forms a crescent around the oocyte nucleus at the dorsal anterior corner starting stage 8. In a UAS-Dmn background (C,D), there is mislocalized grk*GFP in the oocyte cytoplasm accompanied by large aggregates that can also be seen in the nurse cells (arrow). Under these conditions, a significant proportion of grk*GFP is capable of reaching its destination both at the posterior and dorsal-anterior corner. In hypomorphic alleles (E,F), grk*GFP has a very diffuse localization during young stages and accumulates at the anterior cortex in later stages. Similar to (C,D), there are aggregates in both the nurse cell and oocyte cytoplasm. A defined area of fluorescent grk at the posterior and dorsal-anterior was bleached during FRAP experiments. (G) In control early stages (6-7), grk*GFP has on average 67% fluorescence recovery and during mid-stages (l8 to e9) a 46% recovery during FRAP experiments, whereas by contrast a significant reduction occurs in both young and mid stages when dynein activity is compromised. When Dmn is overexpressed, recovery is reduced to 34% in stages 6-7 and 28% in l8-e9. Similarly, in the dynein hypomorphs, recovery is reduced to 24% in stage l8-e9 egg chambers. A minimum of five FRAP experiments were performed for each data point. Error bars represent standard deviations.

 

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