Figure 6.

Ran and Crm1 Spatially Control Nup Condensation and AL Assembly

(A and B) RanGAP and RCC1 distributions in the egg chamber. Top views of fixed wild-type egg chambers stained with antibodies detecting RanGAP (A, A’, B, and B’) and the RanGEF RCC1 (A, A’’, B, and B’’). Shown in (A)–(A’’), the oocyte nucleus is strongly enriched for nucleoplasmic RCC1 (green arrowhead in A’’) but depleted for RanGAP at the NE (green arrowhead in A’), in comparison to nurse cell nuclei. Note that the strong RanGAP spot at the oocyte nucleus’ anterior side is likely an AL at the NE (red arrowhead in A’). Shown in (B) and (B’), RanGAP localizes to the NE throughout the egg chamber (cyan arrowheads in B’) to Nup358 granules in nurse cells (yellow arrowhead in B’) and to AL (red arrowhead in B’). RCC1 is enriched in all nuclei and in the ooplasm (green arrowhead in B’’), compared with nurse cell cytoplasm.

(C–F) Ran controls AL biogenesis and Nup condensation. Shown in (C) is the quantification of the cytosolic volume fraction [% (v/v)] of GFP::Nup358 condensates in egg chambers expressing GFP::Nup358 and Ran (n = 6 egg chambers), RanT24N (n = 9) or RanQ69L (n = 10). Bars represent mean ± STDV. Shown in (D)–(F’’’) are images from fixed egg chambers isolated from flies expressing GFP::Nup358 and HA::Ran (D–D’’’), HA::RanT24N (E–E’’’), or HA::RanQ69L (F–F’’’), respectively and stained for FG-Nups detected by WGA-Alexa647. Compared with HA::Ran (D–D’’), HA::RanT24N expression reduces GFP::Nup358 condensates (E and E’) and AL (E and E’’) whereas HA::RanQ69L, which co-localizes with GFP::Nup358 (F–F’’) augments Nup358 condensates (F and F’) and induces AL clusters at the anterior ooplasm (red arrowhead in F).

(G–J) The crm1 homolog embargoed regulates Nup condensation. Stills from time lapse videos imaging egg chambers from GFP::Nup358 expressing control (G and I–I’’) or embargoed shRNA-treated ovaries (H and J–J’’) and injected with WGA-Alexa647 to label FG-Nups (I–J’’). Shown in (G) and (H) is the depletion of embargoed abolishes condensation of GFP::Nup358 and AL formation (H) compared with controls (G). In (I) and (J), reversed Nup condensation in embargoed shRNA-induced egg chambers GFP::Nup358 condensed in oocytes (J and yellow arrowhead in J’) into granules that were WGA negative (J’’), whereas FG-Nups condensed in nurse cells (J and cyan arrowhead in J’’). GFP- and WGA-positive AL (red arrowheads in I) as in oocytes of control ovaries were missing in egg chambers depleted for embargoed (J–J’’).

See also Figure S6.