Figure 3.
Microtubule-Dependent Transport Promotes Nup Granule Interactions
(A) Nup358 granules migrate from nurse cells to the ooplasm. Shown are a top view image (A) and color-coded time projection (A’) of a video taken of an egg chamber expressing GFP::Nup358 and the plasma membrane marker GAP43::cherry. Nup358 granules move into the oocyte through a ring canal (arrowhead in A), signified by the gap in the GAP43::cherry staining.
(B and C) Interactions between Nup granules and AL. Shown are stills (B and C) or kymographs (B’ and C’) from videos capturing GFP::Nup358 and RFP::Nup107 (B and B’) or GFP::Nup358 (C, and C’) expressing egg chambers, injected with the FG-Nup marker WGA-Alexa647. Kymographs were produced from the boxed regions indicated in (B and C). Shown and (B and B’) is interaction between a Nup358 granules and a RFP::Nup107/FG-Nup-positive, oocyte-specific granule. Shown in (B’), granules stay adjacent with little overlap and controlled material transfer (arrowhead). Shown in (C’) is a representative event of an interaction between a FG-Nup-labeled, oocyte-specific granule and an AL with mixing (arrowheads).
(D and E) Nup granules can occur independent of ER membrane. Kymographs (D–E’) obtained from movies recording a representative stage 10 nurse cell (D–D’’) or oocyte (E and E’) dissected from flies expressing either GFP::Nup358 and the ER marker KDEL::RFP (D–D’’) or KDEL::RFP, injected with WGA-Alexa488 labeling FG-Nups (E and E’). Shown in (D–D’’), KDEL::RFP temporally co-localizes with a GFP::Nup358 (yellow arrowhead in D’’), before KDEL::RFP loses contact from the Nup358 granule. Shown in (E and E’), a FG-Nup-labeled, oocyte-specific granule undergoes a fast directed run independent of KDEL::RFP (cyan arrowhead in E’), before it attaches to membrane.
(F–J) Microtubules are crucial for granule dynamics and mixing. Shown in (F) is a confocal image from a video recorded in ovaries expressing GFP::Nup358 and Tubulin::cherry. Nup358 granules localize to the dense MT network. Shown in (G and H) are color-coded temporal projections (G–H’) or stills (I and I’) of a video recording control (G, H, and I) or Colchicine-treated (G’, H’, and I’) egg chambers that express RFP::Nup107 (G and G’), GFP::Nup358 (H and H’), or both (I and I’). Colchicine treatment reduced NPC precursor granule and AL dynamics (G’ and H’), compared with controls (G and H), and impaired granule mixing (I’) compared with controls (I). Shown in (J) is the quantification of granule separation and AL number in Colchicin-treated (n = 10 egg chambers) and control (n = 8) mid-oogenesis egg chambers: MT de-polymerization increases the percentage of separated GFP::Nup358 and RFP::Nup107 granules and reduces AL number. Data represent means ± STDV.