Figure 1.
AL-NPCs Insert into the Nuclear Envelope
(A and A′) Nuclear growth and NPC distribution during interphase in the Drosophila syncytium: Stills from a time-lapse movie recorded from a GFP::Nup107 expressing embryo imaged right after interphase onset (A) and 5 min later (A′). GFP::Nup107 localizes to the NE and to cytoplasmic foci (arrowheads) in (A), which disappear at t + 5 min (A′).
(B–B″) Cytoplasmic foci of Nup107 fluorescence localize to AL-NPCs. Correlative light and electron microscopy (CLEM) of a RFP::Nup107 expressing interphase embryo. RFP::Nup107 is concentrated along the NE and at foci (boxed in B), that correspond to NPCs along ER membranes (arrowheads in B′ and B″).
(C) NPC density stays constant as nuclei grow. Quantification of normalized nuclear surface increase (blue curves) and the mean GFP::Nup107 fluorescence intensity ± SD at the NE (red curves) during interphases. Values on both graphs are normalized to the earliest measured time point for each movie (n = 71 nuclei in four embryos).
(D–E) The Y-complex protein Nup107 and WGA-labeled transport Nups co-localize at the NE and at AL-NPCs that insert to the NE. Top view still (D–D″) and kymograph (E) from a time-lapse movie imaging a WGA-Alexa555 injected syncytial blastoderm embryo expressing GFP::Nup107 (see also Movie S2). Insertion is captured in the kymograph (E) that spans the region of interest (ROI) boxed in (D).
(F–G) Stills (F–F″) and kymograph (G) of the blue shaded ROI from a time-lapse movie recording photo-converted EosFP::Seh-1 before (0 s, F) or after (6 s, F′, and 145 s, F″) photo-conversion of AL-NPCs adjacent to the NE. NPC transfer from AL to the NE is documented by the lateral dispersion of the converted signal, which starts after ∼100 s (G).
(H) AL-NPC number drops during early interphases. Quantification of the relative AL-NPC number inferred from GFP::Nup107 fluorescence for four embryos live-imaged over interphases. AL-NPCs were counted from 1 μm distant z sections spanning nuclear height in a constant field of view comprising ∼10 nuclei for each embryo.
(I) GFP::Nup107 fluorescence intensity shifts from AL to the NE in the first 25% of interphases, when AL number drops the most (H). Fluorescence intensities were integrated over consecutive confocal slices covering the entire nuclear height at the NE (NE-NPCs) and at AL (AL-NPCs). Cytoplasmic GFP::Nup107 was determined from the mean fluorescence intensity. Analysis was done on two embryos (n = 13 and 11 nuclei, respectively). See Figure 1A for representative image; error bars represent ± SD over multiple ROIs. All images in Figure 1 are acquired from embryos in cycles 10–13 of the syncytial blastoderm stage.