Figure 4.
Crb endocytosis facilitates NB ingression. (A) In late ingressing NBs, Crb is found at the apical membrane (apical, arrowheads) and in large endosomes 0.9 μm below the apical surface (basal, arrowheads). After ingression, Crb is seen in large endosomes in the perinuclear region. Arrowheads in apical sections point to the position of NBs prior to ingression. Arrowheads in the basal sections point to NBs 6 μm below the surface. Yurt labels the basolateral membrane. Scale bars, 5 μm. (B) Single ingressed NBs displaying co-localization of Crb and the endosome markers Hrs, Vps26, Delta, and Ecad. The plasma membrane is labeled with Discs Large (Dlg). Scale bars, 2.5 μm. (C) Frequency of Crb-positive endosomes in NBs after ingression versus adjacent NICs according to endosomal size. Ingressed NBs contain larger Crb-positive endosomes than NICs. N = 2,024 endosomes from 84 NBs and 751 endosomes from 77 NICs (five embryos). (D) Mander’s and Pearson’s co-localization coefficients (M/PCC) of intracellular Crb and markers shown in B in ingressed NBs. See Fig. S3 for positive and negative co-localization controls. 67–109 NBs from 3–7 embryos analyzed per condition. (E) Stills from time-lapse movies of ingressing NBs expressing GFP::Crb (control) or GFP::CrbRR (dots and arrowheads). Control NBs lost their apical domain within ∼35 min (35 NBs, six embryos); GFP::CrbRR expression strongly delayed ingression: 75% of NBs completed ingression after ∼70 min (middle panels; 55 NBs, six embryos) and 25% divided on the embryo surface (lower panels; 18 NBs, six embryos). Note NBs at 40 min showing mitotic rounding. Scale bar, 5 μm. (F) NBs at stage 9 expressing GFP::Crb (84 NBs, four embryos) or GFP::CrbRR (115 NBs, seven embryos) in the absence of endogenous Crb (crb11a22 mutant embryos). GFP::CrbRR is mostly retained cortically (95/115 NBs), while GFP::Crb localizes to endosomes (84/84 NBs). Scale bar, 5 μm. (G) Total GFP::Crb and GFP::CrbRR fluorescence levels at the junctional domain during ingression. N values as in F. Means ± SEM. (H) Mean apical area loss during ingression in GFP::Crb (35 NB, six embryos) or GFP::CrbRR (55 NBs, six embryos) expressing NBs. T = 0 min indicates the onset of ingression. Data presented are means ± SEM. (I and J) Duration and amplitude of individual apical contractions/expansions during ingression in GFP::Crb versus GFP::CrbRR expressing NBs. Median duration for GFP::Crb/GFP::CrbRR of contractions, 64.1/54.6 s, ***, P = 1 × 10−4; expansions, 34/33.6 s, ns, P = 0.51 (KS test); median amplitudes for GFP::Crb/GFP::CrbRR of contractions, 3.2/2.6 μm2/min; **, P = 1 × 10−3; expansions, 1.7/1.8 μm2/min; ns, P = 0.94 (KS test); n = 514–1,553 events per condition.

Crb endocytosis facilitates NB ingression. (A) In late ingressing NBs, Crb is found at the apical membrane (apical, arrowheads) and in large endosomes 0.9 μm below the apical surface (basal, arrowheads). After ingression, Crb is seen in large endosomes in the perinuclear region. Arrowheads in apical sections point to the position of NBs prior to ingression. Arrowheads in the basal sections point to NBs 6 μm below the surface. Yurt labels the basolateral membrane. Scale bars, 5 μm. (B) Single ingressed NBs displaying co-localization of Crb and the endosome markers Hrs, Vps26, Delta, and Ecad. The plasma membrane is labeled with Discs Large (Dlg). Scale bars, 2.5 μm. (C) Frequency of Crb-positive endosomes in NBs after ingression versus adjacent NICs according to endosomal size. Ingressed NBs contain larger Crb-positive endosomes than NICs. N = 2,024 endosomes from 84 NBs and 751 endosomes from 77 NICs (five embryos). (D) Mander’s and Pearson’s co-localization coefficients (M/PCC) of intracellular Crb and markers shown in B in ingressed NBs. See Fig. S3 for positive and negative co-localization controls. 67–109 NBs from 3–7 embryos analyzed per condition. (E) Stills from time-lapse movies of ingressing NBs expressing GFP::Crb (control) or GFP::CrbRR (dots and arrowheads). Control NBs lost their apical domain within ∼35 min (35 NBs, six embryos); GFP::CrbRR expression strongly delayed ingression: 75% of NBs completed ingression after ∼70 min (middle panels; 55 NBs, six embryos) and 25% divided on the embryo surface (lower panels; 18 NBs, six embryos). Note NBs at 40 min showing mitotic rounding. Scale bar, 5 μm. (F) NBs at stage 9 expressing GFP::Crb (84 NBs, four embryos) or GFP::CrbRR (115 NBs, seven embryos) in the absence of endogenous Crb (crb11a22 mutant embryos). GFP::CrbRR is mostly retained cortically (95/115 NBs), while GFP::Crb localizes to endosomes (84/84 NBs). Scale bar, 5 μm. (G) Total GFP::Crb and GFP::CrbRR fluorescence levels at the junctional domain during ingression. N values as in F. Means ± SEM. (H) Mean apical area loss during ingression in GFP::Crb (35 NB, six embryos) or GFP::CrbRR (55 NBs, six embryos) expressing NBs. T = 0 min indicates the onset of ingression. Data presented are means ± SEM. (I and J) Duration and amplitude of individual apical contractions/expansions during ingression in GFP::Crb versus GFP::CrbRR expressing NBs. Median duration for GFP::Crb/GFP::CrbRR of contractions, 64.1/54.6 s, ***, P = 1 × 10−4; expansions, 34/33.6 s, ns, P = 0.51 (KS test); median amplitudes for GFP::Crb/GFP::CrbRR of contractions, 3.2/2.6 μm2/min; **, P = 1 × 10−3; expansions, 1.7/1.8 μm2/min; ns, P = 0.94 (KS test); n = 514–1,553 events per condition.

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