Figure 2.
Figure 2. Sbf associates with Rab35 in apically constricting cells. (A, A′, and A″) Immunogold TEM images of anti-GFP CRISPR:GFP:Rab35 demonstrates that Rab35 is present at apical surfaces and in large endosomal structures. (A′ and A″) Magnification of area marked by dashed lines in A showing apical surface– (A′) or endosomal structure–associated (A′″) Rab35 immunogold compartments (arrows). (B) Quantification of Rab35 localization in A. (C) Time-lapse images of embryo expressing UAS:mCh:Rab35 (red) and UAS:EGFP:Sbf (green). mCh:Rab35 displays similar dynamics as CRISPR:GFP:Rab35 (see Materials and methods; Fig. S1, B and C). (D) The interaction between EGFP:Sbf (green) and mCh:Rab35 (red) over time. Arrowhead indicates colocalized Sbf and Rab35 compartment. (E) Percentage of colocalization between Rab35 and Sbf. n = 241 (Rab35) and 177 (Sbf) compartments. (F) Sbf association with Rab35 over the lifetime of a Rab35 compartment. n = 190 compartments. (G) Tracking of fluorescence intensity during the lifetime of a colocalized Rab35 and Sbf compartment. (H) Still frames of embryos expressing CRISPR:GFP:Rab35 in control or Sbf shRNA backgrounds during either early or late ventral furrow formation. (I) Quantification of the size of Rab35 compartments in control and Sbf shRNA embryos at early (t = 0) and late (t = 8 min) time points. n = 79 (control, 0 min), 101 (Sbf shRNA, 0 min), 171 (control, 8 min), and 169 (Sbf shRNA, 8 min) compartments. (J) Images of embryo expressing CRISPR:GFP:Rab35 in either control or Sbf shRNA background injected with dextran into extracellular space. Very few Rab35 compartments are labeled with dextran when Sbf function is compromised. Yellow dashed line marks the cell outline. (K) Quantification of the labeled Rab35 compartments in J. n = 245 (control) and 312 (sbf shRNA) compartments. (L) Quantification of the average number of Rab35 compartments in control and Sbf shRNA embryos. n = 433 (control, 0 min), 820 (Sbf shRNA, 0 min), 272 (control, 8 min) and 200 (Sbf shRNA, 8 min) compartments. Scale bar in A represents 200 nm, scale bars in H and J represent 5 μm, and scale bars in C and D represent 2.5 μm. Statistical significance was calculated using Student’s t test (K) or Mann–Whitney U test in (E, I, and L). *, P < 0.05; ***, P < 0.0005. In C, D, H, and J, embryos are oriented with anterior up and posterior down.

Sbf associates with Rab35 in apically constricting cells. (A, A′, and A″) Immunogold TEM images of anti-GFP CRISPR:GFP:Rab35 demonstrates that Rab35 is present at apical surfaces and in large endosomal structures. (A′ and A″) Magnification of area marked by dashed lines in A showing apical surface– (A′) or endosomal structure–associated (A′″) Rab35 immunogold compartments (arrows). (B) Quantification of Rab35 localization in A. (C) Time-lapse images of embryo expressing UAS:mCh:Rab35 (red) and UAS:EGFP:Sbf (green). mCh:Rab35 displays similar dynamics as CRISPR:GFP:Rab35 (see Materials and methods; Fig. S1, B and C). (D) The interaction between EGFP:Sbf (green) and mCh:Rab35 (red) over time. Arrowhead indicates colocalized Sbf and Rab35 compartment. (E) Percentage of colocalization between Rab35 and Sbf. n = 241 (Rab35) and 177 (Sbf) compartments. (F) Sbf association with Rab35 over the lifetime of a Rab35 compartment. n = 190 compartments. (G) Tracking of fluorescence intensity during the lifetime of a colocalized Rab35 and Sbf compartment. (H) Still frames of embryos expressing CRISPR:GFP:Rab35 in control or Sbf shRNA backgrounds during either early or late ventral furrow formation. (I) Quantification of the size of Rab35 compartments in control and Sbf shRNA embryos at early (t = 0) and late (t = 8 min) time points. n = 79 (control, 0 min), 101 (Sbf shRNA, 0 min), 171 (control, 8 min), and 169 (Sbf shRNA, 8 min) compartments. (J) Images of embryo expressing CRISPR:GFP:Rab35 in either control or Sbf shRNA background injected with dextran into extracellular space. Very few Rab35 compartments are labeled with dextran when Sbf function is compromised. Yellow dashed line marks the cell outline. (K) Quantification of the labeled Rab35 compartments in J. n = 245 (control) and 312 (sbf shRNA) compartments. (L) Quantification of the average number of Rab35 compartments in control and Sbf shRNA embryos. n = 433 (control, 0 min), 820 (Sbf shRNA, 0 min), 272 (control, 8 min) and 200 (Sbf shRNA, 8 min) compartments. Scale bar in A represents 200 nm, scale bars in H and J represent 5 μm, and scale bars in C and D represent 2.5 μm. Statistical significance was calculated using Student’s t test (K) or Mann–Whitney U test in (E, I, and L). *, P < 0.05; ***, P < 0.0005. In C, D, H, and J, embryos are oriented with anterior up and posterior down.

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