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. 2011 Apr 19;20(4):511–525. doi: 10.1016/j.devcel.2011.03.018

Figure 4.

Figure 4

FRAP Analysis of Junctional Fz-EYFP and Fmi-EGFP

(A) Images showing a 28-hr live wing expressing ActP-fz-EYFP/+ at the resolution used in FRAP experiments (pixel size 139 nm) (A) or higher resolution (pixel size 35 nm) (A′). Ovals indicate a typical bright region containing predominantly puncta (green), a less-bright region containing mostly nonpuncta (white), or an anterior-posterior (AP) boundary where no puncta are seen (blue). Scale bars, 5 μm. (A″) Cartoon of a pupal wing cell. (B–D) Live wings expressing ArmP-fmi-EGFP/+ at 28 hr APF (B) or ActP-fz-EYFP/+ in prepupal wings (C) and at 20 hr APF (D). Scale bars, 2.5 μm. (E–L) FRAP analysis on PD boundary bright regions (green, red), PD boundary less-bright regions (black), or AP boundaries (blue) of junctions in prepupal wings (J and L), 20-hr wings (K), or 28-hr wings (E, F, H, and I) expressing Fz-EYFP, or 28-hr wings expressing Fmi-EGFP (G). (E and F) ActP-fz-EYFP/+, PD-localized (E) or AP localized (F). (G) ArmP-fmi-EGFP/+. (H and I) Ubx-FLP; ActP-FRT-polyA-FRT-fz-EYFP/+ (H) or Ubx-FLP; ActP-FRT-polyA-FRT-fz-EYFP/+; fzP21 (I). Less-bright regions have a significantly smaller stable fraction when activation of fz-EYFP expression is delayed or fz dosage is reduced, p∗∗∗ for ActP-fz-EYFP/+ (E) compared to Ubx-FLP; ActP-FRT-polyA-FRT-fz-EYFP/+ (H), p∗∗∗ for ActP-fz-EYFP/+ (E) compared to Ubx-FLP; ActP-FRT-polyA-FRT-fz-EYFP/+; fzP21 (I), p∗ for Ubx-FLP; ActP-FRT-polyA-FRT-fz-EYFP/+ (H) compared to Ubx-FLP; ActP-FRT-polyA-FRT-fz-EYFP/+; fzP21 (I). (J and K) ActP-fz-EYFP/+ in prepupal (J) and 20 hr APF (K) wings. Dotted lines indicate 28-hr wing data, for comparison (see E). The stable fraction of bright regions is significantly different at each time point: p(prepupal–28 hr)∗∗∗, p(20–28 hr)∗∗∗, p(prepupal–20 hr)∗∗∗; whereas it is similar for less-bright regions: p(prepupal–28 hr)∗, p(20–28 hr)NS, p(prepupal-20 hr)NS. (L) ActP-fz-EYFP/ptc-GAL4, UAS-shits1 in prepupal wings. Dotted lines are FRAP on tissue outside the ptc-GAL4 domain in the same wings, for comparison. The unstable fraction in both bright and less-bright regions is significantly increased in mutant compared to wild-type (p∗∗∗). The half-life of recovery is not significantly different for bright regions (wild-type, 20.44 s; mutant, 25.18s; p = 0.39) or less-bright regions (wild-type, 22.85 s; mutant, 19.82 s; p = 0.69). See also Figure S5.