Regulation and localization of prenylation enzymes and GTPases during AC invasion. (A) Sum intensity z-projected images show high levels of FNTA-1::mNG in the AC at the P6.p 2-cell stage (n = 12/12 animals) and 4-cell stage (n = 12/12). FNTA-1::mNG basal enrichment was observed at the P6.p 4-cell stage (arrows, n = 12/12). Bottom panels show spectral fluorescence-intensity maps, which display the minimum (L, low) and maximum (H, high) pixel value range of the acquired data. (B) Sum intensity z-projected images of ICMT-1::mScarlet in the AC show polarization in a wild-type animal (arrows) and lack of polarization in an unc-6(ev400) mutant animal at the initiation of invasive protrusion formation. Bottom panels show spectral fluorescence-intensity maps, which display the minimum (L, low) and maximum (H, high) pixel value range of the acquired data. Right: Boxplot shows AC basal/apical ratio of ICMT-1::mScarlet in wild-type and unc-6(ev400) animals (n = 31 control and 34 unc-6(ev400) animals, **** P ≤ 0.0001, unpaired two-tailed Student’s t test). (C) Top: Single slice confocal images of mCherry::KDEL (magenta, ER marker), mNG::FCE-1 (cyan), and overlay in the AC show overlap of mNG::FCE-1 and mCherry::KDEL signals (white arrows) and a region of mNG::FCE-1 with no overlap with mCherry::KDEL (yellow arrows). Bottom: Insets highlighting region of mNG::FCE-1 fluorescence where there is no mCherry::KDEL (yellow arrows, similar colocalization observed in n = 13/13 animals). (D) Top: Single slice confocal images of mScarlet::PTS1 (magenta, peroxisome marker), mNG::FCE-1 (cyan), and overlay in the AC shows overlap of mNG::FCE-1 regions and mScarlet::PTS1 (white arrows). Bottom: Insets highlighting region of overlap (white arrows; similar colocalization observed in = 3/3 animals). (E) Top: Central AC DIC images and corresponding AC max intensity z-projected images of sfGFP::KDEL (ER marker, top) and (bottom) DIC image and AMAN-2::GFP (Golgi marker, bottom) at the time of protrusion formation show the ER and Golgi extending into the protrusion (below the BM breach indicated by dotted orange line, n = 12/12 ER and 12/14 Golgi). (F) Top: Sum intensity z-projected image of AC specific expression of sfGFP::KDEL (ER marker) at the P6.p 4-cell stage. Bottom: Line graph of AC basal/apical polarization from the P6.p 1-cell through 4-cell stages (n = 8 1-cell, 8 2-cell, 9 2–4-cell, and 6 4-cell stage animals, ns [not statistically significant], Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test and Kruskal–Wallis test followed by Dunn’s multiple comparisons test). (G) Left: Sum intensity z-projected images showing the time course of GFP::CED-10, GFP::MIG-2, and mNG::RAP-1 polarization from the P6.p 1-cell to 4-cell stage. Right: Line graphs of AC basal/apical polarization of each protein’s fluorescence intensity ([CED-10] n = 10 1-cell, 15 2-cell, 14 2–4-cell, and 15 4-cell stage animals; [MIG-2] n = 10 1-cell, 11 2-cell, 13 2–4-cell, and 17 4-cell stage animals; [RAP-1] n = 11 1-cell, 13 2-cell, 10 2–4-cell, and 19 4-cell stage animals, * P ≤ 0.05, ** P ≤ 0.01, Brown-Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test and Kruskal–Wallis test followed by Dunn’s multiple comparisons test). All data are from two or more replicates. Scale bars, 5 µm.