Figure 8.
ICMT-1 localizes to ER exit sites that dynamically polarize toward the invasive protrusion. (A) Top: Sum intensity z-projected images of ICMT-1::mScarlet (magenta), ER exit marker mNG::SEC-16A.1 (cyan), and overlay in the AC reveal colocalization at ER exit sites (arrows). The average percentage ± SD of colocalization of ER exit sites with ICMT-1 puncta is indicated (n = 17 animals). Bottom: Insets highlight ICMT-1::mScarlet and mNG::SEC-16A.1 colocalization in the ER at the AC invasive front (arrows). (B) Max intensity z-projected fluorescence images of GFP::CED-10 in a control and sar-1 RNAi treated animals shows GFP::CED-10 localized to the invasive plasma membrane (arrow) in the control animal, but predominantly localized in intracellular vesicles in sar-1 RNAi animals (brackets). Right: The relative proportion of animals with CED-10::GFP that localized predominantly to the AC basal plasma membrane or was found predominantly in the cytosol in control and sar-1 RNAi treated animals (n = 38 control and 45 sar-1 RNAi animals, ** P ≤ 0.01, Fisher’s exact test). (C) Fluorescence images showing AC ER exit sites (mNG::SEC-16A.1, Cyan; AC outlined with the plasma membrane marker mCherry::PLCδPH, red), Golgi (AMAN-2::mScarlet), and prenylated GFP::CAAX in the AC from the P6.p1-cell to 4-cell stages. Arrows indicate basal (invasive front) enrichment of each protein. Right: Quantifications of the relative distribution of the ER exit site, Golgi, and plasma membrane markers in the AC from the P6.p 1-cell to 4-cell stages. For mNG::SEC-16A.1 and AMAN-2::mScarlet, the total number of puncta in the AC apical and basal halves (green dashed line) was expressed as a basal/apical ratio. The mean fluorescence intensity of GFP::CAAX was expressed as a basal/apical ratio ([SEC-16A.1] n = 10 1-cell, 10 2-cell, 9 2–4-cell, and 10 4-cell stage animals; [AMAN-2] n = 10 1-cell, 15 2-cell, 14 2–4-cell, and 16 4-cell stage animals; [CAAX] n = 12 1-cell, 11 2-cell, 10 2–4-cell, and 11 4-cell stage animals, * P ≤ 0.05, ** P ≤ 0.01, Mann–Whitney U test, and Brown–Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test). (D) A schematic diagram illustrating where GTPase prenylation occurs, with FCE-1 present in the ER and peroxisomes and ICMT-1, the final prenylation enzyme, enriched in ER exit sites (ERES) that deliver prenylated proteins (i.e., Rac and Ras-related) to the Golgi apparatus for targeting to the invasive protrusion (red arrows). Rac/Ras-related prenylated proteins might also be delivered directly from the ER to the invasive plasma membrane (magenta arrows, inset). All data in the figure are from two or more replicates. Scale bars, 5 µm.

ICMT-1 localizes to ER exit sites that dynamically polarize toward the invasive protrusion. (A) Top: Sum intensity z-projected images of ICMT-1::mScarlet (magenta), ER exit marker mNG::SEC-16A.1 (cyan), and overlay in the AC reveal colocalization at ER exit sites (arrows). The average percentage ± SD of colocalization of ER exit sites with ICMT-1 puncta is indicated (n = 17 animals). Bottom: Insets highlight ICMT-1::mScarlet and mNG::SEC-16A.1 colocalization in the ER at the AC invasive front (arrows). (B) Max intensity z-projected fluorescence images of GFP::CED-10 in a control and sar-1 RNAi treated animals shows GFP::CED-10 localized to the invasive plasma membrane (arrow) in the control animal, but predominantly localized in intracellular vesicles in sar-1 RNAi animals (brackets). Right: The relative proportion of animals with CED-10::GFP that localized predominantly to the AC basal plasma membrane or was found predominantly in the cytosol in control and sar-1 RNAi treated animals (n = 38 control and 45 sar-1 RNAi animals, ** P ≤ 0.01, Fisher’s exact test). (C) Fluorescence images showing AC ER exit sites (mNG::SEC-16A.1, Cyan; AC outlined with the plasma membrane marker mCherry::PLCδPH, red), Golgi (AMAN-2::mScarlet), and prenylated GFP::CAAX in the AC from the P6.p1-cell to 4-cell stages. Arrows indicate basal (invasive front) enrichment of each protein. Right: Quantifications of the relative distribution of the ER exit site, Golgi, and plasma membrane markers in the AC from the P6.p 1-cell to 4-cell stages. For mNG::SEC-16A.1 and AMAN-2::mScarlet, the total number of puncta in the AC apical and basal halves (green dashed line) was expressed as a basal/apical ratio. The mean fluorescence intensity of GFP::CAAX was expressed as a basal/apical ratio ([SEC-16A.1] n = 10 1-cell, 10 2-cell, 9 2–4-cell, and 10 4-cell stage animals; [AMAN-2] n = 10 1-cell, 15 2-cell, 14 2–4-cell, and 16 4-cell stage animals; [CAAX] n = 12 1-cell, 11 2-cell, 10 2–4-cell, and 11 4-cell stage animals, * P ≤ 0.05, ** P ≤ 0.01, Mann–Whitney U test, and Brown–Forsythe and Welch ANOVA tests followed by Dunnett’s T3 multiple comparisons test). (D) A schematic diagram illustrating where GTPase prenylation occurs, with FCE-1 present in the ER and peroxisomes and ICMT-1, the final prenylation enzyme, enriched in ER exit sites (ERES) that deliver prenylated proteins (i.e., Rac and Ras-related) to the Golgi apparatus for targeting to the invasive protrusion (red arrows). Rac/Ras-related prenylated proteins might also be delivered directly from the ER to the invasive plasma membrane (magenta arrows, inset). All data in the figure are from two or more replicates. Scale bars, 5 µm.

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