FIGURE SUMMARY
Title

trappc11 is required for protein glycosylation in zebrafish and humans

Authors
DeRossi, C., Vacaru, A., Rafiq, R., Cinaroglu, A., Imrie, D., Nayar, S., Baryshnikova, A., Milev, M.P., Stanga, D., Kadakia, D., Gao, N., Chu, J., Freeze, H.H., Lehrman, M.A., Sacher, M., Sadler, K.C.
Source
Full text @ Mol. Biol. Cell

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Mutation of trappc11 in zebrafish causes secretory pathway defects. (A) Confocal microscopy on cryosections from 4- and 5-dpf trappc11 mutants and phenotypically WT siblings that ubiquitously express a Golgi apparatus marker (green), Tg(actb2:GalT-GFP), and a hepatocyte-specific ER marker (red), Tg(fabp10:ER-td-TMT). Hepatocytes that show abnormal morphology, that is, fragmented Golgi apparatus and distended ER, are marked with arrows and asterisks, respectively, and quantified (B) as percentage abnormal to total hepatocytes counted (# cells) from number of fish counted (n). The p values were calculated using Fisher’s exact test. (C) Bright-field and fluorescence images of 3- to 5-dpf WT and trappc11 transgenic Tg(fabp10:Gc-EGFP) larvae demonstrate GFP accumulation in the liver of trappc11 mutants, similar to that seen with BFA-treated (1 µg/ml) control larvae, which indicates defective protein secretion. Arrows indicate livers where Gc-EGFP is being produced, and arrowheads show secreted Gc-EGFP in the vasculature. (D) Confocal images of livers from 5-dpf WT and trappc11 mutants that express both the secreted Gc-EGFP (green) protein and the ER marker ER-tdTMT (red) confirms accumulation in the ER of mutant hepatocytes, evidenced by colocalization of both markers. (E) Western analysis of WT and trappc11-mutant livers using anti-GFP to detect Gc-EGFP and anti-Histone H3 as a loading control. Two independent clutches are shown for liver samples. Eight pooled livers and one liverless carcass were loaded. Bars, 10 µm (A, D).

Tm phenocopies trappc11 mutants and synergizes with trappc11 mutation. (A) Transgenic WT and trappc11 mutant larvae that express a hepatocyte-specific marker (Tg(fabp10:dsRed)) were exposed to sublethal doses of Tm at 3 dpf and analyzed at 5 dpf for gross morphology. (B) WT, trappc11 mutants, and WT exposed to 0.5 µg/ml BFA were treated with increasing concentrations of Tm (1, 2, or 3 µg/ml) beginning at 3 dpf and scored for survival at 4 and 5 dpf (three clutches for each Tm concentration). WT, trappc11 mutants, and BFA-treated samples are respectively represented by black, blue, and green lines, with 1, 2, and 3 µg/ml Tm indicated by solid, dashed, and dotted lines, respectively. (C) Cumulative survival at 5 dpf of WT, trappc11 mutants, and WT + 0.5 µg/ml BFA exposed to Tm (1, 2, and 3 µg/ml) between 3 and 5 dpf. Black, blue, and green bars indicate WT, trappc11, and WT + BFA, respectively, with increased shading representing different clutches. Three clutches total were scored for survival. Gray segmented lines mark 100, 200, and 300% levels. (D) Livers from 5-dpf WT and trappc11 mutants treated with DMSO or 0.125 µg/ml Tm were collected and subjected to qPCR analysis for UPR-responsive genes, and PC1 metrics were calculated. Values for each individual sample. (E) Livers from 5-dpf WT and trappc11 mutants treated with DMSO, 0.25 µM thapsigargin, or 0.5 µM thapsigargin were collected and subjected to qPCR analysis for UPR-responsive genes, and PC1 metrics were calculated. The p values in C and D were calculated using Student’s t test. n.s., not significant.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Terpenoid and dolichol synthesis defects in trappc11 mutants. (A) Schematic representation of the terpenoid biosynthetic pathway, with branch points at farnesyl-PP leading to production of dolichol (protein glycosylation), squalene (steroid synthesis), and geranylgeranyl-PP (protein prenylation). Fold changes of expression in trappc11 mutant livers are indicated by color: blue indicates decrease and red indicates increase relative to WT siblings; gray indicates genes not tested. Drugs used are indicated with a red T at their targets. (B) Confocal microscopy of transgenic WT and trappc11-mutant zebrafish expressing a prenylated form of GFP (Tg(actb2:CAAX-GFP)) in hepatocytes. Zebrafish were collected at 5 dpf and analyzed for membrane localization of CAAX-GFP. Samples were counterstained with DAPI (nucleus) and Cy5-streptavidin to stain the biotin-rich hepatocytes (middle; white boxes outline magnified GFP-only images shown on right). Scale bars, 10 µm. (C) Confocal microscopy of WT transgenic zebrafish expressing a prenylated form of GFP in hepatocytes (Tg(fabp10:CAAX-GFP). Zebrafish larvae were treated with DMSO (top) or 0.75 µg/ml BFA (bottom) from 3 to 5 dpf and analyzed at 5 dpf for membrane localization of CAAX-GFP. Samples were counterstained with DAPI (blue nucleus). White boxes outline area shown magnified on right (GFP only). Scale bars, 10 µM. (D) Whole WT and trappc11-mutant larvae were collected at 5 dpf, and total cholesterol was measured and normalized to protein content. trappc11 mutants have a significant decrease in cholesterol compared with WT siblings (p = 0.0008, paired Student’s t test), suggestive of decreased terpenoid biosynthetic activity. Individual clutches are indicated (n = 17 for each), with lines representing clutch pairing.

Atorvastatin is synthetically lethal with trappc11 mutation. (A) Transgenic WT and trappc11-mutant embryos expressing a liver-specific marker (Tg(fabp10:dsRed)) were treated with DMSO or the HMG-CoA reductase inhibitor, Atv, between 3 and 5 dpf at the concentrations indicated and analyzed for gross morphology and lethality. (B) Livers were collected from WT (n = 7 clutches), trappc11 (n = 8 clutches), and 5 µM Atv–treated WT embryos between 3 and 5 dpf (n = 7 clutches) and subjected to qPCR to analyze UPR gene expression used to calculate PC1. p values were calculated with Student’s t test. n.s., not significant. (C) Livers from larvae treated with DMSO or 1 µM Atv from 3 to 5 dpf (n = 8 clutches) were collected and analyzed at 5 dpf for the expression of genes involved in N-linked glycosylation. Atorvastatin-induced fold increases are shown relative to DMSO-treated samples.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

EXPRESSION / LABELING:
Gene:
Antibody:
Fish:
Anatomical Term:
Stage: Day 5

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Mol. Biol. Cell