Abstract
ADP-ribosylation factor GTPases are activated by guanine nucleotide exchange factors including Gbf1 (Golgi brefeldin A-resistant factor 1) and play important roles in regulating organelle structure and cargo-selective vesicle trafficking. However, the developmental role of Gbf1 in vertebrates remains elusive. In this study, we report the zebrafish mutant line tsu3994 that arises from N-ethyl-N-nitrosourea (ENU)-mediated mutagenesis and is characterized by prominent intracerebral and trunk hemorrhage. The mutant embryos develop hemorrhage accompanied by fewer pigments and shorter caudal fin at day 2 of development. The hemorrhage phenotype is caused by vascular breakage in a cell autonomous fashion. Positional cloning identifies a T → G nucleotide substitution in the 23rd exon of the gbf1 locus, resulting in a leucine → arginine substitution (L1246R) in the HDS2 domain. The mutant phenotype is mimicked by gbf1 knockouts and morphants, suggesting a nature of loss of function. Experimental results in mammalian cells show that the mutant form Gbf1(L1246R) is unable to be recruited to the Golgi apparatus and fails to activate Arf1 for recruiting COPI complex. The hemorrhage in tsu3994 mutants can be prevented partially and temporally by treating with the endoplasmic reticulum stress/apoptosis inhibitor tauroursodeoxycholic acid or by knocking down the proapoptotic gene baxb Therefore, endothelial endoplasmic reticulum stress and subsequent apoptosis induced by gbf1 deficiency may account for the vascular collapse and hemorrhage.
Highlights
ADP-ribosylation factor GTPases are activated by guanine nucleotide exchange factors including Gbf1 (Golgi brefeldin A-resistant factor 1) and play important roles in regulating organelle structure and cargo-selective vesicle trafficking
Experimental results in mammalian cells show that the mutant form Gbf1(L1246R) is unable to be recruited to the Golgi apparatus and fails to activate Arf1 for recruiting COPI complex
These results suggest that the Leu 3 Arg mutation disrupts the Golgi targeting property of Gbf1 protein; in other words, the HDS2 domain is required for localization of Gbf1 to the Golgi apparatus
Summary
Tsu3994 Mutant Embryos Exhibit Hemorrhage in the Head and Trunk—In an ENU-mediated mutagenesis screen, we identified a zygotic mutant line, tsu3994. When the cells were treated with BFA or GCA, Flag-Gbf was prominently enriched in the cis-Golgi region (Fig. 5, C, E, and G), whereas less FlagGbf1(L1246R) was recruited to the Golgi apparatus (Fig. 5, D, F, and G) These results suggest that the Leu 3 Arg mutation disrupts the Golgi targeting property of Gbf protein; in other words, the HDS2 domain is required for localization of Gbf to the Golgi apparatus. Numerous Genes Are Up-regulated in Endothelial Cells of tsu3994 Mutants—We analyzed, by RNA-Seq, transcriptional profiles of kdrl:GFPϩ ECs sorted from tsu3994;Tg(kdrl:GFP) mutant or WT sibling embryos at 44 hpf. Inhibition of ER Stress and Apoptosis Compromises Hemorrhage in tsu3994 Mutants—Interestingly, we observed a significant increase of the ER stress marker ddit3/chop, as well as the proapoptotic genes badb and baxb in mutant endothelial cells (Fig. 9, E and F). Defects in pigmentation and fins in tsu3994 were not obviously compromised by TUDCA treatment or/and baxb knockdown (data not shown), suggesting that molecular mechanisms underlying different defects are distinct
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