Abstract
Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure. Here, we report that zebrafish Tmbim3a/Grinaa functions in the prevention of cold-induced endoplasmic reticulum (ER) stress and apoptosis. Using a gene-trapping approach, we obtained a mutant zebrafish line in which the expression of the tmbim3a/grinaa gene is disrupted by a Tol2 transposon insertion. Homozygous tmbim3a/grinaa mutant larvae exhibited time-dependently increased mortality and apoptosis under cold exposure (at 16 °C). Mechanistically, using immunofluorescence, fluorescence-based assessments of intracellular/mitochondrial Ca2+ levels, mitochondrial membrane potential measurements, and Ca2+-ATPase assays, we found that cold exposure suppresses sarcoplasmic/ER Ca2+-ATPase (SERCA) activity and induces the unfolded protein response (UPR) and ER stress. We also found that the cold-induced ER stress is increased in homozygous tmbim3a/grinaa mutant embryos. The cold-stress hypersensitivity of the tmbim3a/grinaa mutants was tightly associated with disrupted intracellular Ca2+ homeostasis, followed by mitochondrial Ca2+ overload and cytochrome c release, leading to the activation of caspase 9- and caspase-3-mediated intrinsic apoptotic pathways. Treatment of zebrafish larvae with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) or with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the calcium-releasing protein inositol 1,4,5-trisphosphate receptor (IP3R), alleviated cold-induced cell death. Together, these findings unveil a key role of Tmbim3a/Grinaa in relieving cold-induced ER stress and in protecting cells against caspase 9- and caspase 3-mediated apoptosis during zebrafish development.
Highlights
Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure
We have generated a tmbim3a/grinaa mutant zebrafish line using a Tol2 transposon– based gene-trapping approach [12], in which the tmbim3a/grinaa expression was interrupted by the insertion of a Tol2 transposon into the second intron
Tmbim3a/Grinaa-deficient embryos at 16 °C extensively suffered from individual death and cell death, which cannot be compensated by Tmbim3b/Grinab, suggesting a specific function of Tmbim3a/ Grinaa in the protection of developing embryos against cold stress
Summary
In a screening for insertion mutants through a Tol transposon–mediated gene-trapping approach [12], we identified a zebrafish line that carries a Tol transposon in the second intron of the tmbim3a/grinaa gene through PCR-based genome walking assays (Fig. 1A). The survival rates of WT and homozygous mutants at 16 °C were not significantly affected by injection of tmbim3b/grinab-MO [3] or tmbim3b/grinab capped mRNA (Fig. S3) These data suggest that Tmbim3a/Grinaa and Tmbim3b/Grinab have differential roles in zebrafish and that Tmbim3a/Grinaa plays a dominant role in protection of developing embryos from cold-induced cell death. The expression levels of UPR-related ER stress genes in homozygous tmbim3a/grinaa mutants were significantly higher than those in WT embryos after exposure to 16 °C for 60 h (from 12 to 72 hpf) (Fig. 5D). Together with inhibited CHOP expression in (Fig. 6D), TUNEL assays revealed that treatment with 4-PBA or CDN1163 markedly attenuated cold-induced apoptosis (Fig. S6) Taken together, these results suggest that cold exposure inhibits the Ca2ϩ-ATPase activity of SERCA, leading to the disrupted Ca2 homeostasis, increased ER stress, and cell death. These results suggest that cold stress can stimulate the release of ER-Ca2ϩ, followed by the elevation of cytosolic Ca2ϩ level and mitochondrial Ca2ϩ overload
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