Maintenance of Melanophore Morphology and Survival Is Cathepsin and vps11 Dependent in Zebrafish
Maintenance of Melanophore Morphology and Survival Is Cathepsin and vps11 Dependent in Zebrafish
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DOI：
10.1371/journal.pone.0065096
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发表时间：
2013-05-27
期刊：
影响因子：
3.7
通讯作者：
Cooper, Cynthia D.
中科院分区：
文献类型：
--
作者：
Clancey, Lauren F.;Beirl, Alisha J.;Cooper, Cynthia D.
在这里，我们描述了一个斑马鱼色素细胞突变体（黑素细胞完整性突变体），它显示了一个缺陷，在维护黑素细胞和虹膜细胞的数量。定位和候选基因分析将黑素细胞完整性突变体突变与液泡蛋白分选11（vps 11（w 66））基因联系起来。在幼虫阶段的vps 11（w 66）色素细胞的定量表明，与野生型同胞相比，数量减少。TUNEL分析和用半胱天冬酶抑制剂zVAD-favorite处理表明vps 11（w 66）色素细胞死亡是半胱天冬酶非依赖性的。突变体裂解物中PARP-1裂解模式的Western印迹分析表明，pH依赖性组织蛋白酶活性的增加参与了在vps 11（w 66）突变体中观察到的色素细胞过早死亡。同样，用ALLM和巴弗洛霉素A1（分别为组织蛋白酶/钙蛋白酶和空泡型H+-ATP酶抑制剂）处理，恢复vps 11（w 66）突变体中正常的黑色素细胞形态和数量。最后，LC 3B蛋白质印迹分析表明，与野生型对照相比，vps 11（w 66）突变体中自噬体标志物LC 3B II增加，但在ALLM或巴弗洛霉素A1处理的突变体中没有增加。综上所述，这些数据表明vps 11通过抑制组织蛋白酶释放和/或活性促进正常黑色素细胞形态和存活。
Here, we characterize a Danio rerio zebrafish pigment cell mutant (melanophore integrity mutant), which displays a defect in maintenance of melanophore and iridophore number. Mapping and candidate gene analysis links the melanophore integrity mutant mutation to the vacuolar protein sorting 11 (vps11(w66)) gene. Quantification of vps11(w66) chromatophores during larval stages suggests a decrease in number as compared to wildtype siblings. TUNEL analysis and treatment with the caspase inhibitor, zVAD-fmk, indicate that vps11(w66) chromatophore death is caspase independent. Western blot analysis of PARP-1 cleavage patterns in mutant lysates suggests that increases in pH dependent cathepsin activity is involved in the premature chromatophore death observed in vps11(w66) mutants. Consistently, treatment with ALLM and Bafilomycin A1 (cathepsin/calpain and vacuolar-type H+-ATPase inhibitors, respectively), restore normal melanophore morphology and number in vps11(w66) mutants. Last, LC3B western blot analysis indicates an increase in autophagosome marker, LC3B II in vps11(w66) mutants as compared to wildtype control, but not in ALLM or Bafilomycin A1 treated mutants. Taken together, these data suggest that vps11 promotes normal melanophore morphology and survival by inhibiting cathepsin release and/or activity.