Knockdown of SCF(Skp2) function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes.
Knockdown of SCF(Skp2) function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes.
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DOI：
10.1371/journal.pone.0079019
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Schulz RA
中科院分区：
文献类型：
--
作者：
Kroeger PT Jr;Shoue DA;Mezzacappa FM;Gerlach GF;Wingert RA;Schulz RA
在果蝇中，循环血细胞在造血的胚胎波中来源于头中胚层。这些细胞被贡献给幼虫，并在蜕变成成虫的过程中持续存在。为了分析这一血细胞群，我们考虑了先前发表的造血生态位RNAi筛选数据，该数据表明SCF复合物的几个成员在淋巴腺发育中发挥作用。eater-Gal4;将UAS-GFP果蝇与UAS-RNAi细胞系杂交，以血浆细胞特异性方式敲除所有已知SCF复合物成员的功能，以确定哪些成员是血浆细胞的新调节剂。这种特殊的SCF复合物包含五个核心成员：Lin-19-like、SkpA、Skp2、Roc1a和复合物激活剂Nedd8。该复合体是由其非常独特的大细胞表型确定的。此外，这些大细胞被染色为抗p1，一种浆细胞特异性抗体。人们还注意到，这些细胞中的DNA似乎过度复制。γ -微管蛋白和DAPI染色提示细胞正在进行再复制，因为它们有多个中心粒和过量的DNA含量。进一步实验表明，增大的细胞brdu呈阳性，表明它们已进入s期。为了确定这些细胞如何变大并进行再复制，用免疫荧光分析细胞周期蛋白。该分析确定了在这些增大的细胞中改变亚细胞定位的三种蛋白质：Cyclin E、Geminin和double - parking。先前的研究表明，双停放必须被降解才能退出s期，否则DNA将进行重新复制。当用过量的双停抑制剂geminin从细胞核中滴定double - parking时，增大的细胞和异常的蛋白质定位表型被部分拯救。本报告中的数据表明，在浆细胞分裂过程中，SCFSkp2复合物对于双停放细胞泛素化是必要的，从而确保正常的细胞周期进程和正常的这种必需血细胞类型的产生。
In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCFSkp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division, ensuring proper cell cycle progression and the generation of a normal population of this essential blood cell type.
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