Characterization of kinesin-like proteins in silkworm posterior silkgland cells
Characterization of kinesin-like proteins in silkworm posterior silkgland cells
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DOI：
10.1038/cr.2010.91
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发表时间：
2010
期刊：
影响因子：
44.1
通讯作者：
Qiao Wang;J. Teng;Birong Shen;Wei Zhang;Yige Guo;Xiaolei Su;Chuanxi Zhang;A. C. Yu;Jianguo Chen
中科院分区：
文献类型：
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作者：
Qiao Wang;J. Teng;Birong Shen;Wei Zhang;Yige Guo;Xiaolei Su;Chuanxi Zhang;A. C. Yu;Jianguo Chen
动蛋白是一种以微管为基础的马达，参与多种细胞内转运。神经元、鞭毛细胞和色素细胞传统上被用作研究动蛋白的细胞功能的模型系统。在这里，我们报道了家蚕后丝腺(PSG)，一种具有广泛的细胞内膜系统的特殊细胞，用于细胞内运输和有效地分泌丝素，作为一种新的运动蛋白研究模型。为了研究动蛋白在PSG细胞中的细胞内转运，我们克隆了5个家蚕动蛋白样蛋白(KLP)：BmKinesin-1、BmKinesin-6、BmKinesin-7、BmKinesin-13和BmKinesin-14A。用相对实时定量聚合酶链式反应和免疫印迹法检测它们的表达模式。免疫荧光显微镜证实它们与微管共存。通过结合下拉实验、LC-MS/MS和Western blotting分析，我们鉴定了PSG中许多潜在的BmKinesin-1，包括含有丝素的颗粒和与Experantia相关的核糖核蛋白(RNP)复合体。此外，BmKinesin-13的过表达破坏了BMN细胞中的微管网络，这与Kinesin-13在其他生物中调节微管动力学的作用是一致的。在这些结果的基础上，我们得出结论，PSG在阐明分泌组织的细胞内转运机制方面具有优势，可以作为一种潜在的运动蛋白研究模型。
Kinesins are microtubule-based motors involved in various intracellular transports. Neurons, flagellated cells, and pigment cells have been traditionally used as model systems to study the cellular functions of kinesins. Here, we report silkworm posterior silkgland (PSG), specialized cells with an extensive endomembrane system for intracellular transport and efficient secretion of fibroin, as a novel model for kinesin study. To investigate kinesin-driven intracellular transport in PSG cells, we cloned five silkworm kinesin-like proteins (KLPs), BmKinesin-1, BmKinesin-6, BmKinesin-7, BmKinesin-13, and BmKinesin-14A. We determined their expression patterns by relative real-time PCR and western blotting. Immunofluorescence microscopy verified their colocalization with microtubules. By combining pull-down assays, LC-MS/MS, and western blotting analysis, we identified many potential cargoes of BmKinesin-1 in PSG, including fibroin-containing granules and exuperantia-associated ribonucleoprotein (RNP) complexes. Moreover, BmKinesin-13 overexpression disrupted the microtubule network in BmN cells, which is consistent with a role of Kinesin-13 in regulating microtubule dynamics in other organisms. On the basis of these results, we concluded that PSG might have advantages in elucidating mechanisms of intracellular transport in secretory tissues and could serve as a potential model for kinesin studies.