Comprehensive study for SNARE involved in the post-Golgi transport in Drosophila photoreceptors

Polarized transport is essential for the construction of multiple plasma membrane domains within cells. Drosophilaphotoreceptors serve as excellent model systems for studying the mechanisms of polarized transport. We conducted a comprehensive SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) screening of the fly genome using RNAi knockdown and CRISPR/Cas9 somatic knockout combined with the CoinFLP system to identify SNAREs involved in post-Golgi trafficking. The results suggest that, in post-Golgi transport, no SNARE is exclusively responsible for transport to a single specific plasma membrane domain. However, each SNARE shows some preference for certain membrane domains: the loss of nSyb, Ykt6, and Snap24/25 results in severe defects in rhabdomere transport, while the loss of Syx1A and Snap29 leads to significant impairments in basolateral transport. Together with the function of Syx1A, Snap25, and nSyb in the fusion of synaptic vesicles with the synaptic pl..., Plasmids were constructed as follows. pU62-wsh1pU62-wsh1 is a plasmid carrying a cassette that works as a \"dominant white-eye\" marker in Drosophila, consist of Drosophila U6-2 promoter and shRNA against white gene. This vector was constructed based on a vector, pAc-sgRNA-Cas9 (Bassett et al., 2014) by inserting a synthetic shRNA unit against white gene, HMS00017, predesigned in TRiP RNAi & CRISPR fly project (Zirin et al., 2020). Four oligonucleotides TTC-wsh1-F1, wsh1-R1p, wsh1-F2p and wsh1-Ap-GTT were ligated into pAc-sgRNA-Cas9 digested with BbsI, to obtain pU62-wsh1. pBIDwsh-UASCpBIDwsh-UASC is a phi31 transgenic vector based on pBID-UASC (Wang et al., 2012) but carrying \"dominant white-eye\" marker instead of miniature-white “red-eye” marker. From pU62-wsh1, U6-wshRNA cassette was amplified with primers Nh-U62 and gCas9puro-F, then digested with NheI and ApaI, and then cloned between NheI and ApaI sites of pBID-UASC to obtain pBID-wsh-UASC. pBIDwsh-Act-coinFLP-Gal4pBIDwsh-Act-c..., , Nucleotide sequences of plasmids and DNA fragments used in the paper. All files are in standard Genbank format, which can be viewed using many DNA handling sofwares such as Ape ([https://jorgensen.biology.utah.edu/wayned/ape/](https://jorgensen.biology.utah.edu/wayned/ape/)). ##### 5x\_longGMR\_promoter.gb * Usage: * The longGMR promoter (Wernet et al., 2003) consists of five copies of enhancer sequence in the upstream of Rh1 promoter and a basal promoter core of hsp70. Since the detail of the construct was not described in the original paper, the sequence was analysed. The region containing longGMR promoter was amplified using PCR from a transgenic fly caring P{longGMR-Gal4} with primers wdelR_Nhe (GGGCTAGCTTCAATGATGTCCAGTGCAG) and Gal4DBD-R2 (GACCTTTTGGTTTTGGGAGAGTAGCG) and sequenced. * Source: * Drosophila strain P{longGMR-Gal4} * Bloomington Drosophila Stock Center #8605 * [http://flybase.org/reports/FBti0058798.htm](http://flybase.org/reports/FBti0058798.htm) ...极性运输（polarized transport）对于细胞内多种质膜结构域的构建至关重要。果蝇光感受器是研究极性运输机制的极佳模型系统。本研究借助RNA干扰（RNA interference，RNAi）敲低、CRISPR/Cas9（成簇规律间隔短回文重复序列相关蛋白9，clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9）体细胞敲除，并联合CoinFLP系统，对果蝇全基因组开展了全面的SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体，soluble N-ethylmaleimide-sensitive factor attachment protein receptor）筛选，以鉴定参与高尔基后运输（post-Golgi trafficking）的SNARE蛋白。 研究结果显示，在高尔基后运输过程中，并无单一SNARE蛋白专门负责向某一特定质膜结构域的物质运输。但每种SNARE蛋白均对特定膜结构域表现出一定的运输偏好：nSyb、Ykt6及Snap24/25的缺失会导致感杆束（rhabdomere）运输出现严重缺陷，而Syx1A与Snap29的缺失则会使基底侧运输受到显著损伤。结合Syx1A、Snap25及nSyb在突触囊泡与突触[原文未竟]融合过程中的功能，本研究按如下方式构建了质粒。 ### pU62-wsh1 pU62-wsh1是一种携带果蝇“显性白眼”筛选标记盒的质粒，该标记盒由果蝇U6-2启动子及靶向white基因的短发夹RNA（short hairpin RNA，shRNA）组成。本载体以pAc-sgRNA-Cas9（Bassett等人，2014）为骨架，插入了TRiP RNAi与CRISPR果蝇项目（Zirin等人，2020）中预先设计的靶向white基因的合成shRNA单元HMS00017构建而成。将四条寡核苷酸TTC-wsh1-F1、wsh1-R1p、wsh1-F2p及wsh1-Ap-GTT连接至经BbsI酶切的pAc-sgRNA-Cas9载体中，即可获得pU62-wsh1。 ### pBIDwsh-UASC pBIDwsh-UASC是一种基于pBID-UASC（Wang等人，2012）的phi31转基因载体，但其筛选标记替换为“显性白眼”标记，而非原载体所使用的微型白眼“红眼”标记。以pU62-wsh1为模板，利用引物Nh-U62与gCas9puro-F扩增得到U6-wshRNA表达盒，经NheI与ApaI酶切后，克隆至pBID-UASC的NheI与ApaI酶切位点之间，即可获得pBID-wsh-UASC。 ### pBIDwsh-Act-coinFLP-Gal4 pBIDwsh-Act-c...（原文截断） 本论文中使用的质粒及DNA片段的核苷酸序列均采用标准Genbank格式存储，可通过多种DNA分析软件（如Ape）查看，相关链接：https://jorgensen.biology.utah.edu/wayned/ape/。 ##### 5x_longGMR_promoter.gb * 用途： longGMR启动子（Wernet等人，2003）包含Rh1启动子上游的五份增强子序列拷贝及hsp70的基础启动子核心区域。由于原始论文未详细描述该构建体的细节，本研究对其序列进行了分析。从携带P{longGMR-Gal4}的转基因果蝇中，利用引物wdelR_Nhe（GGGCTAGCTTCAATGATGTCCAGTGCAG）与Gal4DBD-R2（GACCTTTTGGTTTTGGGAGAGTAGCG）通过PCR扩增得到包含longGMR启动子的区域，并进行了测序。 * 来源： 果蝇品系P{longGMR-Gal4} 布卢明顿果蝇种质中心（Bloomington Drosophila Stock Center）编号#8605 相关链接：http://flybase.org/reports/FBti0058798.htm