Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen.
Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen.
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高内涵阵列 CRISPR 筛选揭示了基于动力蛋白的运输的基因组规模要求。
DOI：
10.1101/2023.03.01.530592
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Bullock,SimonL
中科院分区：
文献类型：
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作者：
Wong,ChunHao;Wingett,StevenW;Qian,Chen;Taliaferro,JMatthew;Ross-Thriepland,Douglas;Bullock,SimonL
细胞质动力蛋白-1(Dynein)马达在细胞组织中起着关键作用，它将多种细胞成分输送到微管的负端。然而，对于马达的生物合成、组装和功能多样性是如何协调的，人们知之甚少。为了解决这个问题，我们在人类细胞中进行了阵列式CRISPR功能丧失筛查，使用动力蛋白连接的过氧化物体和早期内切体的分布作为读数。从针对18,253个基因的引导RNA文库中，恢复了195个有效的命中，并将其解析为影响多个动力蛋白货物的那些，以及那些作用仅限于部分货物的那些。对来自多路复用图像的高维表型指纹进行聚类，揭示了参与许多细胞过程的协同功能基因，包括几个候选的核心动力蛋白功能的新调节因子。对其中一种蛋白质，RNA结合蛋白SUGP1的机械分析提供了证据，表明它通过维持动力蛋白激活剂Lis1的功能表达来促进货物运输。我们的数据集代表了丰富的新假说来源，用于研究基于微管的运输，以及我们的高含量成像捕捉到的细胞组织的其他几个方面。
The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.