Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

Abstract

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.

Spatiotemporal proteomic profiling of cellular responses to NLRP3 agonists

“…However, our data support a model where TGN38/46 dispersion into endosomes is a consequence of cellular stress rather than a feature of NLRP3 activation, in agreement with recent studies [58][59]. Further supporting this conclusion, a phenotypic CRISPR screen demonstrated that many cellular perturbations of transport likely unrelated to NLRP3 activation induce TGN38/46 peripheral redistribution [171].…”

The ORP9-ORP11 dimer promotes sphingomyelin synthesis

“…1A). For optimal gene disruption, we used a lentiviral gene delivery system and 3 guide RNAs per gene – a method also used by others to increase the frequency of gene disruption 16 . We applied the LTP knockout library to MelJuSo, a human melanoma cell line.…”

“…Targeting and non-targeting guide sequences (Supplementary Table 1) were obtained from the Brunello Human CRISPR Knockout Pooled Library [https://www.addgene.org/pooled-library/broadgpp-humanknockout-brunello/ ; 26780180] and cloned into lentiCRISPR v2 plasmid [https://www.addgene.org/52961/ ; 25075903] as described previously 36 . To increase the efficiency of the gene disruption, we used 3 guide RNAs per gene -a method also used by others to increase the frequency of gene disruption 37 . Cloning of guide RNAs were individually confirmed by Sanger sequencing.…”

The intracellular bacteriumOrientia tsutsugamushihijacks the adaptor protein BICD2 for dynein-based motility

“…As a positive control, we used a GFP-BICD2N MTS line expressing the constitutively active N-terminal fragment of BICD2 (residues 1-400, BICD2N), which is known to elicit dynein-based motility and has been used extensively in organelle relocation assays (Supplementary Fig. 1B) 25,26,28 . GFP MTS expression did not alter the distribution of mitochondria, which remained scattered in the cytoplasm as observed in wild type cells (Fig.…”

“…1C) 25,26 . An accumulation of mitochondria at the perinuclear region provides evidence that the candidate protein is a dynein effector 25,27,28 . We generated Flp-in T-REX HeLa cell lines expressing GFP MTS or GFP-ScaC MTS fusion proteins.…”