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A high-content arrayed CRISPR screen reveals genetic requirements for dynein-based trafficking

ABSTRACT: A high-content arrayed CRISPR screen reveals genetic requirements for dynein-based trafficking

PROVIDER: PRJNA902801 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR22325460_1.fastq.gz	Fastqsanger.gz
	SRR22325460_2.fastq.gz	Fastqsanger.gz
	SRR22325461_1.fastq.gz	Fastqsanger.gz
	SRR22325461_2.fastq.gz	Fastqsanger.gz
	SRR22325462_1.fastq.gz	Fastqsanger.gz

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A high-content arrayed CRISPR screen reveals genetic requirements for dynein-based trafficking

Project description:The RNA-binding protein SUGP1 was one of many proteins identified in a CRISPR-based screen for proteins that promote dynein-based cargo trafficking in U-2 OS cells. To elucidate how SUGP1 promotes transport, we investigated changes in gene expression caused by disrupting the SUGP1 gene in this cell line with CRISPR/Cas9. As controls, we disrupted the XCR1 gene, which is not expressed in U-2 OS cells, and treated cells with crRNAs that lack specific targets in the human genome. Sequences were quality trimmed by the commercial provider of the RNA-seq data before being provided to the contributors.

2024-02-29 | GSE218249 | GEO

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

Project description: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.

| S-EPMC10002790 | biostudies-literature

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

Project description:The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are 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 genome-wide gRNA library, 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 revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

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HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1- bound endosomal membranes_BioMass@LMB

Project description:The dynein motor complex mediates polarised trafficking of a wide variety of organelles, intracellular vesicles and macromolecules. These functions are dependent on the dynactin 5 complex, which helps recruit cargoes to dynein’s tail region and activates motor movement. How dynein and dynactin orchestrate trafficking of diverse cargoes is unclear. Here, we identify HEATR5B, an interactor of the AP1 clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B is one of several proteins recovered in a biochemical screen for proteins whose association with the human dynein tail complex is augmented by 10 dynactin. We show that HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. We also demonstrate that the HEATR5B homologue in Drosophila is an essential gene that promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiledcoil architecture typical of dynein adaptors, our data point to a non-canonical process 15 orchestrating motor function on a specific cargo. We additionally show that HEATR5B promotes association of AP1 with endosomal membranes in a dynein-independent manner. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking.

2023-10-20 | PXD043387 | Pride

Antiviral function and viral antagonism of the rapidly evolving dynein activating adapter NINL

Project description:Viruses interact with the intracellular transport machinery to promote viral replication. Such host-virus interactions can drive host gene adaptation, leaving signatures of pathogen-driven evolution in host genomes. Here we leverage these genetic signatures to identify the dynein activating adaptor, ninein-like (NINL), as a critical component in the antiviral innate immune response and as a target of viral antagonism. Unique among genes en¬¬coding for dynein subunits, subunits of its co-factor dynactin, and dynein activating adaptors, NINL has evolved under recurrent positive selection, specifically in its carboxy-terminal cargo binding region. Consistent with a role for NINL in host immunity, NINL knockout cells are more permissive to viral replication as a result of a severe attenuation of interferon stimulated gene (ISG) production following interferon treatment. Moreover, we show that proteases encoded by diverse picornaviruses and coronaviruses cleave and disrupt NINL function in a host- and virus-specific manner. Our work reveals the importance of NINL in the antiviral response and the utility of using signatures of host-virus conflicts to uncover new components of antiviral immunity and targets of viral antagonism.

2022-07-08 | GSE206784 | GEO

An endophyte-derived small molecule targets LIC1 to suppress lung tumor growth by inducing autophagy

Project description:LIC1(DYNC1LI1,DYN), along its homologue LIC2, constitutes subunits of the multiprotein cytoplasmic dynein complex. Although the dynein complex is known to play a role in membrane trafficking, organelle positioning, and microtubule organization, the specific functions of these individual LICs are rarely investigated.Knockdown or small molecule inhibition of LIC1 in NSCLC cells significantly suppressed cell proliferation and induced autophagic cell death.

2025-08-15 | GSE282502 | GEO

Dynlrb1 is Essential for Dynein Mediated Transport and Neuronal Survival

Project description:Dynlrb1 is Essential for Dynein Mediated Transport and Neuronal Survival

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Transcription profiling of multiple human tumour specimens of different anatomical origin arrayed against a common reference

Project description:Multiple tumour specimens of different anatomical origin arrayed against a common reference.

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Ruminal content

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An arrayed CRISPR library for individual, combinatorial and multiplexed gene knockout III

Project description:We have combined a machine-learning approach with other strategies to optimize the efficiency of sgRNAs for CRISPR screens and have constructed a genome-wide, sequence-verified, arrayed CRISPR library. This incorporates expression strategies to facilitate multiplexed or combinatorial screening. By conducting parallel loss-of-function screens, we compare our approach to existing sgRNA design and expression strategies.

2017-07-20 | GSE97433 | GEO

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