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CRISPR-Cas9-mediated labelling of the C-terminus of human laminin ?1 leads to secretion inhibition.

ABSTRACT: OBJECTIVES:The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elegans ortholog of human the LM?1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models. RESULTS:CRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LM?1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LM?1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. Moreover, the edited cells displayed reduced proliferation rates. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LM?1 inhibits LM secretion, and is not a viable approach for use in animal models.

SUBMITTER: Shaw L

PROVIDER: S-EPMC7035763 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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CRISPR-Cas9-mediated labelling of the C-terminus of human laminin β1 leads to secretion inhibition.

Shaw L L Williams R L RL Hamill K J KJ

BMC research notes 20200221 1

<h4>Objectives</h4>The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elega ...[more]

PMID: 32085798

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Project description:BackgroundThe prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic β-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance.MethodsCRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing.ResultsThe major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2.ConclusionThese findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.

Dataset Information

CRISPR-Cas9-mediated labelling of the C-terminus of human laminin ?1 leads to secretion inhibition.

Publications

CRISPR-Cas9-mediated labelling of the C-terminus of human laminin β1 leads to secretion inhibition.

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