20 Jan 2023
by Richa

Summary- paper 9: A genome-wide CRISPR interference screen using an engineered trafficking biosensor reveals a role for RME-8 in opioid receptor regulation

Brandon Novy, Hayden Adoff, Monica De Maria, Martin Kampmann, Nikoleta G. Tsvetanova, Mark von Zastrow, Braden Lobingier

Biorxiv, 2022

Questions/gaps addressed:

  • Post agonist-stimulated-endocytosis trafficking of GPCRs from the cell surface can result in receptor degradation in the lysosome or recycling back to the plasma membrane. The fate depends on a combination of sorting motifs and dynamic post-translation modifications. What are the machineries involved that make these sorting decisions?

Key methods:

  • Chemical functional genomics FACS-based pooled screen using APEX2 driven activation of fluorogenic substrate Amplex UltraRed as a read-out to monitor GPCR trafficking.

  • Delta opioid receptor (DOR) fused to APEX2 at the cytsoplasmic C-terminus (DOR-APEX2). When in the cytosol, APEX2 can convert the non-fluorescent substrate Amplex UltraRed (AUR) to a fluorescent resorufin-like molecule in the presence of hydrogen peroxide.

  • Upon agonist (DADLE) addition, DOR-APEX2 is delivered to the MVB and lysosome lumen, leading to DOR-APEX2 degrdation by proteases at low pH, resulting in loss of fluoresence in lysates. Lysate APEX2/AUR reaction can be quenched with 100-fold molar excess of a competitive substrate (sodium L-ascorbate).

  • Adapting the assay to intact cells: Applying a transient pulse of sodium azide to quench the reaction and bovine serum albumin to scavenge excess dye. RNAi against Arrestin3, blocks DOR endocytosis, resulting in stabilization of fluorescence signal monitored by red laser (APC channel: Ex: 633 nm; Filter: 670/30 nm)

  • Genome-wide CRISPR interference (CRISPRi) dCas9 based screen and second generation libraries encompassing the entire human genome. HEK293 cell line stably expressing dCas9-KRAB and pUBC:DOR-APEX2. Seven sub-libraries consisting of 5 sgRNAs/gene and controls. Estimated sub-library coverage of 500-fold. Following APEX2/AUR reaction, cells sorted to isolate the high and low fluorescence. Genes identified by next generation sequencing.

Major takeaways:

  • The activity of APEX2 that is sensitive to mildly acidic conditions.

  • Identified hits of genes functioning along the endocytic and secretory pathway. Followed-up on some hits that were not known to function with DOR: DNAJC13/ RME-8 (strongest stabilization of fluorescence), WDR91, and SNX24.

  • RME-8 (no yeast homolog), conserved from worms to humans, peripherally associates with EEA1-positive endosomes. RME-8 and endocytosed DOR2 were adjacent not overlapping. RME-8 signal in between DOR2 and VPS35. RME-8 is enriched at sites of endosomal tubulation/recycling?

  • No defect in lysosomal delivery upon KD of other components of the recycling pathway, VPS35 or FAM21A/C. RME-8 appears to be anew player modulating the lysosomal delivery of DOR-APEX2. Mechanism unclear. Cool genetic screen approach.

  • Flexibility of the APEX2 enzyme to mediate oxidation of different chemical substrates allows switching between proximity labeling and fluorogenic activation in the same cell line. Cool strategy!