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Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.

ABSTRACT: Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

SUBMITTER: Levin-Konigsberg R

PROVIDER: S-EPMC8340083 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aim and experimental set-up: Sequencing of small RNA from total RNA fractions. The aim of this experiment was to compare profiles of miRNA expression between the following genetic backgrounds: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter). Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested, and total RNA was prepared by Trizol extraction. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform. Sequencing of small RNA bound to AGO1 in membrane fractions The aim of this experiment was to characterize AGO1-bound small RNAs in membrane fractions, and to answer two specific questions: Can miRNAs be identified whose association with membrane-bound AGO1 is different between the following four genotypes: Col-0 (wild type), era1-2 (farnesyl transferase mutant), j2-2/j3-2 + pJ3:J3 (j2/j3 double knockout expressing transgenic J3 under the control of the endogenous J3 promoter), j2-2/j3-2 + pJ3:J3C417S (j2/j3 double knockout expressing transgenic J3 mutated in the farnesylation site (C417S) under the control of the endogenous J3 promoter) Is the ratio between reads matching miRNA and miRNA* different between the above four genotypes? Seedlings were grown under sterile conditions for 16 days. Two biological replicates of each genotype were harvested. Microsomes were prepared from 2g of seedling tissue, solubilized by 1% deoxycholate and AGO1 was immunoprecipitated with specific antibodies (Agrisera). Immunopurified AGO1 was eluted from Protein A sepharose beads by competitive elution with antigenic AGO1 peptide. Small RNA was extracted by Trizol extraction from eluted AGO1. Small RNA libraries were constructed using NEBNext Small RNA Library Prep Set and sequenced on an Illumina platform.

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Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.

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