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PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles.

PloS one (2017-09-15)
Nathan M Merrill, Joshua L Schipper, Jonathan B Karnes, Audra L Kauffman, Katie R Martin, Jeffrey P MacKeigan
ABSTRACT

Phosphoinositide 3-kinase (PI3K) family members are involved in diverse cellular fates including cell growth, proliferation, and survival. While many molecular details are known about the Class I and III PI3Ks, less is known about the Class II PI3Ks. To explore the function of all eight PI3K isoforms in autophagy, we knock down each gene individually and measure autophagy. We find a significant decrease in autophagy following siRNA-mediated PIK3C2A (encoding the Class 2 PI3K, PI3K-C2α) knockdown. This defective autophagy is rescued by exogenous PI3K-C2α, but not kinase-dead PI3K-C2α. Using confocal microscopy, we probe for markers of endocytosis and autophagy, revealing that PI3K-C2α colocalizes with markers of endocytosis. Though endocytic uptake is intact, as demonstrated by transferrin labeling, PIK3C2A knockdown results in vesicle accumulation at the recycling endosome. We isolate distinct membrane sources and observe that PI3K-C2α interacts with markers of endocytosis and autophagy, notably ATG9. Knockdown of either PIK3C2A or ATG9A/B, but not PI3KC3, results in an accumulation of transferrin-positive clathrin coated vesicles and RAB11-positive vesicles at the recycling endosome. Taken together, these results support a role for PI3K-C2α in the proper maturation of endosomes, and suggest that PI3K-C2α may be a critical node connecting the endocytic and autophagic pathways.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
OptiPrep Density Gradient Medium, used for cell and subcellular organelle isolation
Sigma-Aldrich
Anti-LC3B antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
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Anti-V5 antibody produced in rabbit, IgG fraction of antiserum
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MISSION® esiRNA, targeting human PIK3C2A

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