PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles
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 isoform...
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| creator | Merrill, Nathan M Schipper, Joshua L Karnes, Jonathan B Kauffman, Audra L Martin, Katie R MacKeigan, Jeffrey P |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0184909 |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0184909</identifier><identifier>PMID: 28910396</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>1-Phosphatidylinositol 3-kinase ; Accumulation ; Autophagy ; Biology ; Biology and Life Sciences ; Biomarkers - metabolism ; Cancer ; Cell death ; Cell Line ; Cell Proliferation ; Cell survival ; Clathrin ; Coated vesicles ; Confocal microscopy ; Endocytosis ; Endosomes ; Endosomes - metabolism ; Enzymes ; Gene Knockdown Techniques ; Humans ; Isoforms ; Kinases ; Markers ; Maturation ; Medical research ; Microscopy ; Phagocytosis ; Phosphatidylinositol 3-Kinases - genetics ; Phosphatidylinositol 3-Kinases - metabolism ; Phosphorylation ; Physiology ; Protein turnover ; Proteins ; RNA, Small Interfering - metabolism ; Signal Transduction ; siRNA ; Transferrin ; Transferrins ; Transport Vesicles - metabolism ; Vesicles ; Yeast</subject><ispartof>PloS one, 2017-09, Vol.12 (9), p.e0184909-e0184909</ispartof><rights>2017 Merrill et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Merrill et al 2017 Merrill et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-44cb61ad26bd3c3632fcf48cf2b92fab261d558561df80f7f3ef49e2e99d412c3</citedby><cites>FETCH-LOGICAL-c526t-44cb61ad26bd3c3632fcf48cf2b92fab261d558561df80f7f3ef49e2e99d412c3</cites><orcidid>0000-0002-0198-5679</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599018/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599018/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28910396$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Donaldson, Julie G.</contributor><creatorcontrib>Merrill, Nathan M</creatorcontrib><creatorcontrib>Schipper, Joshua L</creatorcontrib><creatorcontrib>Karnes, Jonathan B</creatorcontrib><creatorcontrib>Kauffman, Audra L</creatorcontrib><creatorcontrib>Martin, Katie R</creatorcontrib><creatorcontrib>MacKeigan, Jeffrey P</creatorcontrib><title>PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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. 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Merrill, Nathan M</au><au>Schipper, Joshua L</au><au>Karnes, Jonathan B</au><au>Kauffman, Audra L</au><au>Martin, Katie R</au><au>MacKeigan, Jeffrey P</au><au>Donaldson, Julie G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2017-09-14</date><risdate>2017</risdate><volume>12</volume><issue>9</issue><spage>e0184909</spage><epage>e0184909</epage><pages>e0184909-e0184909</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28910396</pmid><doi>10.1371/journal.pone.0184909</doi><orcidid>https://orcid.org/0000-0002-0198-5679</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | 1-Phosphatidylinositol 3-kinase Accumulation Autophagy Biology Biology and Life Sciences Biomarkers - metabolism Cancer Cell death Cell Line Cell Proliferation Cell survival Clathrin Coated vesicles Confocal microscopy Endocytosis Endosomes Endosomes - metabolism Enzymes Gene Knockdown Techniques Humans Isoforms Kinases Markers Maturation Medical research Microscopy Phagocytosis Phosphatidylinositol 3-Kinases - genetics Phosphatidylinositol 3-Kinases - metabolism Phosphorylation Physiology Protein turnover Proteins RNA, Small Interfering - metabolism Signal Transduction siRNA Transferrin Transferrins Transport Vesicles - metabolism Vesicles Yeast |
| title | PI3K-C2α knockdown decreases autophagy and maturation of endocytic vesicles |
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