The class II phosphoinositide 3-kinases PI3K-C2α and PI3K-C2β differentially regulate clathrin-dependent pinocytosis in human vascular endothelial cells
- 327 Downloads
Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2β, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)–dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2β were required for clathrin-mediated—but not clathrin-non-mediated—FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2β were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2β, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2β play differential, indispensable roles in clathrin-mediated pinocytosis.
KeywordsClass II PI3K PI3K-C2α PI3K-C2β Pinocytosis Clathrin Endothelial cell
We thank Ms. Chiemi Hirose for secretarial assistance, and also thank the members of Department of Physiology for their support and assistance.
KTA, KY, SA, and YT designed the study. KTA, KY, and SA performed experiments. KI and NT helped experiments. KTA, KY, SA, and YT analyzed the data. KTA, KY, and YT wrote the manuscript. NT helped to draft the manuscript. All authors approved the final version of the manuscript.
This study was supported by grants from the Japan International Cooperation Agency (JICA, J-14-10306 to KTA), the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (25116711 to Y.T.), and the Japan Society for the Promotion of Science (17K08532 to K.Y., 16K18988 to S.A., 16K15409 to K.I., 17K08542 to N.T., 15H04673 to Y.T.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 2.Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78:857–902. https://doi.org/10.1146/annurev.biochem.78.081307.110540 CrossRefGoogle Scholar
- 13.Yoshioka K, Yoshida K, Cui H, Wakayama T, Takuwa N, Okamoto Y, Du W, Qi X, Asanuma K, Sugihara K, Aki S, Miyazawa H, Biswas K, Nagakura C, Ueno M, Iseki S, Schwartz RJ, Okamoto H, Sasaki T, Matsui O, Asano M, Adams RH, Takakura N, Takuwa Y (2012) Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function. Nat Med 18:1560–1569. https://doi.org/10.1038/nm.2928 CrossRefGoogle Scholar
- 14.Posor Y, Eichhorn-Gruenig M, Puchkov D, Schöneberg J, Ullrich A, Lampe A, Müller R, Zarbakhsh S, Gulluni F, Hirsch E, Krauss M, Schultz C, Schmoranzer J, Noé F, Haucke V (2013) Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499:233–237. https://doi.org/10.1038/nature12360 CrossRefGoogle Scholar
- 15.Biswas K, Yoshioka K, Asanuma K, Okamoto Y, Takuwa N, Sasaki T, Takuwa Y (2013) Essential role of class II phosphatidylinositol-3-kinase-C2α in sphingosine 1-phosphate receptor-1-mediated signaling and migration in endothelial cells. J Biol Chem 288:2325–2339. https://doi.org/10.1074/jbc.M112.409656 CrossRefGoogle Scholar
- 18.Rozycka M, Lu YJ, Brown RA, Lau MR, Shipley JM, Fry MJ (1998) cDNA cloning of a third human C2-domain-containing class II phosphoinositide 3-kinase, PI3K-C2γ, and chromosomal assignment of this gene (PIK3C2G) to 12p12. Genomics 54:569–574. https://doi.org/10.1006/geno.1998.5621 CrossRefGoogle Scholar
- 20.Kitatani K, Usui T, Sriaman SK, Toyoshima M, Ishibashi M, Shigeta S, Nagase S, Sakamoto M, Ogiso H, Okazaki T, Hannun YA, Torchilin VP, Yaegashi N (2016) Ceramide limits phosphatidylinositol-3-kinase C2β -controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid. Oncogene 35:2801–2812. https://doi.org/10.1038/onc.2015.330 CrossRefGoogle Scholar
- 27.Hussain NK, Yamabhai M, Ramjaun AR, Guy AM, Baranes D, O’Bran JP, Der CJ, Kay BK, McPherson PS (1999) Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells. J Biol Chem 274:15671–15677. https://doi.org/10.1074/jbc.274.22.15671 CrossRefGoogle Scholar
- 28.Almeida-Souza L, Frank RAW, García-Nafría J, Colussi A, Gunawardana N, Johson CM, Yu M, Howard G, Andrews B, Vallis Y, McMahon HT (2018) A flat BAR protein promotes actin polymerization at the base of clathrin-coated pits. Cell 174:325–337. https://doi.org/10.1016/j.cell.2018.05.020 CrossRefGoogle Scholar
- 31.Tamura N, Hazeki K, Okazaki N, Kametani Y, Murakami H, Takaba Y, Ishikawa Y, Nigorikawa K, Hazeki O (2009) Specific role of phosphoinositide 3-kinase p110α in the regulation of phagocytosis and pinocytosis in macrophages. Biochem J 423:99–108. https://doi.org/10.1042/BJ20090687 CrossRefGoogle Scholar
- 37.Gulluni F, Martini M, De Santis MC, Campa CC, Ghigo A, Margaria JP, Ciraolo E, Franco I, Ala U, Annaratone L, Disalvatore D, Bertalot G, Viale G, Noatynska A, Compagno M, Sigismund S, Montemurro F, Thelen M, Fan F, Meraldi P, Marchio C, Pece S, Sapino A, Chiarle R, Di Fiore PP, Hirsch E (2017) Mitotic spindle assembly and genomic stability in breast cancer require PI3K-C2α scaffolding function. Cancer Cell 32:444–459. https://doi.org/10.1016/j.ccell.2017.09.002 CrossRefGoogle Scholar
- 40.Katso RM, Pardo OE, Palamidessi A, Franz CM, Marinov M, De Laurentiis A, Downward J, Scita G, Ridley AJ, Waterfield MD, Arcaro A (2006) Phosphoinositide 3-Kinase C2 beta regulates cytoskeletal organization and cell migration via Rac-dependent mechanisms. Mol Biol Cell 17:3729–3744. https://doi.org/10.1091/mbc.e05-11-1083 CrossRefGoogle Scholar