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Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes

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Abstract

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.

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References

  • Akilesh S, Huber TB, Wu H, Wang G, Hartleben B, Kopp JB, Miner JH, Roopenian DC, Unanue ER, Shaw AS (2008) Podocytes use FcRn to clear IgG from the glomerular basement membrane. Proc Natl Acad Sci U S A 105:967–972

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bechtel W, Helmstadter M, Balica J, Hartleben B, Kiefer B, Hrnjic F, Schell C, Kretz O, Liu S, Geist F, Kerjaschki D, Walz G, Huber TB (2013) Vps34 deficiency reveals the importance of endocytosis for podocyte homeostasis. J Am Soc Nephrol 24:727–743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cagan RL (2011) The Drosophila nephrocyte. Curr Opin Nephrol Hypertens 20:409–415

    Article  PubMed  Google Scholar 

  • Carson JM, Okamura K, Wakashin H, McFann K, Dobrinskikh E, Kopp JB, Blaine J (2014) Podocytes degrade endocytosed albumin primarily in lysosomes. PLoS One 9:e99771

    Article  PubMed  PubMed Central  Google Scholar 

  • Christensen EI, Birn H (2002) Megalin and cubilin: multifunctional endocytic receptors. Nat Rev Mol Cell Biol 3:256–266

    Article  CAS  PubMed  Google Scholar 

  • Christensen EI, Verroust PJ, Nielsen R (2009) Receptor-mediated endocytosis in renal proximal tubule. Pflugers Arch 458:1039–1048

    Article  CAS  PubMed  Google Scholar 

  • Dobrinskikh E, Okamura K, Kopp JB, Doctor RB, Blaine J (2014) Human podocytes perform polarized, caveolae-dependent albumin endocytosis. Am J Physiol Renal Physiol 306:F941–F951

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dunst S, Kazimiers T, von Zadow F, Jambor H, Sagner A, Brankatschk B, Mahmoud A, Spannl S, Tomancak P, Eaton S, Brankatschk M (2015) Endogenously tagged Rab proteins: a resource to study membrane trafficking in Drosophila. Dev Cell 33:351–365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gorvel JP, Chavrier P, Zerial M, Gruenberg J (1991) Rab5 controls early endosome fusion in vitro. Cell 64:915–925

    Article  CAS  PubMed  Google Scholar 

  • Gorvin CM, Wilmer MJ, Piret SE, Harding B, van den Heuvel LP, Wrong O, Jat PS, Lippiat JD, Levtchenko EN, Thakker RV (2013) Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients. Proc Natl Acad Sci U S A 110:7014–7019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grahammer F, Schell C, Huber TB (2013) The podocyte slit diaphragm—from a thin grey line to a complex signalling hub. Nat Rev Nephrol 9:587–598

    Article  CAS  PubMed  Google Scholar 

  • Han Z, Yi P, Li X, Olson EN (2006) Hand, an evolutionarily conserved bHLH transcription factor required for Drosophila cardiogenesis and hematopoiesis. Development 133:1175–1182

    Article  CAS  PubMed  Google Scholar 

  • Inoue K, Ishibe S (2015) Podocyte endocytosis in the regulation of the glomerular filtration barrier. Am J Physiol Renal Physiol 309:F398–F405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ivy JR, Drechsler M, Catterson JH, Bodmer R, Ocorr K, Paululat A, Hartley PS (2015) Klf15 is critical for the development and differentiation of Drosophila nephrocytes. PLoS One 10:e0134620

    Article  PubMed  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • McLauchlan H, Newell J, Morrice N, Osborne A, West M, Smythe E (1998) A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits. Curr Biol 8:34–45

    Article  CAS  PubMed  Google Scholar 

  • Na J, Cagan R (2013) The Drosophila nephrocyte: back on stage. J Am Soc Nephrol 24:161–163

    Article  CAS  PubMed  Google Scholar 

  • Nebenfuhr A (2002) Vesicle traffic in the endomembrane system: a tale of COPs, Rabs and SNAREs. Curr Opin Plant Biol 5:507–512

    Article  PubMed  Google Scholar 

  • Nielsen R, Christensen EI (2010) Proteinuria and events beyond the slit. Pediatr Nephrol 25:813–822

    Article  PubMed  Google Scholar 

  • Nielsen R, Courtoy PJ, Jacobsen C, Dom G, Lima WR, Jadot M, Willnow TE, Devuyst O, Christensen EI (2007) Endocytosis provides a major alternative pathway for lysosomal biogenesis in kidney proximal tubular cells. Proc Natl Acad Sci U S A 104:5407–5412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pavenstadt H, Kriz W, Kretzler M (2003) Cell biology of the glomerular podocyte. Physiol Rev 83:253–307

    Article  CAS  PubMed  Google Scholar 

  • Pereira-Leal JB, Seabra MC (2001) Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 313:889–901

    Article  CAS  PubMed  Google Scholar 

  • Pfeffer SR (2001) Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol 11:487–491

    Article  CAS  PubMed  Google Scholar 

  • Schwartz SL, Cao C, Pylypenko O, Rak A, Wandinger-Ness A (2007) Rab GTPases at a glance. J Cell Sci 120:3905–3910

    Article  CAS  PubMed  Google Scholar 

  • Scott RP, Quaggin SE (2015) Review series: the cell biology of renal filtration. J Cell Biol 209:199–210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Simons M, Huber TB (2009) Flying podocytes. Kidney Int 75:455–457

    Article  PubMed  Google Scholar 

  • Steed E, Balda MS, Matter K (2010) Dynamics and functions of tight junctions. Trends Cell Biol 20:142–149

    Article  CAS  PubMed  Google Scholar 

  • Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513–525

    Article  CAS  PubMed  Google Scholar 

  • Swiatecka-Urban A (2013) Membrane trafficking in podocyte health and disease. Pediatr Nephrol 28:1723–1737

    Article  PubMed  Google Scholar 

  • Tang VW, Brieher WM (2012) Alpha-actinin-4/FSGS1 is required for Arp2/3-dependent actin assembly at the adherens junction. J Cell Biol 196:115–130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang T, Ming Z, Xiaochun W, Hong W (2011) Rab7: role of its protein interaction cascades in endo-lysosomal traffic. Cell Signal 23:516–521

    Article  PubMed  Google Scholar 

  • Weavers H, Prieto-Sanchez S, Grawe F, Garcia-Lopez A, Artero R, Wilsch-Brauninger M, Ruiz-Gomez M, Skaer H, Denholm B (2009) The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. Nature 457:322–326

    Article  CAS  PubMed  Google Scholar 

  • Yi P, Han Z, Li X, Olson EN (2006) The mevalonate pathway controls heart formation in Drosophila by isoprenylation of Ggamma1. Science 313:1301–1303

    Article  CAS  PubMed  Google Scholar 

  • Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117

    Article  CAS  PubMed  Google Scholar 

  • Zhang F, Zhao Y, Chao Y, Muir K, Han Z (2013a) Cubilin and amnionless mediate protein reabsorption in Drosophila nephrocytes. J Am Soc Nephrol 24:209–216

    Article  CAS  PubMed  Google Scholar 

  • Zhang F, Zhao Y, Han Z (2013b) An in vivo functional analysis system for renal gene discovery in Drosophila pericardial nephrocytes. J Am Soc Nephrol 24:191–197

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhuang S, Shao H, Guo F, Trimble R, Pearce E, Abmayr SM (2009) Sns and kirre, the Drosophila orthologs of nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes. Development 136:2335–2344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Bloomington Drosophila Stock Center and the VDRC for Drosophila stocks. We acknowledge the Microscopy and Image Analysis Laboratory at the University of Michigan for their technical support with transmission electron microscopy. We are especially grateful to Dotty Sorenson and Sasha Meshinchi for their assistance in electron microscopy. Z.H. was supported by grant R01-DK098410 from the NIH.

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    Authors and Affiliations

    1. Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010, USA

      Yulong Fu, Jun-yi Zhu, Adam Richman & Zhe Han

    2. Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, People’s Republic of China

      Fujian Zhang

    3. Department of Nephrology, First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, People’s Republic of China

      Zhanzheng Zhao

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    1. Yulong Fu
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    Correspondence to Zhe Han.

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    Yulong Fu, Jun-yi Zhu and Fujian Zhang contributed equally to this work.

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    Fu, Y., Zhu, Jy., Zhang, F. et al. Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes. Cell Tissue Res 368, 615–627 (2017). https://doi.org/10.1007/s00441-017-2575-2

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