Skip to main content

Protein composition and movements of membrane swellings associated with primary cilia

Cellular and Molecular Life Sciences volume 72, pages 2415–2429 (2015)Cite this article

Abstract

Dysfunction of many ciliary proteins has been linked to a list of diseases, from cystic kidney to obesity and from hypertension to mental retardation. We previously proposed that primary cilia are unique communication organelles that function as microsensory compartments that house mechanosensory molecules. Here we report that primary cilia exhibit membrane swellings or ciliary bulbs, which based on their unique ultrastructure and motility, could be mechanically regulated by fluid-shear stress. Together with the ultrastructure analysis of the swelling, which contains monosialodihexosylganglioside (GM3), our results show that ciliary bulb has a distinctive set of functional proteins, including GM3 synthase (GM3S), bicaudal-c1 (Bicc1), and polycystin-2 (PC2). In fact, results from our cilia isolation demonstrated for the first time that GM3S and Bicc1 are members of the primary cilia proteins. Although these proteins are not required for ciliary membrane swelling formation under static condition, fluid-shear stress induced swelling formation is partially modulated by GM3S. We therefore propose that the ciliary bulb exhibits a sensory function within the mechano-ciliary structure. Overall, our studies provided an important step towards understanding the ciliary bulb function and structure.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. Zimmermann K (1898) Beiträge zur Kenntnis einiger Drüsen und Epithelien. Arch Mikroskop Anat 52:552–706

    Article  Google Scholar 

  2. Tobin JL, Beales PL (2009) The nonmotile ciliopathies. Genetics Med 11(6):386–402. doi:10.1097/GIM.0b013e3181a02882

    Article  CAS  Google Scholar 

  3. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J (2003) Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 33(2):129–137. doi:10.1038/ng1076

    Article  CAS  PubMed  Google Scholar 

  4. Nauli SM, Kawanabe Y, Kaminski JJ, Pearce WJ, Ingber DE, Zhou J (2008) Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1. Circulation 117(9):1161–1171. doi:10.1161/CIRCULATIONAHA.107.710111

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Praetorius HA, Spring KR (2001) Bending the MDCK cell primary cilium increases intracellular calcium. J Membr Biol 184(1):71–79

    Article  CAS  PubMed  Google Scholar 

  6. Jin X, Mohieldin AM, Muntean BS, Green JA, Shah JV, Mykytyn K, Nauli SM (2014) Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli. Cell Mol Life Sci 71(11):2165–2178. doi:10.1007/s00018-013-1483-1

    Article  CAS  PubMed  Google Scholar 

  7. Aboualaiwi WA, Muntean BS, Ratnam S, Joe B, Liu L, Booth RL, Rodriguez I, Herbert BS, Bacallao RL, Fruttiger M, Mak TW, Zhou J, Nauli SM (2014) Survivin-induced abnormal ploidy contributes to cystic kidney and aneurysm formation. Circulation 129(6):660–672. doi:10.1161/CIRCULATIONAHA.113.005746

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. AbouAlaiwi WA, Takahashi M, Mell BR, Jones TJ, Ratnam S, Kolb RJ, Nauli SM (2009) Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades. Circ Res 104(7):860–869. doi:10.1161/CIRCRESAHA.108.192765

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Takaoki M, Murakami N, Gyotoku J (2004) C-fos expression of osteoblast-like MC3T3-E1 cells induced either by cooling or by fluid flow. Uchu Seibutsu Kagaku 18(3):181–182

    PubMed  Google Scholar 

  10. McGlashan SR, Haycraft CJ, Jensen CG, Yoder BK, Poole CA (2007) Articular cartilage and growth plate defects are associated with chondrocyte cytoskeletal abnormalities in Tg737orpk mice lacking the primary cilia protein polaris. Matrix Biol 26(4):234–246. doi:10.1016/j.matbio.2006.12.003

    Article  CAS  PubMed  Google Scholar 

  11. Jensen CG, Poole CA, McGlashan SR, Marko M, Issa ZI, Vujcich KV, Bowser SS (2004) Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ. Cell Biol Int 28(2):101–110. doi:10.1016/j.cellbi.2003.11.007

    Article  CAS  PubMed  Google Scholar 

  12. Masyuk AI, Masyuk TV, Splinter PL, Huang BQ, Stroope AJ, LaRusso NF (2006) Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling. Gastroenterology 131(3):911–920. doi:10.1053/j.gastro.2006.07.003

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Yoshiba S, Shiratori H, Kuo IY, Kawasumi A, Shinohara K, Nonaka S, Asai Y, Sasaki G, Belo JA, Sasaki H, Nakai J, Dworniczak B, Ehrlich BE, Pennekamp P, Hamada H (2012) Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2. Science 338(6104):226–231. doi:10.1126/science.1222538

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. McGrath J, Somlo S, Makova S, Tian X, Brueckner M (2003) Two populations of node monocilia initiate left-right asymmetry in the mouse. Cell 114(1):61–73

    Article  CAS  PubMed  Google Scholar 

  15. Su S, Phua SC, DeRose R, Chiba S, Narita K, Kalugin PN, Katada T, Kontani K, Takeda S, Inoue T (2013) Genetically encoded calcium indicator illuminates calcium dynamics in primary cilia. Nat Methods 10(11):1105–1107. doi:10.1038/nmeth.2647

    Article  CAS  PubMed  Google Scholar 

  16. Rydholm S, Zwartz G, Kowalewski JM, Kamali-Zare P, Frisk T, Brismar H (2010) Mechanical properties of primary cilia regulate the response to fluid flow. Am J Physiol Renal Physiol 298(5):F1096–F1102. doi:10.1152/ajprenal.00657.2009

    Article  CAS  PubMed  Google Scholar 

  17. Xu C, Rossetti S, Jiang L, Harris PC, Brown-Glaberman U, Wandinger-Ness A, Bacallao R, Alper SL (2007) Human ADPKD primary cyst epithelial cells with a novel, single codon deletion in the PKD1 gene exhibit defective ciliary polycystin localization and loss of flow-induced Ca2+signaling. Am J Physiol Renal Physiol 292(3):F930–F945. doi:10.1152/ajprenal.00285.2006

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Nauli SM, Rossetti S, Kolb RJ, Alenghat FJ, Consugar MB, Harris PC, Ingber DE, Loghman-Adham M, Zhou J (2006) Loss of polycystin-1 in human cyst-lining epithelia leads to ciliary dysfunction. J Am Soc Nephrol 17(4):1015–1025. doi:10.1681/ASN.2005080830

    Article  CAS  PubMed  Google Scholar 

  19. Siroky BJ, Ferguson WB, Fuson AL, Xie Y, Fintha A, Komlosi P, Yoder BK, Schwiebert EM, Guay-Woodford LM, Bell PD (2006) Loss of primary cilia results in deregulated and unabated apical calcium entry in ARPKD collecting duct cells. Am J Physiol Renal Physiol 290(6):F1320–F1328. doi:10.1152/ajprenal.00463.2005

    Article  CAS  PubMed  Google Scholar 

  20. Liu W, Murcia NS, Duan Y, Weinbaum S, Yoder BK, Schwiebert E, Satlin LM (2005) Mechanoregulation of intracellular Ca2+ concentration is attenuated in collecting duct of monocilium-impaired orpk mice. Am J Physiol Renal Physiol 289(5):F978–F988. doi:10.1152/ajprenal.00260.2004

    Article  CAS  PubMed  Google Scholar 

  21. Cano DA, Sekine S, Hebrok M (2006) Primary cilia deletion in pancreatic epithelial cells results in cyst formation and pancreatitis. Gastroenterology 131(6):1856–1869. doi:10.1053/j.gastro.2006.10.050

    Article  CAS  PubMed  Google Scholar 

  22. Cano DA, Murcia NS, Pazour GJ, Hebrok M (2004) Orpk mouse model of polycystic kidney disease reveals essential role of primary cilia in pancreatic tissue organization. Development 131(14):3457–3467. doi:10.1242/dev.01189

    Article  CAS  PubMed  Google Scholar 

  23. Hoey DA, Tormey S, Ramcharan S, O’Brien FJ, Jacobs CR (2012) Primary cilia-mediated mechanotransduction in human mesenchymal stem cells. Stem Cells 30(11):2561–2570. doi:10.1002/stem.1235

    Article  PubMed Central  PubMed  Google Scholar 

  24. Gilula NB, Satir P (1972) The ciliary necklace. A ciliary membrane specialization. J Cell Biol 53(2):494–509

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Dilly PN (1977) Further observations of transport within paddle cilia. Cell Tissue Res 185(1):105–113

    Article  CAS  PubMed  Google Scholar 

  26. Dilly PN (1977) Material transport within specialised ciliary shafts on Rhabdopleura zooids. Cell Tissue Res 180(3):367–381

    Article  CAS  PubMed  Google Scholar 

  27. Beninger PG, Potter TM, Stjean SD (1995) Paddle cilia fixation artifacts in pallial organs of adult Mytilus edulis and Placopecten magennanicus. Canadian J Zool 73:610–614

    Article  Google Scholar 

  28. Short G, Tamm SL (1991) On the nature of paddle cilia and discocilia. Biol Bull 180:466–474

    Article  Google Scholar 

  29. Menco BP, Farbman AI (1985) Genesis of cilia and microvilli of rat nasal epithelia during pre-natal development. II. Olfactory epithelium, a morphometric analysis. J Cell Sci 78:311–336

    CAS  PubMed  Google Scholar 

  30. Menco BP, Farbman AI (1985) Genesis of cilia and microvilli of rat nasal epithelia during pre-natal development. I. Olfactory epithelium, qualitative studies. J Cell Sci 78:283–310

    CAS  PubMed  Google Scholar 

  31. Holley A, Mac Leod P (1977) Transduction and coding of olfactory information. Journal de physiologie 73(6):725–848

    CAS  PubMed  Google Scholar 

  32. Roth KE, Rieder CL, Bowser SS (1988) Flexible-substratum technique for viewing cells from the side: some in vivo properties of primary (9 + 0) cilia in cultured kidney epithelia. J Cell Sci 89(Pt 4):457–466

    PubMed  Google Scholar 

  33. Janich P, Corbeil D (2007) GM1 and GM3 gangliosides highlight distinct lipid microdomains within the apical domain of epithelial cells. FEBS Lett 581(9):1783–1787. doi:10.1016/j.febslet.2007.03.065

    Article  CAS  PubMed  Google Scholar 

  34. Yoder BK, Hou X, Guay-Woodford LM (2002) The polycystic kidney disease proteins, polycystin-1, polycystin-2, polaris, and cystin, are co-localized in renal cilia. J Am Soc Nephrol 13(10):2508–2516

    Article  CAS  PubMed  Google Scholar 

  35. Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB (2002) Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr Biol 12(11):R378–R380

    Article  CAS  PubMed  Google Scholar 

  36. Tran U, Zakin L, Schweickert A, Agrawal R, Doger R, Blum M, De Robertis EM, Wessely O (2010) The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity. Development 137(7):1107–1116. doi:10.1242/dev.046045

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Mitchell KA (2013) Isolation of primary cilia by shear force. Current protocols in cell biology/editorial board, Juan S Bonifacino [et al] Chapter 3:Unit 3 42 41–49. doi:10.1002/0471143030.cb0342s59

  38. Berselli P, Zava S, Sottocornola E, Milani S, Berra B, Colombo I (2006) Human GM3 synthase: a new mRNA variant encodes an NH2-terminal extended form of the protein. Biochim Biophys Acta 1759(7):348–358. doi:10.1016/j.bbaexp.2006.07.001

    Article  CAS  PubMed  Google Scholar 

  39. Koulen P, Cai Y, Geng L, Maeda Y, Nishimura S, Witzgall R, Ehrlich BE, Somlo S (2002) Polycystin-2 is an intracellular calcium release channel. Nat Cell Biol 4(3):191–197. doi:10.1038/ncb754

    Article  CAS  PubMed  Google Scholar 

  40. Elofsson R, Andersson A, Falck B, Sjoborg S (1984) The ciliated human keratinocyte. J Ultrastruct Res 87(3):212–220

    Article  CAS  PubMed  Google Scholar 

  41. Dalen H (1981) An ultrastructural study of primary cilia, abnormal cilia and ciliary knobs from the ciliated cells of the guinea-pig trachea. Cell Tissue Res 220(4):685–697

    Article  CAS  PubMed  Google Scholar 

  42. Albrecht-Buehler G, Bushnell A (1980) The ultrastructure of primary cilia in quiescent 3T3 cells. Exp Cell Res 126(2):427–437

    Article  CAS  PubMed  Google Scholar 

  43. Jensen CG, Davison EA, Bowser SS, Rieder CL (1987) Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption. Cell Motil Cytoskelet 7(3):187–197. doi:10.1002/cm.970070302

    Article  CAS  Google Scholar 

  44. Jensen CG, Jensen LC, Rieder CL (1979) The occurrence and structure of primary cilia in a subline of Potorous tridactylus. Exp Cell Res 123(2):444–449

    Article  CAS  PubMed  Google Scholar 

  45. Wood CR, Huang K, Diener DR, Rosenbaum JL (2013) The cilium secretes bioactive ectosomes. Curr Biol 23(10):906–911. doi:10.1016/j.cub.2013.04.019

    Article  CAS  PubMed  Google Scholar 

  46. Hogan MC, Manganelli L, Woollard JR, Masyuk AI, Masyuk TV, Tammachote R, Huang BQ, Leontovich AA, Beito TG, Madden BJ, Charlesworth MC, Torres VE, LaRusso NF, Harris PC, Ward CJ (2009) Characterization of PKD protein-positive exosome-like vesicles. J Am Soc Nephrol 20(2):278–288. doi:10.1681/ASN.2008060564

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. Wang J, Silva M, Haas LA, Morsci NS, Nguyen KC, Hall DH, Barr MM (2014) C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr Biol 24(5):519–525. doi:10.1016/j.cub.2014.01.002

    Article  CAS  PubMed  Google Scholar 

  48. Kaimori JY, Nagasawa Y, Menezes LF, Garcia-Gonzalez MA, Deng J, Imai E, Onuchic LF, Guay-Woodford LM, Germino GG (2007) Polyductin undergoes notch-like processing and regulated release from primary cilia. Hum Mol Genet 16(8):942–956. doi:10.1093/hmg/ddm039

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Hidaka S, Konecke V, Osten L, Witzgall R (2004) PIGEA-14, a novel coiled-coil protein affecting the intracellular distribution of polycystin-2. J Biol Chem 279(33):35009–35016. doi:10.1074/jbc.M314206200

    Article  CAS  PubMed  Google Scholar 

  50. Cai Y, Maeda Y, Cedzich A, Torres VE, Wu G, Hayashi T, Mochizuki T, Park JH, Witzgall R, Somlo S (1999) Identification and characterization of polycystin-2, the PKD2 gene product. J Biol Chem 274(40):28557–28565

    Article  CAS  PubMed  Google Scholar 

  51. Wang L, Eckmann CR, Kadyk LC, Wickens M, Kimble J (2002) A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419(6904):312–316. doi:10.1038/nature01039

    Article  CAS  PubMed  Google Scholar 

  52. Saffman EE, Styhler S, Rother K, Li W, Richard S, Lasko P (1998) Premature translation of oskar in oocytes lacking the RNA-binding protein bicaudal-C. Mol Cell Biol 18(8):4855–4862

    PubMed Central  CAS  PubMed  Google Scholar 

  53. Mahone M, Saffman EE, Lasko PF (1995) Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1. EMBO J 14(9):2043–2055

    PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work partially fulfills the requirements of a PhD degree in Medicinal and Biological Chemistry for Ashraf M. Mohieldin. This work was supported by the Department of Defense PR130153 (SMN) and in part by the National Institute of Health R01DK080640 (SMN), R01DK080745 (OW) and F31DK096870 (AMM).

Author information

Authors and Affiliations

  1. Department of Medicinal and Biological Chemistry, University of Toledo, Health Science Building, 3000 Arlington Ave, Toledo, OH, 43614, USA

    Ashraf M. Mohieldin & Surya M. Nauli

  2. Department of Pharmacology and Experimental Therapeutics, University of Toledo, Health Science Building, Toledo, OH, 43614, USA

    Ashraf M. Mohieldin, Hanan S. Haymour, Shao T. Lo, Wissam A. AbouAlaiwi & Surya M. Nauli

  3. Department of Biomedical and Pharmaceutical Sciences, Chapman University, Harry and Diane Rinker Health Science Campus, 9401 Jeronimo Road, Irvine, CA, 92618-1908, USA

    Kimberly F. Atkinson & Surya M. Nauli

  4. Department of Medicine, The Kidney Institute, University of Kansas Medical Center, Kansas, KS, 66160, USA

    Christopher J. Ward

  5. Liquid Crystal Institute, Kent State University, 1425 University Esplanade, Kent, OH, 44242, USA

    Min Gao

  6. Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195, USA

    Oliver Wessely

Authors
  1. Ashraf M. Mohieldin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hanan S. Haymour
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shao T. Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wissam A. AbouAlaiwi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kimberly F. Atkinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christopher J. Ward
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Min Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Oliver Wessely
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Surya M. Nauli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Surya M. Nauli.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Mohieldin, A.M., Haymour, H.S., Lo, S.T. et al. Protein composition and movements of membrane swellings associated with primary cilia. Cell. Mol. Life Sci. 72, 2415–2429 (2015). https://doi.org/10.1007/s00018-015-1838-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-015-1838-x

Keywords

  • Primary cilia
  • Ciliary bulb
  • Ciliary membrane swelling
  • Fluid-shear stress
  • Bicc-1
  • GM3S
  • PC2