Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux

An Erratum to this article was published on 01 March 2013

This article has been updated

Abstract

Many unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell Caenorhabditis elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1-overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity. AQP-8 is transiently recruited to the lumen by ERM-1, co-localizing in peri-lumenal cuffs interspaced along expanding canals. An ERM-1[++]-mediated increase in the number of lumen-associated canaliculi is reversed by AQP-8 depletion. We propose that the ERM-1/AQP-8 interaction propels lumen extension by translumenal flux, suggesting a direct morphogenetic effect of water-channel-regulated fluid pressure.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: ERM-1 is required to expand the excretory-canal lumenal membrane and its actin undercoat.
Figure 2: The ERM-1[++] cystic-canal phenotype is suppressed by loss of AQP-8.
Figure 3: AQP-8 promotes excretory canal lumen expansion and localizes to canalicular vesicles.
Figure 4: erm-1 and aqp-8 genetically interact in intracellular lumen morphogenesis.
Figure 5: AQP-8 and ERM-1 transiently co-localize during excretory canal development and physically interact in yeast two-hybrid assays.
Figure 6: AQP-8 functions as a water channel in canal morphogenesis.
Figure 7: ERM-1 recruits AQP-8 to the lumen and increases the canalicular–lumenal membrane connection.
Figure 8: Tomographic analysis of the ERM-1[++] effect on the canalicular–lumenal interface and a model of the ERM-1/AQP-8 function in excretory canal lumen extension.

Similar content being viewed by others

Change history

  • 11 February 2013

    In the version of this Article that was originally published, the results section should have read: "Suppression of aqp-8(RNAi) was confirmed in..." The caption for Fig. 1il should have read: "ERM-1 dose-dependently restricts canal extension." The caption for Fig. 1o should have read: "1/4-extended canal with aligning vacuoles."

References

  1. Lubarsky, B. & Krasnow, M. A. Tube morphogenesis: making and shaping biological tubes. Cell 112, 19–28 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Bryant, D. M. & Mostov, K. E. From cells to organs: building polarized tissue. Nat. Rev. Mol. Cell Biol. 9, 887–901 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Nelson, F. K., Albert, P. S. & Riddle, D. L. Fine structure of the Caenorhabditis elegans secretory–excretory system. J. Ultrastruct. Res. 82, 156–171 (1983).

    Article  CAS  PubMed  Google Scholar 

  4. Buechner, M., Hall, D. H., Bhatt, H. & Hedgecock, E. M. Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell. Dev. Biol. 214, 227–241 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Hall, D. H. & Altun, Z. F. C. elegans Atlas (Cold Spring Harbor Laboratory Press, 2008).

    Google Scholar 

  6. Abdus-Saboor, I. et al. Notch and Ras promote sequential steps of excretory tube development in C. elegans. Development 138, 3545–3555 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kolotuev, I., Hyenne, V., Schwab, Y., Rodriguez, D. & Labouesse, M. A pathway for unicellular tube extension depending on the lymphatic vessel determinant Prox1 and on osmoregulation. Nat. Cell Biol.http://dx.doi.org/10.1038/ncb2662 (2013).

  8. Jones, S. J. & Baillie, D. L. Characterization of the let-653 gene in Caenorhabditis elegans. Mol. Gen. Genet. 248, 719–726 (1995).

    Article  CAS  PubMed  Google Scholar 

  9. Suzuki, N. et al. A putative GDP-GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans. EMBO Rep. 2, 530–535 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Berry, K. L., Bulow, H. E., Hall, D. H. & Hobert, O. A C. elegans CLIC-like protein required for intracellular tube formation and maintenance. Science 302, 2134–2137 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Fujita, M. et al. The role of the ELAV homologue EXC-7 in the development of the Caenorhabditis elegans excretory canals. Dev. Biol. 256, 290–301 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Perens, E. A. & Shaham, S. C. elegans daf-6 encodes a patched-related protein required for lumen formation. Dev. Cell 8, 893–906 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Gobel, V., Barrett, P. L., Hall, D. H. & Fleming, J. T. Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1. Dev. Cell 6, 865–873 (2004).

    Article  PubMed  Google Scholar 

  14. Saotome, I., Curto, M. & McClatchey, A. I. Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine. Dev. Cell 6, 855–864 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Fehon, R. G., McClatchey, A. I. & Bretscher, A. Organizing the cell cortex: the role of ERM proteins. Nat. Rev. Mol. Cell Biol. 11, 276–287 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ten Klooster, J. P. et al. Mst4 and Ezrin induce brush borders downstream of the Lkb1/Strad/Mo25 polarization complex. Dev. Cell 16, 551–562 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Zhu, L., Crothers, J. Jr, Zhou, R. & Forte, J. G. A possible mechanism for ezrin to establish epithelial cell polarity. Am. J. Physiol. Cell Physiol. 299, C431–C443 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kerman, B. E., Cheshire, A. M., Myat, M. M. & Andrew, D. J. Ribbon modulates apical membrane during tube elongation through Crumbs and Moesin. Dev. Biol. 320, 278–288 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gervais, L. & Casanova, J. In vivo coupling of cell elongation and lumen formation in a single cell. Curr. Biol. 20, 359–366 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Wang, Y. et al. Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis. Development 137, 3119–3128 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. King, L. S., Kozono, D. & Agre, P. From structure to disease: the evolving tale of aquaporin biology. Nat. Rev. Mol. Cell Biol. 5, 687–698 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Preston, G. M., Jung, J. S., Guggino, W. B. & Agre, P. The mercury-sensitiveresidue at cysteine 189 in the CHIP28 water channel. J. Biol. Chem. 268, 17–20 (1993).

    CAS  PubMed  Google Scholar 

  23. Hachez, C. & Chaumont, F. Aquaporins: a family of highly regulated multifunctional channels. Adv. Exp. Med. Biol. 679, 1–17 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Huang, C. G., Lamitina, T., Agre, P. & Strange, K. Functional analysis of the aquaporin gene family in Caenorhabditis elegans. Am. J. Physiol. Cell Physiol. 292, C1867–C1873 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Buechner, M. Tubes and the single C. elegans excretory cell. Trends Cell Biol. 12, 479–484 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Koppen, M. et al. Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nat. Cell Biol. 3, 983–991 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. MacQueen, A. J. et al. ACT-5 is an essential Caenorhabditis elegans actin required for intestinal microvilli formation. Mol. Biol. Cell 16, 3247–3259 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mah, A. K. et al. Transcriptional regulation of AQP-8, a Caenorhabditis elegans aquaporin exclusively expressed in the excretory system, by the POU homeobox transcription factor CEH-6. J. Biol. Chem. 282, 28074–28086 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Mattingly, B. C. & Buechner, M. The FGD homologue EXC-5 regulates apical trafficking in C. elegans tubules. Dev. Biol. 359, 59–72 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hahn-Windgassen, A. & Van Gilst, M. R. The Caenorhabditis elegansHNF4α homolog, NHR-31, mediates excretory tube growth and function through coordinate regulation of the vacuolar ATPase. PLoS Genet. 5, e1000553 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Liu, J., Xu, J., Gu, S., Nicholson, B. J. & Jiang, J. X. Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50. J. Cell Sci. 124, 198–206 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Agre, P., Bonhivers, M. & Borgnia, M. J. The aquaporins, blueprints for cellular plumbing systems. J. Biol. Chem. 273, 14659–14662 (1998).

    Article  CAS  PubMed  Google Scholar 

  33. Cheong, H. I. et al. Two novel mutations in the aquaporin 2 gene in a girl with congenital nephrogenic diabetes insipidus. J. Korean Med. Sci. 20, 1076–1078 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Folkman, J. & Haudenschild, C. Angiogenesis in vitro. Nature 288, 551–556 (1980).

    Article  CAS  PubMed  Google Scholar 

  35. Kamei, M. et al. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442, 453–456 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Herwig, L. et al. Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo. Curr. Biol. 21, 1942–1948 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Schottenfeld-Roames, J. & Ghabrial, A. S. Whacked and Rab35 polarize dynein-motor-complex-dependent seamless tube growth. Nat. Cell Biol. 14, 386–393 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Deretic, D. et al. Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors. Mol. Biol. Cell 15, 359–370 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Balklava, Z., Pant, S., Fares, H. & Grant, B. D. Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic. Nat. Cell Biol. 9, 1066–1073 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Chirivino, D. et al. The ERM proteins interact with the HOPS complex to regulate the maturation of endosomes. Mol. Biol. Cell 22, 375–385 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kaksonen, M., Toret, C. P. & Drubin, D. G. A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 123, 305–320 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Lamprecht, G., Weinman, E. J. & Yun, C. H. The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3. J. Biol. Chem. 273, 29972–29978 (1998).

    Article  CAS  PubMed  Google Scholar 

  43. Denker, S. P., Huang, D. C., Orlowski, J., Furthmayr, H. & Barber, D. L. Directbinding of the Na–H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation. Mol. Cell 6, 1425–1436 (2000).

    Article  CAS  PubMed  Google Scholar 

  44. Zhou, R. et al. Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation. J. Biol. Chem. 278, 35651–35659 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Tamura, A. et al. Achlorhydria by ezrin knockdown: defects in the formation/expansion of apical canaliculi in gastric parietal cells. J. Cell Biol. 169, 21–28 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cha, B. et al. The NHE3 juxtamembrane cytoplasmic domain directly binds ezrin: dual role in NHE3 trafficking and mobility in the brush border. Mol. Biol. Cell 17, 2661–2673 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Tamma, G. et al. Actin remodeling requires ERM function to facilitate AQP2 apical targeting. J. Cell Sci. 118, 3623–3630 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Desmond, M. E. & Jacobson, A. G. Embryonic brain enlargement requires cerebrospinal fluid pressure. Dev. Biol. 57, 188–198 (1977).

    Article  CAS  PubMed  Google Scholar 

  49. Stewart, M. P. et al. Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature 469, 226–230 (2011).

    Article  CAS  PubMed  Google Scholar 

  50. Vasilyev, A. et al. Collective cell migration drives morphogenesis of the kidney nephron. PLoS Biol. 7, e1000009 (2009).

    Article  PubMed Central  Google Scholar 

  51. Bagnat, M., Cheung, I. D., Mostov, K. E. & Stainier, D. Y. Genetic control of single lumen formation in the zebrafish gut. Nat. Cell Biol. 9, 954–960 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Krupinski, T. & Beitel, G. J. Unexpected roles of the Na-K-ATPase and other ion transporters in cell junctions and tubulogenesis. Physiology (Bethesda) 24, 192–201 (2009).

    CAS  Google Scholar 

  53. Bagnat, M. et al. Cse1l is a negative regulator of CFTR-dependent fluid secretion. Curr. Biol. 20, 1840–1845 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Maurel, C., Verdoucq, L., Luu, D. T. & Santoni, V. Plant aquaporins: membrane channels with multiple integrated functions. Annu. Rev. Plant Biol. 59, 595–624 (2008).

    Article  CAS  PubMed  Google Scholar 

  55. Morishita, Y. et al. Disruption of aquaporin-11 produces polycystic kidneys following vacuolization of the proximal tubule. Mol. Cell Biol. 25, 7770–7779 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Saadoun, S., Papadopoulos, M. C., Hara-Chikuma, M. & Verkman, A. S. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption. Nature 434, 786–792 (2005).

    Article  CAS  PubMed  Google Scholar 

  57. Chen, Y. et al. Aquaporin 2 promotes cell migration and epithelial morphogenesis. J. Am. Soc. Nephrol. 23, 1506–1517 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Tatsumi, K. et al. Drosophila big brain does not act as a water channel, but mediates cell adhesion. FEBS Lett. 583, 2077–2082 (2009).

    Article  CAS  PubMed  Google Scholar 

  59. Wang, Z. & Schey, K. L. Aquaporin-0 interacts with the FERM domain of ezrin/radixin/moesin proteins in the ocular lens. Invest. Ophthalmol. Vis. Sci. 52, 5079–5087 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Gonen, T., Sliz, P., Kistler, J., Cheng, Y. & Walz, T. Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 429, 193–197 (2004).

    Article  CAS  PubMed  Google Scholar 

  61. Cho, S. J. et al. Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles. Proc. Natl Acad. Sci. USA 99, 4720–4724 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Brown, D. The ins and outs of aquaporin-2 trafficking. Am. J. Physiol. Renal Physiol. 284, F893–F901 (2003).

    Article  CAS  PubMed  Google Scholar 

  63. Nozaki, K., Ishii, D. & Ishibashi, K. Intracellular aquaporins: clues for intracellular water transport? Pflugers Arch. 456, 701–707 (2008).

    Article  CAS  PubMed  Google Scholar 

  64. Sugiya, H., Matsuki-Fukushima, M. & Hashimoto, S. Role of aquaporins and regulation of secretory vesicle volume in cell secretion. J. Cell Mol. Med. 12, 1486–1494 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Tung, J. J., Hobert, O., Berryman, M. & Kitajewski, J. Chloride intracellular channel 4 is involved in endothelial proliferation and morphogenesis in vitro. Angiogenesis 12, 209–220 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Timmons, L., Court, D. L. & Fire, A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103–112 (2001).

    Article  CAS  PubMed  Google Scholar 

  68. Fire, A., Harrison, S. W. & Dixon, D. A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans. Gene 93, 189–198 (1990).

    Article  CAS  PubMed  Google Scholar 

  69. Stinchcomb, D. T., Shaw, J. E., Carr, S. H. & Hirsh, D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell Biol. 5, 3484–3496 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Mello, C. C., Kramer, J. M., Stinchcomb, D. & Ambros, V. Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959–3970 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Hadwiger, G., Dour, S., Arur, S., Fox, P. & Nonet, M. L. A monoclonal antibody toolkit for C. elegans. PLoS One 5, e10161 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Harlow, E. & Lane, D. Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988).

    Google Scholar 

  73. Weimer, R. M. Preservation of C. elegans tissue via high-pressure freezing and freeze-substitution for ultrastructural studies and immunocytochemistry. Methods Mol. Biol. 351, 203–221 (2006).

    PubMed  Google Scholar 

  74. Winkler, H. & Taylor, K. A. Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography. Ultramicroscopy 106, 240–254 (2006).

    Article  CAS  PubMed  Google Scholar 

  75. Mastronarde, D. N. Dual-axis tomography: an approach using alignment methods that preserve resolution. J. Struct. Biol. 120, 343–352 (1997).

    Article  CAS  PubMed  Google Scholar 

  76. Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Baillie (Simon Fraser University, Burnaby, British Columbia, Canada), M. Futai (Osaka University, Osaka, Japan), M. Labouesse (IGBMC, France), K. Nehrke (University of Rochester Medical Center, Rochester New York, USA) and J. Simske (Case Western Reserve University School of Medicine, Cleveland, Ohio, USA), and the following C. elegans resource centres: J. Kohara (National Institute of Genetics, Mishima, Japan), S. Mitani (National Bioresource Project, Tokyo Women’s Medical University, Tokyo, Japan), the International C. elegans Gene Knockout Consortium and the Caenorhabditis elegans genetic centre (NIH Center for Research Resources) for providing plasmids and strains. We thank E. Membreno and D. Fernandez for technical support, A. Sengupta for three-dimensional graphics, A. Kim for image editing, F. Solomon for critical reading of the manuscript, and H. Weinstein and A. Walker for ongoing support. This work was supported by NIH grant GM078653 and a Mattina R. Proctor Award to V.G.

Author information

Authors and Affiliations

Authors

Contributions

L.A.K. performed experiments, generated, analysed and assembled most of the data and contributed to project design and manuscript writing. H.Z. and N.A. contributed to the generation of transgenic strains and RNAi experiments. L.S. and D.H.H. performed the TEM and tomographic analyses, and J.T.F. contributed to TEM experiments. M.B. generated the canal-specific endosomal marker strains, and J.T.F. and M.B. contributed to writing of the manuscript. V.G. conceived and directed the project, participated in experiments and wrote the manuscript.

Corresponding author

Correspondence to Verena Gobel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2648 kb)

Supplementary Table 1

Supplementary Information (XLSX 13 kb)

Supplementary Table 2

Supplementary Information (XLS 39 kb)

Supplementary Table 3

Supplementary Information (XLS 40 kb)

Supplementary Table 4

Supplementary Information (XLSX 13 kb)

Supplementary Table 5

Supplementary Information (XLSX 12 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Khan, L., Zhang, H., Abraham, N. et al. Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux. Nat Cell Biol 15, 143–156 (2013). https://doi.org/10.1038/ncb2656

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2656

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing