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Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis

Nature Cell Biology volume 13, pages 1189–1201 (2011)Cite this article

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Abstract

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

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Figure 1: Apicobasal polarity conversion and ectopic lumen formation in the intestines of lipid-biosynthetic-enzyme-depleted animals.
Figure 2: Apicobasolateral membrane and apical junction components in lipid-biosynthetic-enzyme-depleted intestines.
Figure 3: Lipid biosynthesis perturbations reversibly shift apicobasal domain identities and lumen position on expanding intestinal membranes in situ.
Figure 4: Germline mutations in fatty-acid-biosynthetic enzymes cause intestinal tubulogenesis defects that are rescued with exogenous fatty acids.
Figure 5: Tubular polarity requires saturated LCFA biosynthesis.
Figure 6: Fatty-acid biosynthesis determines tubular polarity through sphingolipid synthesis.
Figure 7: Tubular polarity requires CGTs and GSLs.
Figure 8: Subcellular localization of exogenous sphingolipids and the effects of sphingolipid-biosynthesis suppression on vesicular trafficking.

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Acknowledgements

Strains and plasmids were provided by D. Baillie (Fraser University, Burnaby, British Columbia, Canada), A. Croce (IFOM Istituto FIRC di Oncologia Molecolare, Milan, Italy), B. Grant (Rutgers University, Piscataway, New Jersey, USA), K. Kemphues (Cornell University, Ithaca, New York, USA), K. Nehrke (University of Rochester Medical Center, Rochester New York, USA), G. Ruvkun (Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA), K. Strange (Vanderbilt University Medical Center, Salisbury Cove Maine, USA), J. Simske (Case Western Reserve University School of Medicine, Cleveland, Ohio, USA), S. Mitani (National Bioresource Project Japan) and the Caenorhabditis Genetics Center (NIH Center for Research Resources). We thank G. Ruvkun for the lethal RNAi library, and J. Moore (Avanti Polar Lipids); Mary McKee (MGH Microscopy Core/partially funded by the IBD grant DK43351 and BA DE award DK57521) and K. Nygen; Christopher Crocker; and Edward Membreno for contributions to LC/MS; TEM; illustrations and C. elegans maintenance, respectively. We thank F. Solomon and B. Winckler for critical reading of the manuscript and H. Weinstein and A. Walker for ongoing support. This work was supported by NIH grants HD044589 and GM078653 and a Mattina R. Proctor Award to V.G.

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  1. Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA

    Hongjie Zhang, Nessy Abraham, Liakot A. Khan, John T. Fleming & Verena Göbel

  2. Department of Neuroscience, Center for C. elegans Anatomy, Albert Einstein College of Medicine, Bronx, New York 10461, USA

    David H. Hall

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Contributions

H.Z. generated and assembled most of the data and contributed to project design, data analysis and writing of the manuscript. N.A. participated in most experiments, carried out the glycosylation screen and contributed to experimental design and data analysis. L.A.K. contributed to the genetic interaction experiments. D.H.H. and J.T.F. contributed to electron microscopy experiments and J.T.F. to writing of the manuscript. V.G. conceived and directed the project, participated in experiments and wrote the manuscript.

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Zhang, H., Abraham, N., Khan, L. et al. Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis. Nat Cell Biol 13, 1189–1201 (2011). https://doi.org/10.1038/ncb2328

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