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Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells

Nature Cell Biology volume 5, pages 803–811 (2003)Cite this article

Abstract

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8–18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.

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Figure 1: The effect of SPC on keratin organization and phosphorylation.
Figure 2: Specificity of the effect of SPC on keratin organization.
Figure 3: Keratin network architecture of Panc-1 cells.
Figure 5: Differential effect of LPA and SPC on the elastic moduli of Panc-1 and AGS cells.
Figure 4: Effect of SPC on the elastic properties of Panc-1 cells.

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Acknowledgements

U. Nolte is acknowledged for expert technical assistance. M.B., T.S., A.M. and J.S. are supported by the Deutsche Forschungsgemeinschaft (SFB 518/ A5 to M.B. and B3 to T.S., SP520/5 to A.M. and J.S.).

Author information

Author notes

  1. Michael Beil, Alexandre Micoulet, Stephan Paschke, Ulrike Gern, Elke Wolff-Hieber, Guido Adler, Joachim Spatz and Thomas Seufferlein: The groups of the Department. of Internal Medicine I, Ulm and the Department. of Physical Chemistry, Heidelberg contributed equally to this manuscript.

Authors and Affiliations

  1. Department of Internal Medicine I, University of Ulm, Ulm, 89071, Germany

    Michael Beil, Stephan Paschke, Ulrike Gern, Elke Wolff-Hieber, Guido Adler & Thomas Seufferlein

  2. Department of Physical Chemistry, Biophysical Chemistry Section, University of Heidelberg, Heidelberg, 69120, Germany

    Alexandre Micoulet & Joachim Spatz

  3. Department of Biological Sciences, Columbia University, New York, 10027, NY, USA

    Götz von Wichert

  4. Department of Electron Microscopy, University of Ulm, Ulm, 89071, Germany

    Paul Walther

  5. Department of Medicine, VA Palo Alto Health Care System and Stanford University, Palo Alto, California, 94304, CA, USA

    M. Bishr Omary

  6. Afdeling Farmakologie, Katholieke Universiteit Leuven, Leuven, 3000, Belgium

    Paul P. Van Veldhoven

  7. Department of Internal Medicine II, University of Ulm, Ulm, 89071, Germany

    Juliane Eggermann & Johannes Waltenberger

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Supplementary information

Supplementary Information, Fig. S1

High-resolution SEM of keratin filaments in Panc-1 cells A: Unstimulated Panc-1 cells underwent a prefixation treatment to extract individual keratin filaments for visualisation by SEM. These filaments were identified as keratin 8 and 18 by immunogold labelling. 5 nm gold particles identifying keratin 8/18 specific antibodies are presented in black. The scale bar shown represents 200 nm. B: The density of keratin filaments in the perinuclear area in response to 10 μM SPC or solvent was quantified by counting the number of filaments crossing a 5 μm line. In each condition, this line was placed randomly around ten different nuclei. Shown are the means +/- S.D. of three independent experiments. (PDF 398 kb)

Supplementary Information, Fig. S2

SPC-induced keratin reorganisation is independent of other cytoskeletal components A: Immunofluorescence of VASP in Panc-1 cells after treatment with 10 μM SPC or 10 μM LPA for 45 min or solvent (-). B: Panc-1 cells were treated with 1.5 μM cytochalasin D (CytD, +), 50 nM jasplakinolide (Jas, +) for 2h. Cells were subsequently stimulated with 10 µM SPC for 45 min (SPC, +). Keratin immunostaining was performed using the KL1 antibody, F-actin was visualised by Oregon-green-phallodin. C: Panc-1 cells were incubated with 1 µM nocodazol (Noc) or 5 μM taxol (Tax) for 1h and subsequently stimulated with 10 μM SPC for 45 min (SPC, +). Tubulin and keratin immunostaining was performed with anti-a-tubulin and KL1 antibodies, respectively. The scale bars shown represent 5 μm. (PDF 3228 kb)

Supplementary Information, Fig. S3

Experimental set-up for the force measurements (PDF 42 kb)

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Beil, M., Micoulet, A., von Wichert, G. et al. Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells. Nat Cell Biol 5, 803–811 (2003). https://doi.org/10.1038/ncb1037

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