Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

Abstract

Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR– and F-BAR–domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR–domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Endogenous Pinkbar associates with membranes in Caco-2 cells and colocalizes with the small GTPase Rab13.
Figure 2: The BAR domain of Pinkbar promotes the formation of planar membrane structures.
Figure 3: Crystal structure and oligomerization of the BAR domain of Pinkbar.
Figure 4: Mechanism of membrane binding and deformation by the BAR domain of Pinkbar.

Accession codes

Primary accessions

Protein Data Bank

References

  1. 1

    McMahon, H.T. & Gallop, J.L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Campelo, F., Fabrikant, G., McMahon, H.T. & Kozlov, M.M. Modeling membrane shaping by proteins: focus on EHD2 and N-BAR domains. FEBS Lett. 584, 1830–1839 (2010).

    CAS  Article  Google Scholar 

  3. 3

    Frost, A., Unger, V.M. & De Camilli, P. The BAR domain superfamily: membrane-molding macromolecules. Cell 137, 191–196 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Suetsugu, S., Toyooka, K. & Senju, Y. Subcellular membrane curvature mediated by the BAR domain superfamily proteins. Semin. Cell Dev. Biol. 21, 340–349 (2010).

    CAS  Article  Google Scholar 

  5. 5

    Takei, K., Slepnev, V.I., Haucke, V. & De Camilli, P. Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat. Cell Biol. 1, 33–39 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Peter, B.J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Aspenström, P.A. Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton. Curr. Biol. 7, 479–487 (1997).

    Article  Google Scholar 

  8. 8

    Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791–804 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Tsujita, K. et al. Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis. J. Cell Biol. 172, 269–279 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Shimada, A. et al. Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis. Cell 129, 761–772 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Henne, W.M. et al. Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature. Structure 15, 839–852 (2007).

    CAS  Article  Google Scholar 

  12. 12

    Guerrier, S. et al. The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis. Cell 138, 990–1004 (2009).

    CAS  Article  Google Scholar 

  13. 13

    Frost, A. et al. Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Millard, T.H. et al. Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53. EMBO J. 24, 240–250 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Lee, S.H. et al. Structural basis for the actin-binding function of missing-in-metastasis. Structure 15, 145–155 (2007).

    Article  Google Scholar 

  16. 16

    Mattila, P.K., Salminen, M., Yamashiro, T. & Lappalainen, P. Mouse MIM, a tissue-specific regulator of cytoskeletal dynamics, interacts with ATP-actin monomers through its C-terminal WH2 domain. J. Biol. Chem. 278, 8452–8459 (2003).

    CAS  Article  Google Scholar 

  17. 17

    Woodings, J.A., Sharp, S.J. & Machesky, L.M. MIM-B, a putative metastasis suppressor protein, binds to actin and to protein tyrosine phosphatase delta. Biochem. J. 371, 463–471 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Saarikangas, J. et al. ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension. J. Cell Sci. 121, 1444–1454 (2008).

    CAS  Article  Google Scholar 

  19. 19

    Scita, G., Confalonieri, S., Lappalainen, P. & Suetsugu, S. IRSp53: crossing the road of membrane and actin dynamics in the formation of membrane protrusions. Trends Cell Biol. 18, 52–60 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Suetsugu, S. et al. The RAC binding domain/IRSp53-MIM homology domain of IRSp53 induces RAC-dependent membrane deformation. J. Biol. Chem. 281, 35347–35358 (2006).

    CAS  Article  Google Scholar 

  21. 21

    Mattila, P.K. et al. Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism. J. Cell Biol. 176, 953–964 (2007).

    CAS  Article  Google Scholar 

  22. 22

    Saarikangas, J. et al. Molecular mechanisms of membrane deformation by I-BAR domain proteins. Curr. Biol. 19, 95–107 (2009).

    CAS  Article  Google Scholar 

  23. 23

    Bhatia, V.K. et al. Amphipathic motifs in BAR domains are essential for membrane curvature sensing. EMBO J. 28, 3303–3314 (2009).

    CAS  Article  Google Scholar 

  24. 24

    Suetsugu, S. et al. Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac. J. Cell Biol. 173, 571–585 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Disanza, A. et al. Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex. Nat. Cell Biol. 8, 1337–1347 (2006).

    CAS  Article  Google Scholar 

  26. 26

    Lim, K.B. et al. The Cdc42 effector IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics. J. Biol. Chem. 283, 20454–20472 (2008).

    CAS  Article  Google Scholar 

  27. 27

    Millard, T.H., Dawson, J. & Machesky, L.M. Characterisation of IRTKS, a novel IRSp53/MIM family actin regulator with distinct filament bundling properties. J. Cell Sci. 120, 1663–1672 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Kim, M.H. et al. Enhanced NMDA receptor-mediated synaptic transmission, enhanced long-term potentiation, and impaired learning and memory in mice lacking IRSp53. J. Neurosci. 29, 1586–1595 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Sawallisch, C. et al. The insulin receptor substrate of 53 kDa (IRSp53) limits hippocampal synaptic plasticity. J. Biol. Chem. 284, 9225–9236 (2009).

    CAS  Article  Google Scholar 

  30. 30

    Chauhan, B.K. et al. Cdc42- and IRSp53-dependent contractile filopodia tether presumptive lens and retina to coordinate epithelial invagination. Development 136, 3657–3667 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Quinones, G.A., Jin, J. & Oro, A.E. I-BAR protein antagonism of endocytosis mediates directional sensing during guided cell migration. J. Cell Biol. 189, 353–367 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Wu, C. et al. BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol. 10, R130 (2009).

    Article  Google Scholar 

  33. 33

    Hidalgo, I.J., Raub, T.J. & Borchardt, R.T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96, 736–749 (1989).

    CAS  Article  Google Scholar 

  34. 34

    Gallop, J.L. et al. Mechanism of endophilin N-BAR domain-mediated membrane curvature. EMBO J. 25, 2898–2910 (2006).

    CAS  Article  Google Scholar 

  35. 35

    Honda, Y. et al. Thermal unfolding of chitosanase from Streptomyces sp. N174: role of tryptophan residues in the protein structure stabilization. Biochim. Biophys. Acta 1429, 365–376 (1999).

    CAS  Article  Google Scholar 

  36. 36

    Clark, E.H., East, J.M. & Lee, A.G. The role of tryptophan residues in an integral membrane protein: diacylglycerol kinase. Biochemistry 42, 11065–11073 (2003).

    CAS  Article  Google Scholar 

  37. 37

    Morimoto, S. et al. Rab13 mediates the continuous endocytic recycling of occludin to the cell surface. J. Biol. Chem. 280, 2220–2228 (2005).

    CAS  Article  Google Scholar 

  38. 38

    Yamamura, R., Nishimura, N., Nakatsuji, H., Arase, S. & Sasaki, T. The interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of tight junctions and adherens junctions. Mol. Biol. Cell 19, 971–983 (2008).

    CAS  Article  Google Scholar 

  39. 39

    Massari, S. et al. LIN7 mediates the recruitment of IRSp53 to tight junctions. Traffic 10, 246–257 (2009).

    CAS  Article  Google Scholar 

  40. 40

    Saarikangas, J. et al. Missing-in-metastasis (MIM/MTSS1) promotes actin assembly at intercellular junctions and is required for kidney epithelia integrity. J. Cell Sci. 124, 1245–1255 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Weissenhorn, W. Crystal structure of the endophilin-A1 BAR domain. J. Mol. Biol. 351, 653–661 (2005).

    CAS  Article  Google Scholar 

  42. 42

    Reider, A. et al. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation. EMBO J. 28, 3103–3116 (2009).

    CAS  Article  Google Scholar 

  43. 43

    Stimpson, H.E., Toret, C.P., Cheng, A.T., Pauly, B.S. & Drubin, D.G. Early-arriving Syp1p and Ede1p function in endocytic site placement and formation in budding yeast. Mol. Biol. Cell 20, 4640–4651 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Henne, W.M. et al. FCHo proteins are nucleators of clathrin-mediated endocytosis. Science 328, 1281–1284 (2010).

    CAS  Article  Google Scholar 

  45. 45

    Peränen, J., Rikkonen, M., Hyvonen, M. & Kaariainen, L. T7 vectors with modified T7lac promoter for expression of proteins in Escherichia coli. Anal. Biochem. 236, 371–373 (1996).

    Article  Google Scholar 

  46. 46

    Zwart, P.H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008).

    CAS  Article  Google Scholar 

  47. 47

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  48. 48

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

    CAS  Article  Google Scholar 

  49. 49

    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).

    CAS  Article  Google Scholar 

  50. 50

    Uchiyama, K. et al. VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo. J. Cell Biol. 159, 855–866 (2002).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institutes of Health (NIH), National Institute of Mental Health grant MH087950 to R.D. and grants from the Finnish Cancer Foundation and Academy of Finland to P.L. H.Z. was supported by the Academy of Finland, and A.P. and J.S. were supported by fellowships from Viikki Graduate School in Biosciences (VGSB) and Helsinki Graduate Program in Biotechnology and Molecular Biology (GPBM), respectively. Use of the IMCA-CAT beamline 17-BM was supported by the Industrial Macromolecular Crystallography Association through a contract with the Hauptman-Woodward Medical Research Institute. The Advanced Photon Source was supported by Department of Energy Contract W-31-109-Eng-38. We acknowledge A.-L. Nyfors, A. Salminen and A. Strandell for excellent technical assistance.

Author information

Affiliations

Authors

Contributions

A.P., M.B., H.Z., J.S., G.R., M.J., J.H., E.V.K., H.V. and R.D. performed the experiments. A.P., J.P., E.J., M.S., E.I., R.D. and P.L designed and supervised the project. A.P., R.D. and P.L. prepared the manuscript.

Corresponding authors

Correspondence to Roberto Dominguez or Pekka Lappalainen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Methods (PDF 2383 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pykäläinen, A., Boczkowska, M., Zhao, H. et al. Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures. Nat Struct Mol Biol 18, 902–907 (2011). https://doi.org/10.1038/nsmb.2079

Download citation

Further reading