Letter | Published:

Par-3 controls tight junction assembly through the Rac exchange factor Tiam1

Subjects

Abstract

The par (partitioning-defective) genes express a set of conserved proteins that function in polarization and asymmetric cell division1,2. Par-3 has multiple protein-interaction domains, and associates with Par-6 and atypical protein kinase C (aPKC)3,4,5. In Drosophila, Par-3 is essential for epithelial cell polarization6. However, its function in mammals is unclear. Here we show that depletion of Par-3 in mammalian epithelial cells profoundly disrupts tight junction assembly. Expression of a carboxy-terminal fragment plus the third PDZ domain of Par-3 partially rescues junction assembly, but neither Par-6 nor aPKC binding is required. Unexpectedly, Rac is constitutively activated in cells lacking Par-3, and the assembly of tight junctions is efficiently restored by a dominant-negative Rac mutant. The Rac exchange factor Tiam1 (ref. 7) binds directly to the carboxy-terminal region of Par-3, and knockdown of Tiam1 enhances tight junction formation in cells lacking Par-3. These results define a critical function for Par-3 in tight junction assembly, and reveal a novel mechanism through which Par-3 engages in the spatial regulation of Rac activity and establishment of epithelial polarity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accession codes

Accessions

BINDPlus

References

  1. 1

    Kemphues, K. J., Priess, J. R., Morton, D. G. & Cheng, N. S. Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52, 311–320 (1988).

  2. 2

    Doe, C. Q. & Bowerman, B. Asymmetric cell division: fly neuroblast meets worm zygote. Curr. Opin. Cell Biol. 13, 68–75 (2001).

  3. 3

    Izumi, Y. et al. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J. Cell Biol. 143, 95–106 (1998).

  4. 4

    Joberty, G., Petersen, C., Gao, L. & Macara, I. G. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nature Cell Biol. 2, 531–539 (2000).

  5. 5

    Lin, D. et al. A mammalian PAR-3–PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nature Cell Biol. 2, 540–547 (2000).

  6. 6

    Tepass, U., Tanentzapf, G., Ward, R. & Fehon, R. Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35, 747–784 (2001).

  7. 7

    Mertens, A. E., Roovers, R. C. & Collard, J. G. Regulation of Tiam1–Rac signalling. FEBS Lett. 546, 11–16 (2003).

  8. 8

    Macara, I. G. Parsing the polarity code. Nature Rev. Mol. Cell Biol. 5, 220–231 (2004).

  9. 9

    Nelson, W. J. Adaptation of core mechanisms to generate cell polarity. Nature 422, 766–774 (2003).

  10. 10

    Perez-Moreno, M., Jamora, C. & Fuchs, E. Sticky business: orchestrating cellular signals at adherens junctions. Cell 112, 535–548 (2003).

  11. 11

    Gibson, M. C. & Perrimon, N. Apicobasal polarization: epithelial form and function. Curr. Opin. Cell Biol. 15, 747–752 (2003).

  12. 12

    Hirose, T. et al. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J. Cell Sci. 115, 2485–2495 (2002).

  13. 13

    Matter, K. & Balda, M. S. Functional analysis of tight junctions. Methods 30, 228–234 (2003).

  14. 14

    Furuse, M., Furuse, K., Sasaki, H. & Tsukita, S. Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells. J. Cell Biol. 153, 263–272 (2001).

  15. 15

    Gao, L., Joberty, G. & Macara, I. G. Assembly of epithelial tight junctions is negatively regulated by Par6. Curr. Biol. 12, 221–225 (2002).

  16. 16

    Benton, R. & Johnston, D. S. A conserved oligomerization domain in Drosophila Bazooka/PAR-3 is important for apical localization and epithelial polarity. Curr. Biol. 13, 1330–1334 (2003).

  17. 17

    Mizuno, K. et al. Self-association of PAR-3-mediated by the conserved N-terminal domain contributes to the development of epithelial tight junctions. J. Biol. Chem. 278, 31240–31250 (2003).

  18. 18

    Wodarz, A., Ramrath, A., Grimm, A. & Knust, E. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts. J. Cell Biol. 150, 1361–1374 (2000).

  19. 19

    Ebnet, K. et al. The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). EMBO J. 20, 3738–3748 (2001).

  20. 20

    Takekuni, K. et al. Direct binding of cell polarity protein PAR-3 to cell–cell adhesion molecule nectin at neuroepithelial cells of developing mouse. J. Biol. Chem. 278, 5497–5500 (2003).

  21. 21

    Kovacs, M., Toth, J., Hetenyi, C., Malnasi-Csizmadia, A. & Sellers, J. R. Mechanism of blebbistatin inhibition of myosin II. J. Biol. Chem. 279, 35557–35563 (2004).

  22. 22

    Kosako, H. et al. Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow. Oncogene 19, 6059–6064 (2000).

  23. 23

    Totsukawa, G. et al. Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibres and focal adhesions in 3T3 fibroblasts. J. Cell Biol. 150, 797–806 (2000).

  24. 24

    Noren, N. K., Niessen, C. M., Gumbiner, B. M. & Burridge, K. Cadherin engagement regulates Rho family GTPases. J. Biol. Chem. 276, 33305–33308 (2001).

  25. 25

    Malliri, A., van Es, S., Huveneers, S. & Collard, J. G. The Rac exchange factor Tiam1 is required for the establishment and maintenance of cadherin-based adhesions. J. Biol. Chem. 279, 30092–30098 (2004).

  26. 26

    Stam, J. C. et al. Targeting of Tiam1 to the plasma membrane requires the cooperative function of the N-terminal pleckstrin homology domain and an adjacent protein interaction domain. J. Biol. Chem. 272, 28447–28454 (1997).

  27. 27

    Knoblich, J. A. Asymmetric cell division during animal development. Nature Rev. Mol. Cell Biol. 2, 11–20 (2001).

  28. 28

    Jan, Y. N. & Jan, L. Y. Asymmetric cell division in the Drosophila nervous system. Nature Rev. Neurosci. 2, 772–779 (2001).

  29. 29

    Brummelkamp, T. R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).

  30. 30

    Ren, X. D. & Schwartz, M. A. Determination of GTP loading on Rho. Methods Enzymol. 325, 264–272 (2000).

Download references

Acknowledgements

We thank J. G. Collard for providing Tiam1 C1199 plasmid and anti-Tiam1 antibody, M. A. Schwartz for pGEX-RBD and pGEX-PBD plasmids, and A. Spang (Tübingen) and members of the Macara laboratory for helpful comments. This work was supported by grants GM070902 and CA40042 from the National Institutes of Health, DHHS.

Author information

Competing interests

The authors declare no competing financial interests.

Correspondence to Xinyu Chen.

Supplementary information

  1. Supplementary information

    Supplementary figures S1, S2 and S3 (PDF 554 kb)

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

  • Received

  • Accepted

  • Published

  • Issue Date

DOI

https://doi.org/10.1038/ncb1226

Further reading

Figure 1: Par-3 loss severely disrupts tight junction formation in MDCK II cells.
Figure 2: Rescue of tight junction assembly in Par-3-knockdown cells.
Figure 3: Inhibition of the Rho pathway rescues junction assembly.
Figure 4: Constitutive activation of Rac in Par-3-knockdown cells.
Figure 5: Par-3 regulates tight junction formation through interaction with Tiam1.