Microtubule transport, concentration and alignment in enclosed microfluidic channels

Abstract

The kinesin-microtubule system has emerged as a versatile model system for biologically-derived microscale transport. While kinesin motors in cells transport cargo along static microtubule tracks, for in vitro transport applications it is preferable to invert the system and transport cargo-functionalized microtubules along immobilized kinesin motors. However, for efficient cargo transport and to enable this novel transport system to be interfaced with traditional microfluidics, it is important to fabricate enclosed microchannels that are compatible with kinesin motors and microtubules, that enable fluorescence imaging of microtubule movement, and that provide fluidic connections for sample introduction. Here we construct a three-tier hierarchical system of microfluidic channels that links microscale transport channels to macroscopic fluid connections. Shallow microchannels (5 μm wide and 1 μm deep) are etched in a glass substrate and bonded to a cover glass using PMMA as an adhesive, while intermediate channels (∼100 μm wide) serve as reservoirs and connect to 250 μm deep microchannels that hold fine gauge tubing for fluid injection. To demonstrate the utility of this device, we first show the performance of a directional rectifier that redirects 96% of moving microtubules and, because any microtubules that detach rapidly rebind to the motor-coated surface, suffers no microtubule loss over time. Second, we develop an approach, using a headless kinesin construct, to eliminate gradients in motor adsorption and microtubule binding in the enclosed channels, which enables precise control of kinesin density in the microchannels. Finally, we show that a 60 μm diameter circular ring functionalized with motors concentrates and aligns bundles of ∼3000 uniformly oriented microtubules, while suffering negligible ATP depletion. These aligned isopolar microtubules are an important tool for microscale transport applications and can be employed as a model in vitro system for studying kinesin-driven microtubule organization in cells.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. D.C.S. Bien, P.V. Rainey, S.J.N. Mitchell, and H.S. Gamble, J. Micromech. Microeng. 13, S34 (2003).

    Article  Google Scholar 

  2. B. Bilenberg, T. Nielsen, B. Clausen, and A. Kristensen, J. Micromech. Microeng. 14, 814 (2004).

    Article  Google Scholar 

  3. T.B. Brown and W.O. Hancock, Nano Lett. 2, 1131 (2002).

    Article  Google Scholar 

  4. C. Brunner, K.H. Ernst, H. Hess, and V. Vogel, Nanotechnology 15, S540 (2004).

    Article  Google Scholar 

  5. M.Q. Bu, T. Melvin, G.J. Ensell, J.S. Wilkinson, and A.G.R. Evans, Sensors and Actuators A-Physical 115, 476 (2004).

    Article  Google Scholar 

  6. L.J. Cheng, M.T. Kao, E. Meyhöfer, and J. Guo, Small 1, 409 (2005).

    Article  Google Scholar 

  7. J. Clemmens, H. Hess, R. Doot, C.M. Matzke, G.D. Bachand, and V. Vogel, Lab. Chip. 4, 83 (2004).

    Article  Google Scholar 

  8. J. Clemmens, H. Hess, R. Lipscomb, Y. Hanein, K. Bohringer, C. Matzke, G. Bachand, B. Bunker, and V. Vogel, Langmuir 19, 10967 (2003).

    Article  Google Scholar 

  9. D.L. Coy, M. Wagenbach, and J. Howard, J. Biol. Chem. 274, 3667 (1999).

    Article  Google Scholar 

  10. S. Farrens, V. Dragoi, R. Pelzer, M. Wimplinger, and P. Lindner, 207th Meeting of the Electrochemical Society (Quebec, Canada, Electrochemical Society Inc., Pennington, NJ, 2005).

  11. F. Gibbons, J.F. Chauwin, M. Desposito, and J.V. Jose, Biophys. J. 80, 2515 (2001).

    Google Scholar 

  12. F. Gittes, B. Mickey, J. Nettleton, and J. Howard, J. Cell Biol. 120, 923 (1993).

    Article  Google Scholar 

  13. L.S. Goldstein and A.V. Philp, Annu. Rev. of Cell Dev. Biol. 15, 141 (1999).

    Article  Google Scholar 

  14. D.D. Hackney, J. Biol. Chem. 269, 16508 (1994).

    Google Scholar 

  15. W.O. Hancock, Protein-based nanotechnology: Kinesin-microtubule driven systems for bioanalytical applications. Nanodevices for Life Sciences. (C. Kumar. Weinheim, Germany, Wiley-VCH, 2006) vol: 4, p. 241.

  16. W.O. Hancock, and J. Howard, J. Cell Biol. 140, 1395 (1998).

    Article  Google Scholar 

  17. H. Hess, G.D. Bachand, and V. Vogel, Chemistry 10, 2110 (2004).

    Article  Google Scholar 

  18. H. Hess, J. Clemmens, C. Matzke, G. Bachand, B. Bunker, and V. Vogel, Appl. Phys. A-Mater. Sci. & Process. 75, 309 (2002).

    Article  Google Scholar 

  19. Y. Hiratsuka, T. Tada, K. Oiwa, T. Kanayama, and T.Q. Uyeda, Biophys. J. 81, 1555 (2001).

    Google Scholar 

  20. N. Hirokawa, Y. Noda, and Y. Okada, Curr. Opin. Cell Biol. 10, 60 (1998).

    Article  Google Scholar 

  21. J. Howard, A.J. Hudspeth, and R.D. Vale, Nature 342, 154 (1989).

    Article  Google Scholar 

  22. Y.M. Huang, M. Uppalapati, W.O. Hancock, and T.N. Jackson, IEEE Adv. Packaging 28, 564 (2005).

    Article  Google Scholar 

  23. A.J. Hunt and J. Howard, Proc. Nat. Acad. Sci. USA 90, 11653 (1993).

    Google Scholar 

  24. A. Hyman, D. Drechsel, D. Kellogg, S. Salser, K. Sawin, P. Steffen, L. Wordeman, and T. Mitchison, Methods Enzymol. 196, 478 (1991).

    Article  Google Scholar 

  25. L. Jia, S.G. Moorjani, T.N. Jackson, and W.O. Hancock, Biomedical Microdevices 6, 67 (2004).

    Article  Google Scholar 

  26. L. Limberis, J.J. Magda, and R.J. Stewart, Nano Letters 1, 277 (2001).

    Article  Google Scholar 

  27. C.-T. Lin, M.-T. Kao, K. Kurabayashi, and E. Meyhöfer, Small 2, 281 (2006).

    Article  MATH  Google Scholar 

  28. E. Meyhöfer, and J. Howard, Proc. Nat. Acad. Sci. USA 92, 574 (1995).

    Article  Google Scholar 

  29. S.G. Moorjani, L. Jia, T.N. Jackson, and W.O. Hancock, Nano Letters 3, 633 (2003).

    Article  Google Scholar 

  30. I. Prots, R. Stracke, E. Unger, and K.J. Bohm, Cell Biol. Int. 27, 251 (2003).

    Article  Google Scholar 

  31. T.K. Rostovtseva and S.M. Bezrukov, Biophys. J. 74, 2365 (1998).

    Article  Google Scholar 

  32. W.R. Schief, R.H. Clark, A.H. Crevenna, and J. Howard, Proc. Nat. Acad. Sci. USA 101, 1183 (2004).

    Article  Google Scholar 

  33. D.J. Sharp, G.C. Rogers, and J.M. Scholey, Biochi. Et. Biophys. Acta. 1496, 128 (2000).

    Article  Google Scholar 

  34. M.F. Stock and D.D. Hackney, Methods Mol. Biol. 164, 43 (2001).

    Google Scholar 

  35. R. Stracke, K.J. Bohm, L. Wollweber, J.A. Tuszynski, and E. Unger, Biochem. Biophys. Res. Commun 293, 602 (2002).

    Article  Google Scholar 

  36. K. Svoboda, C.F. Schmidt, B.J. Schnapp, and S.M. Block, Nature 365, 721 (1993).

    Article  Google Scholar 

  37. M.G. van den Heuvel, C.T. Butcher, R.M. Smeets, S. Diez, and C. Dekker, Nano. Lett. 5, 1117 (2005).

    Article  Google Scholar 

  38. M.G. van den Heuvel, M.P. de Graaff, and C. Dekker, Sci 312, 910 (2006).

    Article  Google Scholar 

  39. R.C. Williams, Jr. and J.C. Lee, Methods Enzymol 85 Pt B, 376 (1982).

    Google Scholar 

  40. J.T. Yang, R.A. Laymon, and L.S. Goldstein, Cell 56, 879 (1989).

    Article  Google Scholar 

Download references

Acknowledgments

This project was funded by the Penn State Center for Nanoscale Science (NSF MRSEC DMR0213623) and by an NSF Biophotonics Grant (0323024) to W.O.H. and T.N.J. funded jointly by NSF and NIH/NIBIB.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to William O. Hancock or Thomas N. Jackson.

Additional information

Ying-Ming Huang and Maruti Uppalapati contributed equally to this work.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Huang, YM., Uppalapati, M., Hancock, W.O. et al. Microtubule transport, concentration and alignment in enclosed microfluidic channels. Biomed Microdevices 9, 175–184 (2007). https://doi.org/10.1007/s10544-006-9019-1

Download citation

Keywords

  • Kinesin
  • Microtubule
  • Motor proteins
  • Molecular motors
  • Microfabrication
  • Microfluidics
  • Active transport