Abstract
Although the mechanical behavior of single-motor protein molecules such as kinesin has been carefully studied in buffer, the mechanical behavior of motor-driven vesicles in cells is much less understood. We have tracked single vesicles in neurites of PC12 cells with a spatial precision of ±30 nm and a time resolution of 120 ms. Because the neurites are thin, long, straight, and attached to the surface of planar cover glasses, the velocity of individual vesicles could be measured for times as long as 15 s and distances as long as 15 μm. The velocity of anterograde vesicles was in most cases constant for periods of 1–2 s, then changed in a step-like fashion to a new constant velocity. The viscoelastic modulus felt by the vesicles within live PC12 cells was determined from the Brownian motion, using Mason’s generalization of the Stokes–Einstein equation. From Stokes’ law, the drag force at the smallest sustained velocity was 4.2±0.6 pN for vesicles of radius 0.30–0.40 μm, about half the maximum force which conventional kinesin can develop during bead assays in buffer. We interpret the observed velocity steps as changes of ±1 or occasionally ±2 in the number of active motor proteins dragging that vesicle along a microtubule. Assuming that the motor is conventional kinesin, which hydrolyzes one ATP per 8 nm step along the microtubule, the motor protein efficiency in PC12 neurites is approximately 35%.
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Acknowledgements
We thank Professors Carol Milligan and Dan Johnson, and Jason Newbern, for help with growth of PC12. We are grateful to Jennifer Shuler and Anita McCauley for guidance with immuno-labeling. We thank Hamamatsu Photonic Systems for the loan of a camera. Finally, we are grateful to Wake Forest University for support of this project through a Cross-Campus Collaboration Grant.
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Hill, D.B., Plaza, M.J., Bonin, K. et al. Fast vesicle transport in PC12 neurites: velocities and forces. Eur Biophys J 33, 623–632 (2004). https://doi.org/10.1007/s00249-004-0403-6
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DOI: https://doi.org/10.1007/s00249-004-0403-6