Hall K, Cole D, Yeh Y, Baskin R J
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Biophys J. 1996 Dec;71(6):3467-76. doi: 10.1016/S0006-3495(96)79542-5.
To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility we have developed a centrifuge microscope sperm-gliding motility assay. The average (extrapolated) value of maximum isometric force at low kinesin density was 0.90 +/- 0.14 pN. Furthermore, in the experiments at low kinesin density, sperm pulled off before stall at forces between 0.40 and 0.75 pN. To further characterize our kinesin-demembranated sperm assay we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34 +/- 1.5 pN was the maximum force. Using values of axoneme stiffness available from other studies, we concluded that, in our centrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall. In addition, drag of the distal portion of the axoneme is increased by the centrifugal force (because the axoneme is rotated into closer proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.
为了测量动力蛋白驱动运动中力的产生并表征力与速度之间的关系，我们开发了一种离心显微镜精子滑行运动测定法。在低动力蛋白密度下，最大等长力的平均（外推）值为0.90±0.14皮牛。此外，在低动力蛋白密度的实验中，精子在力为0.40至0.75皮牛之间时，在失速前就脱离了。为了进一步表征我们的去膜动力蛋白精子测定法，我们使用基于激光阱的测定法估计了最大等长力。在低动力蛋白密度下，最大力为4.34±1.5皮牛。利用其他研究中可得的轴丝刚度值，我们得出结论，在我们基于离心显微镜的测定法中，精子轴丝起到杠杆臂的作用，放大离心力并导致在失速前脱离。此外，轴丝远端部分的阻力因离心力而增加（因为轴丝旋转得更靠近玻璃表面），这代表了动力蛋白马达必须克服的额外力。
