Asbury Charles L, Gestaut Daniel R, Powers Andrew F, Franck Andrew D, Davis Trisha N
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9873-8. doi: 10.1073/pnas.0602249103. Epub 2006 Jun 15.
Kinetochores remain attached to microtubule (MT) tips during mitosis even as the tips assemble and disassemble under their grip, allowing filament dynamics to produce force and move chromosomes. The specific proteins that mediate tip attachment are uncertain, and the mechanism of MT-dependent force production is unknown. Recent work suggests that the Dam1 complex, an essential component of kinetochores in yeast, may contribute directly to kinetochore-MT attachment and force production, perhaps by forming a sliding ring encircling the MT. To test these hypotheses, we developed an in vitro motility assay where beads coated with pure recombinant Dam1 complex were bound to the tips of individual dynamic MTs. The Dam1-coated beads remained tip-bound and underwent assembly- and disassembly-driven movement over approximately 3 microm, comparable to chromosome displacements in vivo. Dam1-based attachments to assembling tips were robust, supporting 0.5-3 pN of tension applied with a feedback-controlled optical trap as the MTs lengthened approximately 1 microm. The attachments also harnessed energy from MT disassembly to generate movement against tension. Reversing the direction of force (i.e., switching to compressive force) caused the attachments to disengage the tip and slide over the filament, but sliding was blocked by areas where the MT was anchored to a coverslip, consistent with a coupling structure encircling the filament. Our findings demonstrate how the Dam1 complex may contribute directly to MT-driven chromosome movement.
在有丝分裂过程中，动粒始终附着于微管（MT）末端，即便末端在其掌控下进行组装和拆卸，使得细丝动态变化产生力并推动染色体移动。介导末端附着的特定蛋白质尚不明确，且依赖微管产生力的机制也未知。近期研究表明，Dam1复合物作为酵母动粒的重要组成部分，可能直接有助于动粒与微管的附着及力的产生，或许是通过形成环绕微管的滑动环来实现。为验证这些假设，我们开发了一种体外运动分析方法，将涂有纯重组Dam1复合物的珠子与单个动态微管的末端相连。涂有Dam1的珠子始终附着于末端，并在大约3微米的距离内进行由组装和拆卸驱动的移动，这与体内染色体的位移相当。基于Dam1的与组装末端的附着很强健，当微管延长约1微米时，能够承受用反馈控制的光镊施加的0.5 - 3皮牛顿的张力。这些附着还利用微管拆卸产生的能量来产生对抗张力的移动。将力的方向反转（即切换为压缩力）会导致附着脱离末端并在细丝上滑动，但滑动会被微管固定在盖玻片上的区域所阻断，这与环绕细丝的耦合结构相符。我们的研究结果证明了Dam1复合物可能如何直接促成微管驱动的染色体移动。
