Dissecting Recruitment of Actin into the Contractile Ring

科学研究：动物细胞的胞质分裂是由肌动球蛋白收缩环的组装引起的，该收缩环将细胞收缩在纺锤体赤道周围。 收缩环的组织和收缩的分子基础是有据可查的，但肌动蛋白丝如何重新分布和组装成环的机制仍然知之甚少。 本计画旨在借由了解微管如何与肌动蛋白丝相互作用，以及肌动蛋白丝如何整合到收缩环中，来阐明收缩环组装的机制。从该项目中获得的知识可以应用于类似的细胞过程，例如单细胞的极化生长和伤口愈合。工作模式如下。 在分裂后期早期，动态的星状体微管将肌动蛋白丝从两极排除，最终导致肌动蛋白在赤道皮层的积累。 同时，在相对稳定的中央纺锤体微管的正端，肌动蛋白丝重新组装，并通过横向张开的微管束传递到赤道皮层。 在那里，它们与被排斥在极地皮层之外的肌动蛋白结合，组装成收缩环。 基于这种微管诱导模型，该项目旨在剖析微管介导的预先存在的肌动蛋白丝的运输机制。 微管将这些细丝从两极排除可能通过两种不同的机制发生。 如果肌动蛋白丝被驱动向赤道皮质微管伸长，微管和肌动蛋白丝之间的机械相互作用将被证明和扰动活细胞动力学细胞。 在缺乏纺锤体的细胞中，微针将被用作微管的替代物以募集肌动蛋白丝，以拯救沟槽定位。 另一方面，如果肌动蛋白丝由马达蛋白沿着与皮层相邻的纺锤体微管运输，则将在活的细胞动力学细胞中直接观察它们在微管上的运动。该项目将使用一种新开发的方法，采用多模式显微技术通过外科手术重塑纺锤体和细胞，从而机械解剖微管介导的收缩环组装。这种独特的方法将经典的显微操作和显微注射与现代激光微束手术相结合-在配备数字增强偏振和旋转圆盘共聚焦显微镜的显微镜上。因此，该方法允许直接显微操作的未标记和荧光标记的细胞骨架成分在活细胞和在vitro.Broader影响：本研究项目的一般影响将在多个方面增加。在实现具体目标的同时，一至两名研究生和一名博士后将接受设计和进行实验、在科学会议上展示数据以及在知名期刊上发表论文的培训。 该研究还将被用作科学教育的有效教学工具在各种情况下-在本科和研究生细胞生物学课程，HHMI支持的本科夏季研究计划，NSF支持的OSU外展计划GK-12学生和教师，HHMI支持的科学数学调查和学习经验（SMILE）计划，并与公众沟通。这项研究通过尖端技术产生了引人注目的、美学上引人注目的数据，使其非常适合教育推广。 此外，由于这项基础研究使用蚱蜢精母细胞作为理解细胞分裂的模型，它可能会引导应用研究走向抑制农业破坏性昆虫繁殖的新方法。

Scientific Research: Cytokinesis in animal cells is brought about by assembly of an actomyosin contractile ring that constricts the cell around the spindle equator. The molecular basis underlying the organization and constriction of the contractile ring is well documented, yet the mechanics of how actin filaments redistribute and assemble into the ring remain poorly understood. This project aims to elucidate the mechanics of contractile ring assembly by learning how microtubules interact with actin filaments and how the filaments incorporate into the ring. Knowledge gained from this project can be applied to analogous cellular processes, such as polarized growth and wound healing of a single cell. The working model is as follows. During early anaphase, dynamic astral microtubules exclude actin filaments from the poles, ultimately resulting in actin accumulation at the equatorial cortex. Meanwhile, at the plus ends of the relatively stable central-spindle microtubules, actin filaments are assembled de novo, and delivered to the equatorial cortex by laterally splaying microtubule bundles. There they coalesce with the actin excluded from the polar cortex to assemble the contractile ring. Based on this Microtubule Induction model, the project aims to dissect the mechanics of microtubule-mediated transport of preexisting actin filaments. Exclusion of these filaments from the poles by microtubules may occur via two distinct mechanisms. If actin filaments are driven towards the equatorial cortex by elongating microtubules, mechanical interactions between microtubules and actin filaments will be demonstrated and perturbed in living cytokinetic cells. In cells deprived of the spindle apparatus, a microneedle will be used as a surrogate for microtubules to recruit actin filaments, in order to rescue furrow positioning. Alternatively, if actin filaments are transported by motor proteins along spindle microtubules that are contiguous to the cortex, direct observations of their movement on microtubules will be made in living cytokinetic cells.The project will use a newly-developed approach that employs multimode micro-techniques to surgically remodel spindles and cells so as to mechanically dissect microtubule-mediated assembly of the contractile ring. This unique approach combines classic micromanipulation and microinjection with modern laser microbeam surgery - on a microscope equipped with both digital-enhanced polarization and spinning disc confocal microscopy. Thus, the approach permits direct micromanipulation of both unlabeled and fluorescently labeled cytoskeletal components in living cells and in vitro.Broader Impacts: The general impact of this research project will be increased in multiple ways. Concomitant with achieving the specific aim, one to two graduate students and one postdoctoral fellow will be trained in designing and conducting experiments, presenting data at scientific conferences, and publishing papers in respected journals. The research will also be used as an effective teaching tool for science education in a variety of contexts - in undergraduate and graduate Cell Biology courses, the HHMI-supported undergraduate summer research program, the NSF-supported OSU outreach programs to GK-12 students and teachers, the HHMI-supported Science & Math Investigative and Learning Experiences (SMILE) program, and in communicating with the general public. This research generates striking, aesthetically compelling data via cutting edge technology, making it ideally suited for educational outreach. Furthermore, because this basic research uses grasshopper spermatocytes as a model for understanding cell division, it may steer applied research toward novel ways of suppressing reproduction in agriculturally destructive insects.