Dissecting Recruitment of Actin into the Contractile Ring

科学研究：动物细胞的细胞质分裂是由肌动球蛋白收缩环的组装引起的，该收缩环将细胞围绕纺锤体赤道收缩。收缩环的组织和收缩的分子基础已被充分记录，然而肌动蛋白丝如何重新分配和组装成环的机制仍然知之甚少。该项目旨在通过了解微管如何与肌动蛋白丝相互作用以及丝如何融入环来阐明收缩环组装的机制。从这个项目中获得的知识可以应用于类似的细胞过程，如单细胞的极化生长和伤口愈合。工作模型如下：在早期后期，动态星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积聚。与此同时，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，并通过侧展的微管束传递到赤道皮层。在那里，它们与被排除在极皮层外的肌动蛋白结合，组装成收缩环。基于这种微管诱导模型，该项目旨在剖析预先存在的肌动蛋白丝的微管介导运输的机制。微管通过两种不同的机制将这些细丝排除在极外。如果肌动蛋白丝通过伸长的微管被推向赤道皮层，微管和肌动蛋白丝之间的机械相互作用将在活的细胞动力学细胞中被证明和扰乱。在失去纺锤体的细胞中，微针将被用作微管的替代物来招募肌动蛋白丝，以挽救沟定位。或者，如果肌动蛋白丝由运动蛋白沿着与皮层相邻的纺锤体微管运输，则可以在活的细胞动力学细胞中直接观察其在微管上的运动。该项目将采用一种新开发的方法，采用多模微技术对纺锤体和细胞进行手术改造，从而机械地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，该方法允许在活细胞和体外对未标记和荧光标记的细胞骨架成分进行直接显微操作。更广泛的影响：本研究项目的总体影响将以多种方式增加。在达到具体目标的同时，将培训一至两名研究生和一名博士后设计和进行实验，在科学会议上展示数据，并在权威期刊上发表论文。这项研究也将被用作各种情况下科学教育的有效教学工具——在本科和研究生细胞生物学课程中，在hhmi支持的本科生暑期研究项目中，在nsf支持的俄勒冈州立大学面向g12学生和教师的推广项目中，在hhmi支持的科学与数学调查和学习经验（SMILE）项目中，以及在与公众的交流中。这项研究通过尖端技术产生引人注目的、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，它可能引导应用研究寻找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.