Ultra-Precise Laser Surgery to study Cell Biomechanics

描述（由申请人提供）：干细胞因其分化成各种不同组织类型的能力而引人注目，并在组织生长，维持和修复中发挥核心作用。为了发挥这一作用，干细胞必须在生长和发育过程中扩大其储存库，在成年期维持组织稳态，并在损伤时增殖。干细胞中调控良好的有丝分裂确保了其自我延续（即维持其库）和产生分化细胞（即补充组织）的卓越能力。然而，基本的规则并没有被很好地理解。我们将使用激光显微手术工具和我们小组开发的延时实时成像技术来研究细胞结构和几何形状在调节干细胞分裂中的作用。有几条证据表明，有丝分裂纺锤体的方向对干细胞分裂至关重要，纺锤体在调节干细胞分裂和分化中的作用将通过纺锤体结构的实时成像来表征，并结合使用激光显微手术切割或去除已建立模型系统中的关键结构的靶向扰动。选择性地消融或切割细胞结构的能力是这一提议的核心。我们小组和其他人的工作导致了“结构敲除”技术的发展，通过这种技术，亚细胞结构可以在空间中被靶向，并使用紧密聚焦的超短激光脉冲快速消融。因为这项技术是基于高度非线性的光学击穿（与加热或普通吸收相反），它可以产生目标亚细胞消融，限制在比光波长小得多的区域；我们已经展示了细胞中100纳米区域的选择性消融。我们将应用这项技术选择性地破坏细胞结构，包括细胞骨架和有丝分裂纺锤体的组成部分，以检查它们在干细胞生长和分化中的作用。

DESCRIPTION (provided by applicant): Stem cells are remarkable for their ability to differentiate into diverse different tissue types, and play a central role in tissue growth, maintenance, and repair. To fulfill this role stem cells must expand their pool (during growth and development), sustain tissue homeostasis (during adulthood), and proliferate in response to injury. Well regulated mitotic divisions in stem cells ensure their remarkable ability to self-perpetuate (i.e., maintain their pool) and generate differentiated cells (i.e., replenish tissue). However, the underlying regulation is not well understood. We will investigate the roles of cellular structure and geometry in regulating stem cell division using laser microsurgery tools and time-lapse live-imaging techniques developed in our group. Several lines of evidence suggest that the orientation of the mitotic spindle is critical to stem cell divisions, and the role of the spindle in regulating stem cell divisions and differentiation will be characterized by live-imaging of spindle structures, combined with targeted perturbations using laser microsurgery to cut or remove critical structures in established model systems. The ability to selectively ablate or cut cellular structures is central to this proposal. Work in our group and others has led to the development of "structural-knockout" technology, whereby subcellular structures can be targeted in space and quickly ablated using tightly focused ultrashort laser pulses. Because this technique is based on highly non-linear optical breakdown (as opposed to heating or ordinary absorption) it can produce targeted sub-cellular ablations confined to regions considerably smaller than the wavelength of light; and we have demonstrated selective ablation of regions 100 nm across in cells. We will apply this technology to selectively disrupt cellular structures, including components of the cytoskeleton and mitotic spindle, to examine their role in stem cell growth and differentiation. PUBLIC HEALTH RELEVANCE: The results of proposed studies will yield a better understanding of the role of centrosomes and spindle orientation in stem cell regulation and differentiation, and through understanding these fundamental processes will greatly facilitate the development of stem cell-based therapeutics. Broad impact across the biomedical sciences will be achieved by advancing transformative laser nanosurgery technology.