Ultra-Precise Laser Surgery to study Cell Biomechanics

描述（由申请人提供）：干细胞因其分化成多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。为了实现这一作用，干细胞必须扩大其库（在生长和发育期间），维持组织稳态（在成年期间），并在损伤时增殖。干细胞中良好调节的有丝分裂确保了它们自我延续的显著能力（即，维持它们的池）并产生分化的细胞（即，补充组织）。然而，基本的规则并没有得到很好的理解。我们将研究细胞结构和几何形状在调节干细胞分裂中的作用，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。有几条证据表明，有丝分裂纺锤体的方向是至关重要的干细胞分裂，和纺锤体在调节干细胞分裂和分化的作用将其特征在于通过纺锤体结构的实时成像，结合有针对性的扰动，使用激光显微手术切割或删除关键结构在建立的模型系统。 选择性消融或切割细胞结构的能力是该提议的核心。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，亚细胞结构可以在太空中被靶向，并使用紧密聚焦的超短激光脉冲快速消融。因为这种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），它可以产生有针对性的亚细胞消融，局限于比光波长小得多的区域;我们已经证明了在细胞中100 nm区域的选择性消融。我们将应用这项技术来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成分，以研究它们在干细胞生长和分化中的作用。 公共卫生相关性：拟议的研究结果将产生一个更好的理解的中心体和纺锤体的方向在干细胞的调节和分化的作用，并通过了解这些基本过程将大大促进基于干细胞的治疗的发展。通过推进变革性的激光纳米外科技术，将对生物医学科学产生广泛影响。

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.