Ultra-Precise Laser Surgery to study Cell Biomechanics

超精密激光手术研究细胞生物力学

• 批准号: 8153109
• 负责人: ALAN J HUNT
• 金额: $ 28.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8153109
• 关键词: Ablation; Adult; Biological Models; Biomechanics; Cell Differentiation process; Cell Polarity; Cell Therapy; Cells; Cellular Structures; Centrosome; Cyst; Cytoskeleton; Daughter; Development; Drosophila genus; Ensure; Failure; Genes; Genome; Glass; Growth; Growth and Development function; Heating; Histocompatibility Testing; Homeostasis; Image; Imaging Techniques; Interphase; Knock-out; Laser Surgery; Lasers; Life; Light; Maintenance; Microsurgery; Mitosis; Mitotic; Mitotic Checkpoint; Mitotic Spindle Apparatus; Mitotic spindle; Molecular Genetics; Mothers; Nomenclature; Optics; Organism; Pattern; Phenotype; Physiologic pulse; Play; Process; Proliferating; Publishing; Quartz; Regulation; Relative (related person); Role; Sapphire; Science; Stem Cell Development; Stem cells; Structure; Subcellular structure; Techniques; Technology; Testing; Testis; Therapeutic; Time; Tissues; Work; absorption; base; cell growth; migration; nanosurgery; public health relevance; repaired; response to injury; stem; stem cell differentiation; stem cell division; stem cell fate; stem cell niche; success; tool; Ablation; Adult; Biological Models; Biomechanics; Cell Differentiation process; Cell Polarity; Cell Therapy; Cells; Cellular Structures; Centrosome; Cyst; Cytoskeleton; Daughter; Development; Drosophila genus; Ensure; Failure; Genes; Genome; Glass; Growth; Growth and Development function; Heating; Histocompatibility Testing; Homeostasis; Image; Imaging Techniques; Interphase; Knock-out; Laser Surgery; Lasers; Life; Light; Maintenance; Microsurgery; Mitosis; Mitotic; Mitotic Checkpoint; Mitotic Spindle Apparatus; Mitotic spindle; Molecular Genetics; Mothers; Nomenclature; Optics; Organism; Pattern; Phenotype; Physiologic pulse; Play; Process; Proliferating; Publishing; Quartz; Regulation; Relative (related person); Role; Sapphire; Science; Stem Cell Development; Stem cells; Structure; Subcellular structure; Techniques; Technology; Testing; Testis; Therapeutic; Time; Tissues; Work; absorption; base; cell growth; migration; nanosurgery; public health relevance; repaired; response to injury; stem; stem cell differentiation; stem cell division; stem cell fate; stem cell niche; success; tool

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.

描述（由申请人提供）：干细胞具有分化为多种不同组织类型的能力，并在组织生长，维护和修复中起着核心作用。为了履行这一角色，干细胞必须扩大其池（在生长和发育过程中），维持组织稳态（在成年期间），并因损害而增殖。干细胞中受良好调节的有丝分裂划分确保了它们的显着自我维持能力（即保持池）并产生分化的细胞（即补充组织）。但是，基本的法规尚不清楚。我们将使用激光显微外科手术工具以及我们组开发的延时生效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。几种证据表明，有丝分裂主轴的方向对于干细胞分裂至关重要，并且纺锤体在调节干细胞分裂和分化中的作用将以实用纺锤体结构的现场成像以及使用激光微医师在固定模型系统中切割或去除关键结构的靶向扰动来表征。 该提案有选择性烧蚀或切割细胞结构的能力至关重要。我们小组和其他人的工作导致了“结构敲除”技术的发展，从而可以将亚细胞结构靶向空间，并使用紧密聚焦的超短激光脉冲快速消融。因为该技术基于高度非线性的光学分解（与加热或普通吸收相反），因此可以产生限制在光波长小于光波长的区域的靶向亚细胞消融。我们已经证明了细胞中100 nm区域的选择性消融。我们将将这项技术应用于有选择地破坏细胞结构，包括细胞骨架和有丝分裂主轴的成分，以检查它们在干细胞生长和分化中的作用。 公共卫生相关性：拟议研究的结果将更好地理解中心体和主轴取向在干细胞调节和分化中的作用，并且通过了解这些基本过程将极大地促进基于干细胞的治疗剂的发展。生物医学科学的广泛影响将通过推进变革性激光纳米外科技术来实现。