Biophysics of development buffering: Temperature as a tool to study how the cytos

发育缓冲的生物物理学：温度作为研究细胞如何发育的工具

• 批准号: 7976887
• 负责人: LANCE A. DAVIDSON
• 金额: $ 21.7万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-07 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7976887
• 关键词: Actins; Affect; Animals; Biochemical Process; Biochemical Reaction; Biological Assay; Biophysics; Buffers; Cell Communication; Cells; Complement; Congenital Abnormality; Coupled; Cytoskeletal Proteins; Cytoskeleton; Defect; Dependence; Development; Embryo; Environmental Exposure; Environmental Health; Exhibits; F-Actin; Face; Fever; Generations; Genetic; Genetic Transcription; Goals; Health; Health Hazards; Heat Stress Disorders; Human; In Vitro; Incidence; Mechanics; Methods; Molecular Genetics; Morphogenesis; Movement; Myosin ATPase; Pathway interactions; Population; Process; Property; Proteins; Reporter; Resistance; Risk Factors; Role; Shapes; Speed; Stress; Substrate Interaction; Temperature; Teratogens; Testing; Tissues; Variant; Xenopus; base; crosslink; design; egg; embryo cell; genetic regulatory protein; improved; in vivo; mechanical behavior; novel; polymerization; public health relevance; research study; response; stressor; tool; viscoelasticity

DESCRIPTION (provided by applicant): To decipher the causes developmental defects and to improve methods of identifying potential teratogens, we need to understand the processes that buffer development against environmental stresses. Temperature affects all biochemical processes, so the mechanisms that allow coordinated development across a broad range of temperatures are likely to buffer against other types of perturbations as well. Furthermore heat stress, whether due to fever or other environmental exposures, is a significant risk factor in many birth defects (Milunsky et al., 1992; Chambers et al., 1998; Acs et al., 2005; Edwards, 2006). Development occurs successfully despite many environmental and genetic stressors, yet it can fail dramatically in response to other stresses, or to stresses above a threshold (Hamdoun and Epel, 2007). Here we propose a novel biophysical study of the mechanisms that buffer morphogenesis against perturbations. Morphogenesis is a mechanical process in that cell-generated forces move and deform tissues to drive shape change, and these forces are resisted by the viscoelastic properties of the developing tissues (Koehl, 1990; Hutson et al., 2003). The mechanics of embryo is likely to be a readout of the expression, localization and activity of a very large network of interacting gene products, including actin, myosin, regulatory proteins, and many others, most of which are highly conserved among animal species (Davidson et al., 2009). Hence we expect embryo mechanical properties to be sensitive reporters of potential teratogenicity and other effects relevant to human health. Temperature-dependence of the speed of morphogenetic movements (Bachmann, 1969), and temperature-dependence of cell mechanics (Sunyer et al., 2009b) strongly suggest that temperature- dependence of cellular force generation and of the mechanical resistance to those forces must have a role in the temperature-dependence of development. To determine how the mechanics of the embryo is regulated in the face of temperature variation we propose the following two aims: Aim 1: Identify biophysical contributions to developmental buffering. We will test whether tissue viscoelasticity and force generation exhibit coupled responses to a broad-spectrum environmental perturbation, temperature. Morphogenetic rates increase rapidly with temperature in ectothermic animals. We will test whether temperature-dependence of force generation and viscoelastic resistance can explain the temperature- dependence of morphogenetic movements. We will then test whether temperature affects the sensitivity of morphogenesis to perturbations in cytoskeletal function. Aim 2: Identify the role of specific cytoskeletal proteins in the temperature dependence of viscoelasticity and contractility. We will use cytoplasmic extracts from Xenopus eggs to investigate the contribution of the cytoskeleton - in the absence of transcription, and cell-cell or cell-substrate interactions - to the temperature dependence of viscoelasticity and force generation. We will use in vitro and in vivo experiments to test the contribution of myosin, actin crosslinking, and F-actin polymerization state to the temperature dependence of force and resistance. Together these aims constitute a novel biophysical approach to understanding how cells and embryos are buffered against environmental perturbations. These aims will help in understanding the contributions of the cytoskeleton and cellular biophysics to birth defects. In particular it will aid in understanding the mechanisms by which hyperthermia causes birth defects. Since temperature effects all biochemical reactions, these same mechanisms are likely to contribute to developmental defects caused by other stresses. These aims will also help us design novel bioassays based on the mechanical behavior of embryos to identify potential environmental health hazards. This approach complements the extensive body of existing studies on the genetics and molecular pathways involved morphogenesis and birth defects. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to decipher the causes developmental defects and to improve methods of identifying potential teratogens. To achieve this goal we need to understand the processes that buffer development against environmental stresses such as temperature by identifying biophysical contributions to developmental buffering and identifying the role of specific cytoskeletal proteins in the temperature dependence of viscoelasticity and contractility. The significance of this proposal is the mechanistic analysis of birth defects from physical principles and understanding the incidence of birth defects in terms of population variation and interaction of cell- and cytoskeletal-mechanics with external risk factors such as maternal fever.

描述（由申请人提供）：为了破译导致发育缺陷并改善识别潜在破坏者的方法，我们需要了解缓冲抗环境压力的过程的过程。温度会影响所有生化过程，因此允许在广泛温度上进行协调发展的机制也可能会缓冲其他类型的扰动。此外，无论是由于发烧还是其他环境暴露，热应激是许多先天缺陷的重要危险因素（Milunsky等，1992; Chambers等，1998; Acs等，2005; Edwards，2006）。尽管有许多环境和遗传压力源，但仍会成功地发生发展，但是它可能会对其他压力或高于阈值的压力发生巨大失败（Hamdoun和Epel，2007）。 在这里，我们提出了一项新的生物物理研究，以缓解反对扰动的机制。形态发生是一种机械过程，因为细胞生成的力移动并变形组织以驱动形状变化，并且这些力被发育组织的粘弹性特性所抵抗（Koehl，1990; Hutson等，2003）。胚胎的机制可能是对非常大的相互作用基因产物网络的表达，定位和活性的读数，包括肌动蛋白，肌球蛋白，调节蛋白等，其中大多数是在动物物种中高度保守的（Davidson等，2009）。因此，我们预计胚胎机械性能将成为潜在致致致造性和其他与人类健康相关的其他影响的敏感记者。 形态发生运动速度的温度依赖性（Bachmann，1969）和细胞力学的温度依赖性（Sunyer等，2009b）强烈表明，细胞力产生和对这些力的机械抗性的温度依赖性必须在发育的温度依赖性中起作用。为了确定面对温度变化的胚胎的力学如何调节，我们提出以下两个目的：目标1：确定生物物理对发育缓冲的贡献。我们将测试组织粘弹性和力产生是否表现出对广谱环境扰动，温度的耦合反应。随着温度热动物的温度，形态发生率迅速增加。我们将测试力产生和粘弹性抗性的温度依赖性是否可以解释形态发生运动的温度依赖性。然后，我们将测试温度是否影响形态发生对细胞骨架功能中扰动的敏感性。 目标2：确定特定细胞骨架蛋白在粘弹性和收缩性温度依赖性中的作用。我们将使用爪蟾卵中的细胞质提取物来研究细胞骨架的贡献 - 在没有转录的情况下以及细胞细胞或细胞 - 底物相互作用 - 对粘弹性和力产生的温度依赖性。我们将使用体外和体内实验来测试肌球蛋白，肌动蛋白交联和F-肌动蛋白聚合态对力和耐药性温度依赖性的贡献。 这些目标共同构成了一种新型的生物物理方法，可以理解细胞和胚胎如何缓冲环境扰动。这些目标将有助于理解细胞骨架和细胞生物物理学对先天缺陷的贡献。特别是它将有助于理解高温导致先天缺陷的机制。由于温度影响所有生化反应，因此这些相同的机制可能导致其他应力引起的发育缺陷。这些目标还将帮助我们根据胚胎的机械行为来设计新型的生物测定，以识别潜在的环境健康危害。这种方法补充了有关遗传学和分子途径的广泛研究，涉及形态发生和出生缺陷。 公共卫生相关性：该提案的目的是破译导致发育缺陷的原因，并改善识别潜在破坏者的方法。为了实现这一目标，我们需要通过确定对发育缓冲的生物物理贡献，并确定特定细胞骨架蛋白在粘弹性和收缩性的温度依赖性中的作用，从而了解缓冲剂对环境应力（例如温度）的过程。该提案的重要性是从物理原理中对先天缺陷的机械分析，并了解人口变化以及细胞和细胞骨架机力与外部危险因素（例如母体发烧）的相互作用的发生率。