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）：为了解释发育缺陷的原因并改进识别潜在致畸因素的方法，我们需要了解缓冲发育免受环境压力的过程。温度影响所有生化过程，因此允许在广泛温度范围内协调发展的机制也可能缓冲其他类型的扰动。此外，热应激，无论是由于发烧还是其他环境暴露，都是许多出生缺陷的重要危险因素（Milunsky 等，1992；Chambers 等，1998；Acs 等，2005；Edwards，2006）。尽管存在许多环境和遗传压力，但发育仍能成功进行，但在应对其他压力或超过阈值的压力时，发育可能会急剧失败（Hamdoun 和 Epel，2007）。 在这里，我们提出了一项新颖的生物物理学研究，研究缓冲形态发生免受扰动的机制。形态发生是一个机械过程，细胞产生的力使组织移动并使组织变形以驱动形状变化，并且这些力受到发育组织的粘弹性特性的抵抗（Koehl，1990；Hutson 等，2003）。胚胎的机制很可能是一个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，这些基因产物包括肌动蛋白、肌球蛋白、调节蛋白和许多其他蛋白，其中大多数在动物物种中高度保守（Davidson et al., 2009）。因此，我们预计胚胎机械特性将成为潜在致畸性和与人类健康相关的其他影响的敏感报告者。 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 等人，2009b）强烈表明，细胞力产生的温度依赖性以及对这些力的机械阻力必须在发育的温度依赖性中发挥作用。为了确定胚胎的力学在面对温度变化时如何受到调节，我们提出以下两个目标： 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的产生是否表现出对广谱环境扰动、温度的耦合响应。变温动物的形态发生率随着温度的升高而迅速增加。我们将测试力产生和粘弹性阻力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的温度依赖性中的作用。我们将使用非洲爪蟾卵的细胞质提取物来研究细胞骨架（在没有转录和细胞-细胞或细胞-基质相互作用的情况下）对粘弹性和力产生的温度依赖性的贡献。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-肌动蛋白聚合状态对力和阻力的温度依赖性的贡献。 这些目标共同构成了一种新颖的生物物理方法，用于了解细胞和胚胎如何缓冲环境扰动。这些目标将有助于了解细胞骨架和细胞生物物理学对出生缺陷的影响。特别是，它将有助于了解高热导致出生缺陷的机制。由于温度会影响所有生化反应，因此这些相同的机制可能会导致其他压力引起的发育缺陷。这些目标还将帮助我们设计基于胚胎机械行为的新型生物测定法，以识别潜在的环境健康危害。这种方法补充了有关形态发生和出生缺陷的遗传学和分子途径的现有研究的广泛内容。 公共健康相关性：该提案的目标是破译发育缺陷的原因并改进识别潜在致畸因素的方法。为了实现这一目标，我们需要通过确定生物物理对发育缓冲的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，了解缓冲发育以应对环境压力（例如温度）的过程。该提案的意义在于从物理原理对出生缺陷进行机制分析，并从人口变异以及细胞和细胞骨架力学与产妇发烧等外部危险因素的相互作用方面了解出生缺陷的发生率。