Nuclear Mechanics varies with Tissue Mechanics & Regulates Cytoskeleton

 描述（由申请人提供）：胚胎组织的生物力学方面知之甚少，特别是核力学。众所周知，非常早期的胚胎非常柔软，核结构蛋白层粘连蛋白A的水平非常低，我们通过单细胞操作表明，这意味着细胞核比几乎任何成年细胞都要柔软[Swift Science 2013]。组织的初始分化开启了心脏中的核纤层蛋白A转录，这似乎很重要，因为敲除小鼠表现出“心脏发育缺陷”并在出生后不久死亡[Kubben Nucleus 2011]，但如本文所研究的，在intac胚胎组织中缺乏核纤层蛋白A蛋白表征。Lamin-A突变导致一系列不同发病年龄的疾病，包括扩张型心肌病（DCM）和影响心脏的加速衰老（早衰症）。Lamin-A也已知会影响分化和细胞存活-所有这些都促使研究在跳动的心脏中观察和干扰Lamina。 对于成体组织和原代细胞，我们发现层粘连蛋白A水平与组织硬度E几乎成比例[Swift Science 2013]。相对坚硬的结缔组织承受高的机械应力，例如骨骼甚至心脏，并且它们具有高的层粘连蛋白A，这表明坚硬的细胞核抵抗应力。与此相反， 非常软的组织，如脑和骨髓，几乎不承受应力，表达低层粘连蛋白-A。B型核纤层蛋白在实体组织中相对恒定，因此核纤层蛋白-A：B化学计量比似乎是成人组织中刚度和应力的机械传感器。我们已经完成了一个简单的机械生物学基因电路的数学计算，它适合成人细胞和组织的发现。我们的假设是，正常胚胎中的核纤层在发育过程中对机械应力做出了反应。我们的目标是确定和干扰发育中胚胎核纤层蛋白基因回路的机械调节，重点是什么发展成为相对于液体血液的僵硬心脏。根据我们最近的研究，我们专注于简易鸡胚系统，这些研究证明了跳动的心脏对基质弹性的急性敏感性[Majkut Curr Biol 2013]。鸡有其优点，包括鸡红细胞具有核纤层蛋白，但我们将在最后与发育中的小鼠组织进行比较。 首先，我们将量化核纤层蛋白质水平在整个发展的质谱，我们将评估其应力和刚度的灵敏度与新的测量和扰动。我们将把胚胎纤层测量与核流变学联系起来，并扰动水平以验证关系和分子机制。初步数据显示，跳动的鸡心脏很容易转染，因此核纤层蛋白启动子-报告基因构建体可以作为应力和刚度的原位机械传感器进行测试。纤层还增强成熟和分化，并且成体细胞的初始数据表明对细胞骨架基因表达和治疗相关性的类维生素A途径的反馈。我们的研究应该最终揭示核纤层作为一个多因素，胚胎应力传感器，反馈到更广泛的结构调节。16

 DESCRIPTION (provided by applicant): Biomechanical aspects of embryonic tissues are poorly understood, especially nuclear mechanics. Very early embryos are well-known to be very soft and have very low levels of the nuclear structure protein lamin-A, which we have shown by single cell manipulations means that the nucleus is softer than in almost any adult cell [Swift Science 2013]. Initial differentiation to tissue turns on lamin-A transcription in heart, which seems important because knockout mice exhibit 'developmental defects of the heart' and die shortly after birth [Kubben Nucleus 2011], but lamin-A protein characterization is lacking in intac embryonic tissues as studied here. Lamin-A mutations cause a range of diseases with various ages of onset, including dilated cardiomyopathy (DCM) and accelerated aging (Progeria) affecting heart. Lamin-A is also known to affect differentiation and cell survival - all of which motivates studies to see & perturb the lamina in beating hearts. With adult tissue and primary cells, we have found that lamin-A levels are nearly proportional to tissue stiffness E [Swift Science 2013]. Relatively stiff connective tissues bear high mechanical stress, such as bone and even heart, and they have high lamin-A, suggesting stiff nuclei resist the stress. In contrast, very soft tissues such as brain and marrow that bear little stress express low lamin-A. B-type lamins are comparatively constant in the solid tissues, so that lamin-A:B stoichiometry seems a mechanosensor of stiffness and stress in adult tissues. We have worked through the mathematics of a simple mechanobiological gene circuit that fits findings for adult cells and tissues. Our hypothesis here is that Lamins in normal embryos adjust developmentally in response to mechanical stresses. Our goal is to determine and perturb mechano- regulation of lamin gene circuits in developing embryos, with a focus on what develops into a stiff heart relative to fluid blood. We focus on the facile chick embryo system per our recent studies that demonstrate acute sensitivity of beating heart to matrix elasticity [Majkut Curr Biol 2013]. Chick has advantages including the fact that chick erythrocytes have lamins, but we will at the end compare to developing mouse tissues. First we will quantify lamin protein levels throughout development by Mass Spec, and we will assess their stress and stiffness sensitivity with novel measurements and perturbations. We will relate embryonic lamina measurements to nuclear rheology and perturb the levels to validate relationships and molecular mechanisms. Preliminary data shows that beating chick hearts are easily transfected, so that Lamin Promoter- Reporter constructs can be tested as in situ mechanosensors of stress and stiffness. The lamina also enhances maturation and differentiation, and initial data with adult cells indicates feedback to cytoskeletal gene expression and the retinoid pathway of therapeutic relevance. Our studies should ultimately reveal the nuclear lamina as a multi-factorial, embryonic stress sensor that feeds back into broader structural regulation. 16