Nuclear Mechanics varies with Tissue Mechanics & Regulates Cytoskeleton

 描述(申请人提供)：对胚胎组织的生物力学方面了解甚少，尤其是核力学。众所周知，非常早期的胚胎非常柔软，核结构蛋白lamin-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]。向组织的初始分化会在心脏中启动lamin-A转录，这似乎很重要，因为基因敲除的小鼠表现出“心脏发育缺陷”并在出生后不久死亡[Kubben Nucleus 2011]，但在这里研究的Intac胚胎组织中缺乏lamin-A的蛋白特征。Lamin-A突变会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(Progeria)。Lamin-A也被认为影响分化和细胞存活--所有这些都促使研究发现并扰乱心脏跳动中的细胞膜。在成人组织和原代细胞中，我们发现层蛋白-A水平几乎与组织硬度E成正比[SWIFT Science 2013]。相对坚硬的结缔组织承受着很高的机械应力，如骨骼甚至心脏，它们含有高层粘连蛋白-A，这表明坚硬的细胞核可以抵抗压力。相比之下， 非常软的组织，如大脑和骨髓，承受的压力很小，表达低层粘连蛋白-A。B型板层在固体组织中相对恒定，因此层蛋白-A：B化学计量比似乎是成人组织中刚性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运算，它符合成人细胞和组织的研究结果。我们的假设是，正常胚胎中的板层在发育过程中会对机械压力做出调整。我们的目标是确定和扰乱胚胎发育中Lamin基因回路的机械调节，重点是相对于液态血液，是什么发育成僵硬的心脏。根据我们最近的研究，我们将重点放在灵活的鸡胚系统上，这些研究表明心脏跳动对基质弹性的敏感度很高[Majkut Curr Biol 2013]。鸡有优势，包括鸡的红细胞有板层的事实，但我们最终会与发育中的小鼠组织进行比较。首先，我们将通过质谱仪对整个发育过程中的层蛋白水平进行量化，并使用新的测量和扰动来评估它们的应力和硬度敏感性。我们将把胚胎叶片测量与核流变学联系起来，并扰乱水平以验证相互关系和分子机制。初步数据显示，跳动的鸡心很容易被转基因，因此Lamin Promoter-Reporter结构可以作为应力和硬度的原位机械传感器进行测试。椎板也促进了成熟和分化，成年细胞的初始数据表明细胞骨架基因表达和与治疗相关的维甲酸途径的反馈。我们的研究最终应该揭示出核层是一个多因素的胚胎应力传感器，它可以反馈到更广泛的结构调节中。16年

 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