Elucidating the Mechanisms Behind Basement Membrane Stretching

摘要 基底膜是一种致密的细胞外基质，包裹在组织周围并在结构上支持组织。一个 BMS的主要成分是IV型胶原，它形成了一个保护BMS和组织的交联网 来自机械压力。尽管IV型胶原在结构支持中的关键作用已经确定，但这是如何 在机械活动的组织中调节BM伸展的网络是未知的。黑石伸展至关重要 在心血管系统中很重要，血管必须扩张和收缩以适应脉搏 来自血液的力量。与这一重要作用相一致的是，IV型胶原网络的中断可能导致 血管疾病，如糖尿病血管增厚、脑微出血和出血性中风。 尽管BM拉伸具有重要意义，但由于缺乏体内模型可供研究，我们对BM拉伸的理解一直受到限制 BMS如何动态扩展。为了满足这一需求，我建立了视觉和基因上的性腺BM 易驯化的模型生物秀丽线虫作为一种新的BM伸展的体内模型。我发现这片区域 专门用于受精/排卵的性腺BM，即精膜BM，被戏剧性地拉伸(~2倍) 在排卵期间。使用原子力显微镜(Afm)，我发现性腺内有一个僵硬梯度。 受精囊是最硬的。此外，20个黑石组件已经贴上了mNeonGreen的标签 或者使用CRISPR/Cas-9的mRuby，使线虫成为唯一一种所有主要BM成分都 内生标记的。使用这些菌株，我发现：(1)在拉伸过程中，IV型胶原的水平增加 排卵；(2)有高水平的过氧化物素-1，一种负向调节IV型胶原交叉的蛋白质。 连接，在精囊BM中；和(3)纤维蛋白，一种被认为维持IV型胶原的BM蛋白质，富含 受精卵。我的总体假设是IV型胶原的水平和交联度 调节以允许胶原网络保持BM/组织的完整性，同时使BM/组织 伸展身体。在目标1中，我将使用遗传分析、条件基因敲除、实时成像和AFM来测试 假设IV型胶原水平在BM拉伸中起关键作用(低水平促进减少 僵硬、拉伸增加和最终断裂；高水平会增加僵硬并限制拉伸)和 确定IV型胶原α1和α2人类疾病突变对线虫的影响是否源于 与BM伸展相关的独特区域中的水平或位置改变。目标2将检验这一假设 过氧化物酶介导的低IV型胶原交联通过降低骨髓硬度促进骨髓拉伸 在这个动态的环境中，纤毛蛋白维持着IV型胶原的水平。在目标3中，我将确定是否 其他组件通过进行生物信息学驱动的RNAi筛选来调节BM伸展，以识别 影响骨髓伸展的血管疾病相关基因。最终，我希望我的工作将显示出 IV型胶原水平和交联度对于平衡组织支持和骨髓伸展至关重要，而这些 机制在人类血管疾病中受到干扰，使其成为更有效治疗的理想靶点。

Abstract Basement membranes (BMs) are dense extracellular matrices that surround and structurally support tissues. A primary component of BMs is type IV collagen, which forms a cross-linked network that protects BMs and tissues from mechanical stress. Although the key role of type IV collagen in structural support is established, how this network accommodates BM stretching in mechanically active tissues is unknown. BM stretching is critically important in the cardiovascular system, where vessels must expand and contract to accommodate pulsatile forces from the blood. Consistent with this important role, disruptions in the type IV collagen network can lead to vascular diseases such as vascular thickening in diabetes, cerebral microbleeds, and hemorrhagic stroke. Despite its significance, our understanding of BM stretching has been limited by a lack of in vivo models to study how BMs dynamically expand. To fill this need, I have established the gonadal BM of the visually and genetically tractable model organism C. elegans as a new in vivo model for BM stretching. I discovered that the region of the gonadal BM specialized for fertilization/ovulation, the spermathecal BM, is stretched dramatically (~2-fold) during ovulation. Using atomic force microscopy (AFM), I found that there is a stiffness gradient within the gonad with the spermatheca being the least stiff. Furthermore, 20 BM components have been tagged with mNeonGreen or mRuby using CRISPR/Cas-9, making C. elegans the only animal where all major BM components are endogenously tagged. Using these strains, I found: (1) Increased levels of type IV collagen limit stretching during ovulation; (2) there are high levels of peroxidasin-1, a protein that negatively regulates type IV collagen cross- linking, in the spermathecal BM; and (3) fibulin, a BM protein thought to maintain type IV collagen, is enriched in the spermatheca. My overall hypothesis is that levels and cross-linking of type IV collagen are precisely regulated to allow the collagen network to maintain BM/tissue integrity while enabling BM/tissue stretching. In Aim 1, I will use genetic analysis, conditional knockdown, live imaging, and AFM to test the hypothesis that type IV collagen levels play a crucial role in BM stretching (low levels promote decreased stiffness, increased stretching and eventual rupture; high levels increase stiffness and restrict stretching) and determine whether the effects of type IV collagen α1 and α2 human disease mutations in C. elegans are due to altered levels or location in unique domains associated with BM stretching. Aim 2 will test the hypothesis that low type IV collagen cross-linking, mediated by peroxidasins, promotes BM stretching by reducing BM stiffness and that fibulin maintains type IV collagen levels in this dynamic environment. In Aim 3, I will determine if additional components regulate BM stretching by conducting a bioinformatics-driven RNAi screen to identify vascular disease associated genes that affect BM stretching. Ultimately, I expect my work will show that precise type IV collagen levels and cross-linking are critical for balancing tissue support and BM stretching and that these mechanisms are perturbed in human vascular disease, making them ideal targets for more effective therapies.