EAGER: Exploring Cell-Cell Gap as a Critical Parameter in Biological Phase Changes

当细胞数量变得足够大时，生物系统有时会迅速改变行为。这个早期概念探索性研究拨款(AGERGE)项目基于初步数据，表明对于一种哺乳动物细胞，触发不同行为的是细胞之间的距离。这是在一种特殊类型的肌肉细胞中观察到的。当细胞之间相距100微米时，就会自动形成肌肉“纤维”。当它们之间的距离超过100微米时，它们就不会。这一观察结果将在成纤维细胞中得到进一步检验。成纤维细胞对细胞外基质的压缩是创面愈合和器官原始形态发生的基础。该项目将确定这个100微米的门槛是否也适用于小鼠、猴子和人类的成纤维细胞。作为对观察广度的一种测试，100微米的门槛也将使用在某些条件下具有聚集成更大结构的特性的Protista Dictyostoma进行测试。如果实验结果在不同类型的细胞中具有可比性，该项目将显著支持在成纤维细胞和网状网茎中的聚集中存在激活组织“相变”的阈值距离。这可以提高我们对各种条件的理解，在这些条件下，组织明显表现出行为的相变，包括胚胎发生、癌症发生、体外组织形成、细菌生物膜形成以及微生物世界中的各种集体现象。识别临界距离条件将有助于预测具有细胞和细胞外基质的3D打印组织的形态发生路径。这项研究将由一名研究生和两名本科生进行。UG的一名学生将来自生物系，其他学生将来自工程系。因此，学生将接受多学科领域的培训。将特别努力从少数族裔和代表性不足的群体中招募学生进行这项研究。该项目的灵感来自最近的实验发现，在3D培养中，彼此100微米以内的肌肉细胞压缩ECM并形成肌管(Pi的实验室)，彼此100微米以内的内皮细胞形成血管系统，胶原蛋白中的成纤维细胞紧凑胶原蛋白。当细胞-细胞间隙超过100微米时，这些过程都不会发生。关键距离的假设将使用来自多个物种(小鼠、猴子和人类)的成纤维细胞在胶原基质中与荧光珠混合进行验证，以光学跟踪粘液霉菌细胞(Dictyostelius)的致密化和集群形成。为了深入了解阈值电池-电池间隙的实验观察，将建立一个数学模型来模拟实验观察。每个细胞通过附着在基质上，并在细胞丝状基座产生收缩力量时拉动纤维来重塑它们周围的基质。每个单元在拉伸下形成一个具有较高刚度的重塑矩阵区域。如果细胞相隔很远，它们的区域就保持彼此独立。在一个有许多细胞的系统中，这样的非相互作用区保持全局对称性。当单元格彼此靠近时，它们的区域重叠，对称性被破坏。细胞之间的硬度变得更高，细胞之间形成了更坚硬的纤维桥。与其他方向相比，更多的丝状伪足倾向于沿着桥延伸，细胞变得极化和拉长，同时保持收缩。这会在细胞之间产生吸引人的相互作用，从而使基质紧凑。来自小鼠(胚胎成纤维细胞，NIH/3T3)、猴子(肾脏成纤维细胞)和人(肺)的三种类型的成纤维细胞将被用来检验关于基质紧凑的假说，而黏液霉菌细胞(盘基网柄菌)将被用来检验其普遍性。所有这些类型的细胞都有类似的大小，直径约10微米。因此，我们为所有类型的细胞预测相似的临界距离。

Biological systems sometimes rapidly change behavior when the number of cells becomes large enough. This EArly-concept Grant for Exploratory Research (EAGER) project is based on preliminary data showing that for one type of mammalian cell, that it is the distance between the cells that triggers the different behavior. This was observed for a particular type of muscle cell. When the cells are 100 micrometers apart, the form into muscle "fibers" automatically. When they are further than 100 micrometers apart, they do not. This observation will be further examined in fibroblasts. The compression of extracellular matrix by fibroblasts is fundamental to the healing of wounds and also to the original morphogenesis of organs. The project will determine whether this threshold of 100 micrometers is also true for fibroblasts from mice, monkeys and humans. As a test of the breadth of the observation, the 100 micrometer threshold will also be tested using the Protista Dictyostelium that has the property of aggregating into larger structures under some conditions. If the results of the experiments are comparable among the different cell types, the project will have developed significant support for the existence of a threshold distance for the activation of a tissue "phase change" in the aggregation of fibroblasts and of Dictyostelium. This could improve our understanding of a variety of conditions where tissues apparently show a phase change in behavior including embryogenesis, cancer tumorigenesis, in vitro tissue formation, bacterial biofilm formation and a variety of collective phenomena in the microbial world. Identification of critical distance conditions will help with predicting the path of morphogenesis of 3D printed tissues with cells and extracellular matrix. The research will be carried out by a graduate and two undergraduate students. One of the UG students will be from the Department of Biology, the others from Engineering. Thus, the students will be trained in multidisciplinary fields. Special effort will be made to recruit students from minorities and under-represented groups to conduct the research.The project is inspired by recent experimental findings suggesting that muscle cells within 100 micrometers of each other in 3D culture compact the ECM and form myotubes (PI's lab), endothelial cells within 100 micrometers of each other form vasculature, fibroblasts in collagen within 100 micrometers of each other compact the collagen. When the cell-cell gap exceeds 100 micrometers, none of these processes occur. The hypothesis of a critical distance will be tested using fibroblasts from multiple species (mouse, monkey and human) in a collagen matrix mixed with fluorescent beads for optically tracking compaction and cluster formation by slime mold cells (Dictyostelium). In order to gain insight on the experimental observations of the threshold cell-cell gap, a mathematical model will be created to simulate the experimental observations. Each cell remodels the matrix around them by adhering to the matrix and by pulling the fibers as the cell filopodia generate contractile forces. Each cell forms a zone of remodeled matrix with higher stiffness under tension. If cells are far apart, their zones remain independent of each other. In a system with many cells, such non-interacting zones maintain global symmetry. When the cells are close to each other, their zones overlap, and symmetry is broken. The stiffness between the cells becomes higher, and the cells form a stiffer fibrous bridge between them. More filopodia tend to extend along the bridge compared to other directions, the cells become polarized and elongated while remaining contractile. This results in an attractive interaction between the cells that will compact the matrix. Three types of fibroblasts from mouse (embryonic fibroblast, NIH/3T3), monkey (kidney fibroblasts) and human (lung) will be used to test hypothesis on matrix compaction, and slime mold cells (Dictyostelium discoideum) will be used to test for generality. All of these cell types have comparable size, ~ 10 micrometer in diameter. Hence, we predict similar critical distances for all cell types.