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

当细胞数量足够大时，生物系统有时会迅速改变行为。这个探索性研究（EAGER）项目的早期概念资助是基于初步数据，这些数据表明，对于一种哺乳动物细胞来说，细胞之间的距离会触发不同的行为。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，它们就会自动形成肌肉“纤维”。当它们之间的距离超过100微米时，它们就不存在了。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伤口愈合和器官原始形态形成的基础。该项目将确定100微米的阈值是否也适用于小鼠、猴子和人类的成纤维细胞。作为对观察广度的测试，100微米的阈值也将使用Protista Dictyostelium进行测试，Protista Dictyostelium在某些条件下具有聚集成更大结构的特性。如果实验结果在不同细胞类型之间具有可比性，那么该项目将为成纤维细胞和盘基骨柱聚集中组织“相变”激活的阈值距离的存在提供重要支持。这可以提高我们对多种条件的理解，其中组织明显表现出行为的相变，包括胚胎发生、癌症肿瘤发生、体外组织形成、细菌生物膜形成和微生物世界中的各种集体现象。关键距离条件的识别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。将特别努力招收少数民族和代表性不足群体的学生进行研究。该项目的灵感来自最近的实验发现，表明在3D培养中，肌肉细胞彼此相距100微米，形成ECM并形成肌管（PI的实验室），内皮细胞彼此相距100微米，形成脉管，胶原蛋白中的成纤维细胞彼此相距100微米，形成胶原蛋白。当细胞-细胞间隙超过100微米时，这些过程都不会发生。临界距离的假设将在胶原基质中使用来自多个物种（小鼠、猴子和人类）的成纤维细胞进行测试，该基质中混合了荧光珠，用于光学跟踪黏菌细胞（Dictyostelium）的压实和簇形成。为了深入了解阈值细胞-细胞间隙的实验观察结果，将创建一个数学模型来模拟实验观察结果。当细胞丝状足产生收缩力时，每个细胞通过粘附基质和拉动纤维来重塑周围的基质。每个单元在张力作用下形成一个具有较高刚度的重塑矩阵区。如果细胞相距很远，它们的区域就保持彼此独立。在有许多细胞的系统中，这种不相互作用的区域保持全局对称性。当细胞彼此靠近时，它们的区域重叠，对称性被打破。细胞之间的硬度变得更高，细胞之间形成更硬的纤维桥。与其他方向相比，更多的丝状伪足倾向于沿桥延伸，细胞变得极化和伸长，同时保持收缩。这导致细胞之间的相互作用产生吸引力，从而使基质致密。将使用小鼠（胚胎成纤维细胞，NIH/3T3）、猴子（肾成纤维细胞）和人（肺）的三种成纤维细胞来验证基质压实的假设，并使用黏菌细胞（盘状盘状体）来验证其一般性。所有这些类型的细胞都有相似的大小，直径约为10微米。因此，我们预测所有细胞类型的临界距离相似。

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.