Resolving the Mechano-Chemical Regulation of Microbial Populations in Microfluidic Devices

 描述(申请人提供)：当细胞生长和分裂形成密集的群体时，它们在化学和物理上都相互作用。例如，生长中的肿瘤或微生物/真菌生物膜中的细胞竞争营养和空间，从而相互施加化学和物理压力。尽管近年来已经发现了哺乳动物组织中决定命运和生长速度的机械调节的隐藏层，但对于机械约束对单细胞微生物的影响知之甚少，这主要是由于缺乏适当的培养技术。拟议研究的目的是通过量化空间受限微生物群落对明确定义的化学和物理压力的细胞和多细胞反应来填补这一空白。为此，P.I.提出了严格控制的微流体实验和新颖的生物物理模拟和理论，以弥合单个细胞和整个种群之间在时空尺度上的差距。这项拟议的研究利用理论和实验之间的持续反馈，根据单个细胞的联合行动，实现对微生物种群中的自组织的预测性理解。这些结果将极大地促进我们对生物膜形成的时空方面的理解，并具体阐明细胞群体如何对物理和化学线索的组合做出反应，这是合理设计策略以对抗微生物和真菌生物膜生长并限制它们进化耐药性的能力的关键。此外，计划中的新型微流控装置和计算机模拟将对生物物理界具有广泛的实用价值，以实现解剖微生物种群的集体性质的目标。私家侦探有三个明确的目标。首先，他将开发一种新的微流控培养设备设计--一种可以严格控制化学和机械条件的微流控机械恒化器。其次，他将结合生物物理建模来探索细胞对机械力化学线索的反应，首先将重点放在单细胞真菌和细菌上。第三，他将根据微流体种群的测量结果进行外推，开发理论和模拟，根据单个细胞的联合作用来预测种群的行为。目标1使用最先进的微流控技术，以突破缺乏物理控制的微流控培养设备的限制。目标2的实验方法是基于对微流体室中细胞的自动时空跟踪和报告基因表达变化的荧光标记。为Aim 3开发的模拟将现代种群生物学理论与物理场和化学场的分子动力学相结合。

 DESCRIPTION (provided by applicant): When cells grow and divide to form dense populations, they interact both chemically and physically. For instance, cells in growing tumors or microbial/fungal biofilms compete for nutrients and space, thereby exerting chemical and physical stresses on each other. Although recent years have uncovered a previously hidden layer of mechanical regulation of fate determination and growth rates in mammalian tissues, little is known about the consequences of mechanical constraints on single-celled microbes, largely, due to a lack of appropriate culturing techniques. The objective of the proposed research is to fill this gap by quantifying the cellular and multi- cellular response of spatially confined microbial communities to well-defined chemical and physical stresses. To this end, the P.I. proposes tightly-controlled microfluidic experiments and novel biophysical simulations and theory that bridges the gap in spatio-temporal scales between single cells and entire populations. The proposed research leverages a continual feedback between theory and experiments to achieve a predictive understanding of self-organization in microbial populations in terms of the joint actions of individual cells. The results will significantly advance our understanding of spatio-temporal aspects of biofilm formation, and elucidate specifically how cellular populations respond to combinations of physical and chemical cues, which is key to the rational design of strategies to battle microbial and fungal biofilm growth and to limit their abilty to evolve drug resistance. Further, the planned novel microfluidic devices and computer simulations will be of broad utility to the biophysics community for the goal of dissecting collective properties of microbial populations. The P.I. has three specific aims. First, he will develop a novel design for microfluidic culturing devices, a microfluidic mechano-chemostat, in which chemical and mechanical conditions can be tightly controlled. Second, he will use this device in conjunction with biophysical modeling to explore cellular response to mechano-chemical cues, focusing at first on single-celled funghi and bacteria. Third, extrapolating from microfluidic population measurements, he will develop theory and simulations to predict the behavior of populations from the joint action of individual cells. Aim 1 uses state-of-the-art microfluidic techniques to transcend the limitations of microfluidic culturing devices, which lack physical control. The experimental approaches to Aim 2 are based on automated spatio-temporal tracking of cells in microfluidic chambers and fluorescence markers reporting changes in gene expression. The simulations developed for Aim 3 synthesize modern population biology theory with the molecular dynamics of physical and chemical fields.