Marinkovic Zoran S, Vulin Clément, Acman Mislav, Song Xiaohu, Di Meglio Jean Marc, Lindner Ariel B, Hersen Pascal
Laboratoire Matière et Systèmes Complexes, UMR 7057 CNRS & Université de Paris, 10 rue Alice Domon et Léonie Duquet, 75013 Paris, France.
U1284 INSERM and.
Bio Protoc. 2020 Jul 5;10(13):e3668. doi: 10.21769/BioProtoc.3668.
The natural environment of microbial cells like bacteria and yeast is often a complex community in which growth and internal organization reflect morphogenetic processes and interactions that are dependent on spatial position and time. While most of research is performed in simple homogeneous environments (, bulk liquid cultures), which cannot capture full spatiotemporal community dynamics, studying biofilms or colonies is complex and usually does not give access to the spatiotemporal dynamics at single cell level. Here, we detail a protocol for generation of a microfluidic device, the "yeast machine", with arrays of long monolayers of yeast colonies to advance the global understanding of how intercellular metabolic interactions affect the internal structure of colonies within defined and customizable spatial dimensions. With as a model yeast system we used the "yeast machine" to demonstrate the emergence of glucose gradients by following expression of fluorescently labelled hexose transporters. We further quantified the expression spatial patterns with intra-colony growth rates and expression of other genes regulated by glucose availability. In addition to this, we showed that gradients of amino acids also form within a colony, potentially opening similar approaches to study spatiotemporal formation of gradients of many other nutrients and metabolic waste products. This approach could be used in the future to decipher the interplay between long-range metabolic interactions, cellular development, and morphogenesis in other same species or more complex multi-species systems at single-cell resolution and timescales relevant to ecology and evolution.
像细菌和酵母这样的微生物细胞的自然环境通常是一个复杂的群落，其中生长和内部组织反映了形态发生过程以及依赖于空间位置和时间的相互作用。虽然大多数研究是在简单的均匀环境（如批量液体培养）中进行的，这种环境无法捕捉完整的时空群落动态，但研究生物膜或菌落很复杂，而且通常无法在单细胞水平上获取时空动态。在这里，我们详细介绍了一种微流控装置“酵母机器”的生成方案，该装置具有酵母菌落长单层阵列，以促进对细胞间代谢相互作用如何在定义的和可定制的空间维度内影响菌落内部结构的全面理解。以[具体酵母系统名称]作为模型酵母系统，我们使用“酵母机器”通过追踪荧光标记的己糖转运蛋白的表达来证明葡萄糖梯度的出现。我们进一步用菌落内生长速率和受葡萄糖可用性调节的其他基因的表达来量化表达空间模式。除此之外，我们还表明菌落内也会形成氨基酸梯度，这可能为研究许多其他营养物质和代谢废物产物梯度的时空形成开辟类似的方法。这种方法未来可用于在与生态和进化相关的单细胞分辨率和时间尺度上，解读其他同一物种或更复杂的多物种系统中远程代谢相互作用、细胞发育和形态发生之间的相互作用。
