| Observing Nutrient Gradients, Gene Expression and Growth Variation Using the "Yeast Machine" Microfluidic Device
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Author:
Date:
2020-07-05
[Abstract] 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 (e.g., 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 ...
[摘要] [摘要 ] 微生物细胞（如细菌和酵母菌）的自然环境通常是一个复杂的社区，在该社区中，生长和内部组织反映了形态发生过程和相互作用，这些过程和相互作用取决于空间位置和时间。虽然大多数研究是在无法捕获完整时空群落动态的简单同质环境（例如，大量液体培养）中进行的，但研究生物膜或菌落却很复杂，通常无法在单个细胞水平上获得时空动态。在这里，我们详细介绍了一种用于生成微流控设备（“酵母机器”）的协议，该协议带有酵母菌落的长单层阵列，以推进对细胞间代谢相互作用如何影响已定义和可定制的空间尺寸内菌落内部结构的全球了解。以酿酒酵母作为模型酵母系统，我们使用“酵母机器”通过追踪荧光标记的己糖转运蛋白来证明葡萄糖梯度的出现。我们进一步量化了菌落内生长速率的表达空间模式和葡萄糖可利用性调控的其他基因的表达。除此之外，我们显示出氨基酸的梯度也在菌落内形成，潜在地打开了类似的方法来研究许多其他营养物和代谢废物的梯度的时空形成。该方法将来可用于在与生态学和进化有关的单细胞分辨率和时标下，破译其他相同物种或更复杂的多物种系统中的远程代谢相互作用，细胞发育和形态发生之间的相互作用。
[背景 ] ...
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| A Microfluidic Device for Massively Parallel, Whole-lifespan Imaging of Single Fission Yeast Cells
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Author:
Date:
2018-04-05
[Abstract] Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and incompatible with fluorescence microscopy. Here, we describe assembly and operation of the multiplexed-Fission Yeast Lifespan Microdissector (multFYLM), a high-throughput microfluidic device for rapidly acquiring single-cell whole-lifespan imaging. multFYLM captures approximately one thousand rod-shaped fission yeast cells from up to six different genetic backgrounds or treatment regimens. The immobilized cells are fluorescently imaged for over a ...
[摘要] 整个寿命的单细胞分析极大地增加了我们对细胞老化等基本细胞过程的理解。为了观察整个寿命期间的个体细胞，必须从生长培养基中移除所有后代，通常通过手动显微切割。然而，手动显微切割费力，低通量，并且与荧光显微镜不兼容。在这里，我们描述了多路复用裂变酵母寿命显微解剖器（multFYLM）的组装和操作，这是一种用于快速获取单细胞全寿命成像的高通量微流体装置。 multFYLM从多达六种不同的遗传背景或治疗方案中捕获约一千个杆状裂殖酵母细胞。将固定的细胞荧光成像超过一周，而将子代细胞从装置中取出。得到的数据集产生记录每个细胞复制寿命的高分辨率多通道图像。我们预计multFYLM将广泛适用于裂殖酵母（Schizosaccharomyces pombe）和其他对称分裂的单细胞生物的单细胞整个寿命研究。
【背景】细胞衰老导致细胞功能的累积下降，最终导致死亡。大多数关于细胞衰老的研究侧重于模型单细胞生物的复制寿命，例如出芽酵母酿酒酵母（Nyström和Liu，2014; Wasko和Kaeberlein，2014; Wierman和Smith，2014; Ruetenik和Barrientos ，2015）。细胞的复制寿命（RLS）被定义为母细胞在其生命过程中产生的女儿的数量（Henderson和Gottschling，2008; Sutphin等人，2014）。 ...
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