Controlling gas/liquid exchange using microfluidics for real-time monitoring of flagellar length in living Chlamydomonas at the single-cell level
Controlling gas/liquid exchange using microfluidics for real-time monitoring of flagellar length in living Chlamydomonas at the single-cell level
复制标题
使用微流体控制气体/液体交换，在单细胞水平上实时监测活衣藻的鞭毛长度
DOI：
10.1039/c2lc40638a
复制
发表时间：
2012-01-01
期刊：
影响因子：
6.1
通讯作者：
Luo, Guoan
中科院分区：
文献类型：
--
作者：
Ai, Xiaoni;Liang, Qionglin;Luo, Guoan
莱茵衣藻被广泛用于研究纤毛/鞭毛，这些细胞器对人类健康和疾病至关重要。由于传统方法耗时的培养基更换以及需要对整个细胞进行固定，在单个活细胞中原位监测鞭毛组装/拆卸动力学一直很困难。在此，我们开发了一种聚二甲基硅氧烷/玻璃混合微流控装置，用于实时追踪莱茵衣藻单个活细胞中的鞭毛长度。通过连续的气 - 液塞可精确控制培养基更换，并且在数秒内即可完成完全的培养基替换。快速的培养基更换能够捕捉到瞬时的鞭毛动态。我们发现莱茵衣藻细胞对酸性培养基更换有反应并会去鞭毛化。然而，两条鞭毛可能不同步脱落。在随后更换培养基后，细胞会重新长出全长的鞭毛。细胞在受到细胞外刺激后也会被诱导缩短其鞭毛。芯片上整个细胞群体的鞭毛再生和拆卸的长期动力学与传统方法相当；然而，单个细胞表现出不均匀的反应动力学。我们还发现鞭毛生长速率取决于鞭毛长度。该装置为持续监测与鞭毛长度变化相关的分子活动以及捕捉鞭毛丢失和鞭毛组装/拆卸起始时的瞬时分子变化提供了一个潜在的平台。
Chlamydomonas reinhardtii is widely used for studying cilia/flagella, organelles important for human health and disease. In situ monitoring of flagellar assembly/disassembly kinetics in single living cells has been difficult with conventional methods because of time-consuming media exchange and the requirement of whole cell fixation. Here, we develop a PDMS/glass hybrid microfluidic device for real-time tracking of flagellar length in single living cells of Chlamydomonas. Media exchange is precisely controlled by sequential gas-liquid plugs and complete medium replacement occurs within seconds. Rapid medium exchange allows the capture of transient flagellar dynamics. We show that Chlamydomonas cells respond to acidic medium exchange and deflagellate. However, the two flagella may shed asynchronously. After subsequent medium exchange, cells regenerate full-length flagella. Cells are also induced to shorten their flagella after being exposed to extracellular stimuli. The long-term kinetics of flagellar regeneration and disassembly for the whole cell population on the chip are comparable to those from conventional methods; however, individual cells display non-uniform response kinetics. We also find that flagellar growth rate is dependent on flagellar length. This device provides a potential platform to continuously monitor molecular activities associated with changes in flagellar length and to capture transient molecular changes upon flagellar loss, and initiation of flagellar assembly/disassembly.