Biomolecular Nanotechnology Center, Berkeley Sensor & Actuator Center, Department of Bioengineering, University of California-Berkeley, 408C Stanley Hall, CA 94720-1762, USA.
Integr Biol (Camb). 2010 Mar;2(2-3):130-8. doi: 10.1039/b919392h. Epub 2010 Feb 9.
Gap junctions are protein channels between cells that allow direct electrical and metabolic coupling via the exchange of biomolecules and ions. Their expression, though ubiquitous in most mammalian cell types, is especially important for the proper functioning of cardiac and neuronal systems. Many existing methods for studying gap junction communication suffer from either unquantifiable data or difficulty of use. Here, we measure the extent of dye spread and effective diffusivities through gap junction connected cells using a quantitative microfluidic cell biology platform. After loading dye by hydrodynamic focusing of calcein/AM, dye transfer dynamics into neighboring, unexposed cells can be monitored via timelapse fluorescent microscopy. By using a selective microfluidic dye loading over a confluent layer of cells, we found that high expression of gap junctions in C6 cells transmits calcein across the monolayer with an effective diffusivity of 3.4 x 10(-13) m(2)/s, which are highly coupled by Cx43. We also found that the gap junction blocker 18alpha-GA works poorly in the presence of serum even at high concentrations (50 microM); however, it is highly effective down to 2.5 microM in the absence of serum. Furthermore, when the drug is washed out, dye spread resumes rapidly within 1 min for all doses, indicating the drug does not affect transcriptional regulation of connexins in these Cx43+ cells, in contrast to previous studies. This integrated microfluidic platform enables the in situ monitoring of gap junction communication, yielding dynamic information about intercellular molecular transfer and pharmacological inhibition and recovery.
间隙连接是细胞间的蛋白质通道，通过生物分子和离子的交换允许直接的电和代谢偶联。尽管它们在大多数哺乳动物细胞类型中普遍表达，但对于心脏和神经元系统的正常功能尤其重要。许多现有的研究间隙连接通讯的方法要么数据不可量化，要么使用困难。在这里，我们使用定量微流控细胞生物学平台测量通过间隙连接连接的细胞中染料扩散的程度和有效扩散系数。在通过 calcein/AM 的流体动力学聚焦加载染料后，可以通过延时荧光显微镜监测染料进入相邻未暴露细胞的转移动力学。通过在细胞的汇合层上选择性地进行微流体染料加载，我们发现 C6 细胞中高表达的间隙连接以 3.4 x 10(-13) m(2)/s 的有效扩散系数将 calcein 传递穿过单层，这是由 Cx43 高度偶联的。我们还发现，即使在高浓度（50 microM）下，间隙连接阻滞剂 18alpha-GA 在存在血清的情况下也效果不佳；然而，在没有血清的情况下，它在低至 2.5 microM 的浓度下就非常有效。此外，当药物被冲洗掉时，所有剂量的染料扩散在 1 分钟内迅速恢复，表明药物不会影响这些 Cx43+细胞中连接蛋白的转录调节，与之前的研究相反。这种集成的微流控平台能够原位监测间隙连接通讯，提供关于细胞间分子转移和药理学抑制及恢复的动态信息。
