Bazou Despina, Dowthwaite Gary P, Khan Ilyas M, Archer Charles W, Ralphs James R, Coakley W Terence
Cardiff University, School of Biosciences, Cardiff, Wales, UK.
Mol Membr Biol. 2006 Mar-Apr;23(2):195-205. doi: 10.1080/09687860600555906.
Particles or cells suspended in an appropriately designed ultrasound standing wave field can be aggregated at a node to form a single monolayer in a plane that can be interrogated microscopically. The approach is applied here to investigate the temporal development of F-actin and Cx43 distribution and of gap junctional intercellular communication in 2-D chondrocyte aggregates (monolayers) rapidly and synchronously formed and held in suspension in an ultrasound trap. Development of the F-actin cytoskeleton in the confluent single layer of 'cuboidal' cells forming the aggregate was completed within 1 h. Chondrocytes levitated in the trap synchronously formed functional gap junctions (as assessed by CMFDA dye transfer assays) in less than 1 h of initiation of cell-cell contact in the trap. It was shown that Cx43 gene expression was retained in isolated chondrocytes in suspension. Preincubation of cells with the protein synthesis inhibitor cycloheximide caused a six-fold decrease in Cx43 accumulation (as assessed by immunofluorescence) at the interfaces of chondrocytes in the aggregate. It is shown that the ultrasound trap provides an approach to studying the early stages of cytoskeletal and gap junction development as cells progress from physical aggregation, through molecular adhesion, to display the intracellular consequences of receptor interactions.
悬浮于精心设计的超声驻波场中的颗粒或细胞可在波节处聚集，在一个平面上形成单层，以便于进行显微镜观察。本文采用该方法，快速且同步地研究了二维软骨细胞聚集体（单层）中F-肌动蛋白和Cx43分布以及缝隙连接细胞间通讯的时间发展过程，这些聚集体在超声阱中悬浮形成并保持。在形成聚集体的“立方形”细胞融合单层中，F-肌动蛋白细胞骨架的发育在1小时内完成。在超声阱中，悬浮的软骨细胞在细胞-细胞接触开始不到1小时内同步形成功能性缝隙连接（通过CMFDA染料转移试验评估）。结果表明，悬浮的分离软骨细胞中保留了Cx43基因表达。用蛋白质合成抑制剂环己酰亚胺对细胞进行预孵育，导致聚集体中软骨细胞界面处的Cx43积累（通过免疫荧光评估）下降了六倍。结果表明，超声阱为研究细胞从物理聚集、分子黏附到展示受体相互作用的细胞内后果这一过程中细胞骨架和缝隙连接发育的早期阶段提供了一种方法。
