Leonova E V, Raphael Y
Kresge Hearing Research Institute, Department of Otolaryngology, The University of Michigan, Ann Arbor 48109-0506, USA.
Hear Res. 1999 Apr;130(1-2):137-54. doi: 10.1016/s0378-5955(99)00004-0.
We adapted a method of platinum replica to study the cytoskeleton of isolated cells of the guinea pig organ of Corti. This technique combined high image resolution with the ability to visualize the three-dimensional organization of the cytoskeleton of a whole cell. The procedure includes: isolation of hair cells and supporting cells using collagenase digestion, attachment of the cells to a coverslip, detergent extraction, chemical fixation, critical point drying, platinum/carbon coating, and transmission electron microscopy analysis. By using the method of platinum replica, we confirmed the existence of structural domains in the cortical lattice of outer hair cells. Based on the analysis of the partly destroyed cortical lattice, we propose that circumferential filaments are underlined with a thin flexible network. In addition, we established that the base of each stereocilium had a cone-like expansion of actin filaments and was surrounded by a thin bundle of filaments. We also produced replicas of the protrusion of the cuticular plate into the cytoplasm (infracuticular network) and the reticular lamina cytoskeleton. Our data indicated that the platinum replica method is useful for studying structural interactions among different cytoskeletal elements in the reticular lamina, as well as the cortex of outer hair cells and the cytoskeleton of supporting cells.
我们采用了一种铂复型方法来研究豚鼠柯蒂氏器分离细胞的细胞骨架。该技术将高图像分辨率与可视化整个细胞细胞骨架三维结构的能力相结合。该程序包括：使用胶原酶消化分离毛细胞和支持细胞，将细胞附着到盖玻片上，去污剂提取，化学固定，临界点干燥，铂/碳涂层，以及透射电子显微镜分析。通过使用铂复型方法，我们证实了外毛细胞皮质晶格中结构域的存在。基于对部分破坏的皮质晶格的分析，我们提出圆周细丝下面有一个薄的柔性网络。此外，我们确定每个静纤毛的基部有肌动蛋白丝的锥形扩张，并被一束细丝包围。我们还制作了角质板突入细胞质（表皮下网络）和网状板细胞骨架的复制品。我们的数据表明，铂复型方法可用于研究网状板中不同细胞骨架元件之间的结构相互作用，以及外毛细胞的皮质和支持细胞的细胞骨架。
