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Development of Evanescence Microscope and Test of the Quantal Hypothesis for Exocytosis

倏逝显微镜的研制及胞吐作用量子假说的检验

基本信息

批准号：
10557003
负责人：
TERAKAWA Susumu
金额：
$ 7.55万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Aiming at development of a user-friendly high-performance evanescence microscope based on an ultra-high NA lens, we compared three methods of introducing the illumination light to the microscope. A fiber-illumination method was advantageous in safety, field size, and easy handling, over a direct-illumination method, but was costly and slightly unstable. Using an evanescence microscope, we examined the exocytosis in chromaffin cells and beta cells. In both types of the cell, a fluorescent probe was released from vesicles with a flash response. The flash response was variable in amplitude depending on vesicles. It reflected water secretion from the vesicles. By laser-trap forcemetry, we demonstrated a small force exerted on water by the cell upon stimulation. The contents of vesicles are released not by a simply diffusion but by a water ejection from the vesicles. The strength of the ejecting force varied with vesicles as the density of Cl channels on the vesicle membrane examined by an immuno-staining technique varied. Cl channel blockers suppressed the flash response without suppressing the exocytosis. In beta cells, vesicle moved along the membrane immediately after its exocytotic response, suggesting a continuous release of vesicle contents. These findings are incompatible with the so-called quantal hypothesis. In endocrine cells, modulation for the secretory function is evolved in a highly complex manner so that fine adaptations to inner and outer environments are made possible.

为了开发基于超高NA透镜的用户友好型高性能消逝显微镜，我们比较了将照明光引入显微镜的三种方法。光纤照明方法在安全性、视野大小和易于操作方面优于直接照明方法，但成本高且稍不稳定。用渐逝光显微镜观察嗜铬细胞和β细胞的胞吐作用。在这两种类型的细胞中，荧光探针从囊泡中释放，并产生闪光反应。闪光反应的幅度是可变的，这取决于囊泡。它反映了水从囊泡分泌。通过激光捕获力谱，我们证明了细胞在刺激时对水施加的小力。囊泡的内容物不是通过简单的扩散而是通过囊泡中的水喷射而释放。喷射力的强度随囊泡的不同而变化，因为通过免疫染色技术检查的囊泡膜上的Cl通道的密度不同。Cl通道阻断剂抑制闪光反应，但不抑制胞吐作用。在β细胞中，囊泡在其胞吐反应后立即沿膜沿着移动，表明囊泡内容物的持续释放。这些发现与所谓的量子假说不相容。在内分泌细胞中，分泌功能的调节是以高度复杂的方式进化的，以便能够对内部和外部环境进行良好的适应。