Mechanisms of secretion studied with two-photon excitation imaging and uncaging

利用双光子激发成像和笼化化合物释放技术，研究了上皮细胞的胞吐、液分泌和突触功能。1)利用cage -Ca^<2+>化合物的双光子释放，我们开发了Ca^<2+>激活通道的功能映射，由于cage -Ca^<2+>本身引起的Ca^<2+>域效应，该通道具有非凡的空间分辨率。在小鼠胰腺腺泡制备中，我们发现Ca^<2+>-激活的Cl^-通道分布在根尖膜和基底膜，但在外侧膜中不存在。这种分布防止分泌到管腔的Cl^-被再吸收到侧膜，并允许Cl^-从基膜有效地单向运输到管腔。外侧膜缺乏功能性通道或转运体可能用于其他上皮运输，并支持啮齿动物胰腺液体分泌的推拉机制。2)我们还开发了一种新的成像应用程序，更多的方法是基于细胞外极性示踪剂的双光子成像（TEP成像）来可视化完整组织中的胞外分泌。TEP成像显示外分泌胰腺的连续胞吐，其中单个囊泡的Ω-shaped轮廓通过快速涂覆f -肌动蛋白而稳定维持。f -肌动蛋白包被损伤导致液泡形成，这是急性胰腺炎的早期征兆。3)我们发现，在豚鼠鼻腺中也发生了连续的胞外分泌，但与液体分泌的开始有很大的延迟。4) TEP成像显示完整朗格汉斯胰岛中单个胰岛素囊泡的胞吐，提示胰岛素囊泡融合孔从一开始就是由脂质构成的。5)我们开发了一种光学方法，利用笼状谷氨酸化合物的双光子释放，以亚微米空间分辨率刺激神经元。我们发现棘头体积与谷氨酸敏感性密切相关，且棘头增大是长期增强的基础。少

Exocytosis and fluid-secretion in epithelial cells and synaptic functions were studied with two-photon excitation imaging and uncaging of caged-compounds.1) We have developed functional mapping of Ca^<2+>-activated channels using two-photon uncaging of caged-Ca^<2+> compound, which has an extraordinary spatial resolution because of the Ca^<2+> domain effect caused by caged-Ca^<2+> itself. In mouse pancreatic acinar preparations, we found that Ca^<2+>-activated Cl^- channel distributed both in the apical and basal membranes, but was absent in the lateral membrane. This distribution prevents the reuptake of Cl^- secreted into the lumen to the lateral membrane, and allows effective unidirectional transport of Cl^-from the basal membrane into the lumen. The lack of functional channels or transporters in the lateral membrane might be utilized in other epithelial transports, and supports the push-pull mechanism of fluid secretion in rodent pancreas.2) We have also developed a new imaging app … More roach to visualize exocytosis in intact tissue based on two-photon imaging of extracellular polar tracer (TEP imaging). TEP imaging revealed the sequential exocytosis in exocrine pancreas, where Ω-shaped profiles of individual vesicles were stably maintained by rapid coating of the vesicles with F-actin. Impairment of F-actin coating resulted in vacuole formation, which is an early sign of acute pancreatitis.3) We have found that sequential exocytosis also occurred in the guinea pig nasal gland, but with a substantial delay from the onset of fluid secretion.4) TEP imaging visualized exocytosis of individual insulin vesicles in the intact islets of Langerhans, and suggested that the fusion pore of insulin vesicles is made of lipid from its very beginning.5) We have developed an optical approach to stimulate neurons with submicron spatial resolution using two-photon uncaging of a caged-glutamate compound. We found that spine-head volumes tightly correlated with glutamate sensitivity, and enlargement of spine head underlay long-term potentiation. Less