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

本研究采用双光子激发成像技术和笼状化合物的释放技术研究了上皮细胞的胞吐、液体分泌和突触功能：1）利用笼状Ca ^<2+>化合物的双光子释放技术，建立了Ca^<2+>激活通道的功能成像，由于笼状Ca ^<2 +>自身引起的Ca^<2 +>结构域效应，该技术具有很高的空间分辨率。在小鼠胰腺腺泡标本中，我们发现Ca^2+激活的Cl^-通道分布于顶膜和基底膜，但在侧膜中不存在。这种分布阻止了分泌到腔中的Cl^-被再摄取到侧膜，并允许Cl^-从基膜有效地单向转运到腔中。侧膜中功能通道或转运蛋白的缺乏可能被用于其他上皮转运，并支持啮齿动物胰腺中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 ...更多信息 roach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吐作用。TEP成像揭示了胰腺外分泌中的连续胞吐作用，其中单个囊泡的Ω形轮廓通过用F-肌动蛋白快速包被囊泡而稳定维持。F-肌动蛋白包被的损伤导致空泡形成，这是急性胰腺炎的早期症状。3）我们发现豚鼠鼻腺中也发生了连续的胞吐作用，但与液体分泌的开始相比有相当大的延迟。4）TEP成像可视化了完整胰岛中单个胰岛素囊泡的胞吐作用，并提出胰岛素囊泡的融合孔是由脂质从其本身的脂肪。5）我们已经开发了一种光学方法来刺激神经元亚微米空间分辨率使用双光子uncaging的笼状谷氨酸化合物。我们发现，脊髓头部的体积与谷氨酸敏感性密切相关，脊髓头部的扩大与长时程增强密切相关。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