The Significance of Cytoskeleton in Exocytosis: Control of Apical Plasma Membrane-Evoked Amylase Release from Secretory Granules Isolated from the Rat Parotid Gland

ラット耳下腺から分離精製した頂端側形質膜と分泌顆粒からなるin vitro 開口分泌モデル系を用い、分泌時の細胞骨格系の役割を検討した。細胞分画で得られた両細胞内小器官の内、細胞骨格系タンパク質(チュブリン、アクチン)は頂端側形質膜に局在していた。開口分泌モデル系に微小管構造阻害剤のコルヒチンを添加するとアミラーゼ分泌活性が約2倍上昇した。この現象は蛍光プローブR18を用いた蛍光消光解消法による解析から膜融合の結果と一致していた。マイクロフィラメント構造阻害剤のサイトカラシンDでは活性上昇傾向は認められなかった。しかし、F-アクチンを添加すると分泌活性はほぼ100%抑制された。これらの結果は、唾液分泌時に細胞骨格系(マイクロフィラメント、微小管)は頂端側形質膜と相互作用をし、膜融合をともなう唾液分泌に直接、抑制的に作用していることが示唆された。一方、分泌顆粒膜との直接相互作用はほとんど無いと考えられた。To investigate the significance of cytoskeletal proteins in the exocytotic in vitro interaction system, we observed the effects of two types of inhibitors, colchicine and cytochalasin D, which depolymerize the microtuble structure consisting of tubulin and microfilament consisting of actins, respectively. The apical plasma membrane-evoked amylase release was increased approximately two times by colchicine. These findings suggest that secretory granules could interact with microtubles coexist via microfilament in the apical plasma membranes and the exocytotic membrane fusion might be suppressed by microtubules. When R18-labeled secretory granules were incubated with apical plasma membranes under the same conditions, de-quenching clearly occurred. In addition, de-quenching was amplified by the addition of colchicine. In the intact cells, colchicine may induce disassembly of the microtubule resulting the inhibition of granule transport to the apical plasma membrane by motor proteins. Based on in vitro observation, it is suggested that the microtubles, which also facilitate cross talk with actin, may modulate access of secretory granules to the apical plasma membrane. On the other hands, cytochalasin D, an inhibitor of actin polymerization, in the cell-free reconstitution system did not affect apical plasma membrane-evoked amylase release. When fiber actin (F-actin) was added to the apical plasma membrane-evoked amylase release system, it decreased amylase release in a concentration-dependent manner. Isolated apical plasma membranes contain microfilaments but the concentration may be not sufficient to function as a barrier secretory granule access. These findings suggest that simplification of the exocytotic interaction system allows observation the direct effects of the cytoskeleton and that cytoskeletal protein inhibitors induce suppressive modulation of exocytotic membrane fusion