首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Direct immunogold labeling of aquaporin-4 in square arrays ofastrocyte and ependymocyte plasma membranes in rat brain and spinalcord
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Direct immunogold labeling of aquaporin-4 in square arrays ofastrocyte and ependymocyte plasma membranes in rat brain and spinalcord

机译:直接免疫金标记水通道蛋白4的方形阵列大鼠和脊髓中的星形胶质细胞和表皮细胞质膜脐带

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摘要

Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freeze-fracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed immunogold labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled ≈33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasmamembrane that were devoid of square arrays. Based on this evidence thatAQP4 is concentrated in glial square arrays, freeze-fracture methodsmay now provide biophysical insights regarding neuropathological statesin which abnormal fluid shifts are accompanied by alterations in theaggregation state or the molecular architecture of square arrays.
机译:水通道蛋白(AQP)水通道在大脑和脊髓中丰富,据信AQP1和AQP4在水代谢和渗透调节中起主要作用。最近对大脑进行的免疫细胞化学分析显示,AQP4具有高度极化的分布,并在星形细胞末端的毛细血管周围形成胶质限脂的末端表达显着。但是,AQP4的结构组织仍然未知。在冷冻断裂的复制物中,星形胶质细胞的末端包含大量平行于AQP4分布的膜内颗粒的正方形阵列。为了确定星形胶质细胞和表皮细胞正方形阵列是否包含AQP4,我们采用SDS洗涤的冷冻断裂复制品的免疫金标记和组织结合的立体确认。 AQP4抗体直接标记了大鼠脑和脊髓星形胶质细胞和表皮细胞质膜中约33%的正方形阵列。总体而言,正方形阵列下方有84%的标签; 11%的粒子簇类似于固定或分裂过程中已改变的正方形阵列; 5%位于神经胶质血浆的更大区域之下没有正方形阵列的膜。基于这一证据,AQP4集中在胶质方阵中,采用冷冻断裂法现在可以提供有关神经病理状态的生物物理学见解其中异常的流体转移伴随着聚集状态或正方形阵列的分子结构。

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