Cholinergic stimulation of lacrimal acinar cells promotes redistribution of membrane-associated kinesin and the secretory protein, beta-hexosaminidase, and increases kinesin motor activity
Cholinergic stimulation of lacrimal acinar cells promotes redistribution of membrane-associated kinesin and the secretory protein, beta-hexosaminidase, and increases kinesin motor activity
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DOI：
10.1006/exer.1996.0198
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发表时间：
1997-02-01
影响因子：
3.4
通讯作者：
Mircheff, AK
中科院分区：
文献类型：
--
作者：
HammAlvarez, SF;DaCosta, S;Mircheff, AK
以微管为基础的运动蛋白在静息和刺激的兔泪腺腺泡细胞膜转运中的作用已被研究。用微管亲和纯化的方法从对照和氨甲酰胆碱处理的腺泡提取物中分离出微管依赖的马达，并用视频增强的差示干涉对比显微镜测定其微管滑动的活性。通过微管亲和纯化和GTP释放分离得到的组分中，卡巴胆碱处理使依赖GTP的微管滑行频率增加2.2倍，这表明激动素是卡巴胆碱诱导的刺激的靶点。通过在山梨醇密度梯度上分离静息细胞的膜，然后在葡聚糖-聚乙二醇双相体系中进行分配分析，发现膜相关的动蛋白与高尔基体衍生的膜、高尔基体后分泌室、反高尔基网状室的膜、内质网和一组可能含有组织蛋白B的溶酶体膜共分布，经氨甲酰胆碱处理的腺泡蛋白的类似分离表明，刺激引起膜相关动蛋白、分泌酶β-己糖苷酶和半乳糖基转移酶的重新分布，似乎反映了高尔基复合体内的重组和物质从分泌途径返回高尔基复合体。我们的研究发现，卡巴胆碱促进泪液腺泡动蛋白的激活以及分泌途径中动蛋白-膜结合的重大变化，这表明动蛋白在泪腺的分泌囊泡组装、顶端分泌和/或分泌囊泡膜循环中起主要作用。(C)1997年学术出版社有限公司。
The role of the microtubule-based motor, kinesin, in membrane trafficking has been investigated in resting and stimulated acinar cells from rabbit lacrimal gland, a cholinergically controlled secretory tissue. Microtubule-dependent motors from extracts of control and carbachol-treated acini were isolated by microtubule-affinity purification and their activity was determined using a video-enhanced differential interference contrast microscopy assay for microtubule gliding. The observation that carbachol treatment resulted in a 2.2-fold stimulation of the frequency of GTP-dependent microtubule gliding in fractions isolated by microtubule-affinity purification and GTP release suggested that kinesin was a target of carbachol-induced stimulation. Resolution of membranes from resting cells by fractionation on a sorbitol density gradient followed by partitioning analysis in a dextran-polyethyleneglycol two-phase system revealed that membrane-associated kinesin codistributed with Golgi-derived membranes, a post-Golgi secretory compartment designated Hex1, membranes from a trans Golgi network-like compartment, endoplasmic reticulum and a group of putative lysosomal membranes containing cathepsin B. Comparable fractionation of carbachol-treated acini showed that stimulation caused redistributions of membrane-associated kinesin, the secretory enzyme beta-hexosaminidase, and galactosyltransferase that appeared to reflect both a reorganization within the Golgi complex and a return of material to the Golgi complex from the secretory pathway. Our findings that carbachol promotes activation of lacrimal acinar kinesin as well as major shifts in kinesin-membrane association within the secretory pathway suggests that kinesin plays a major role in secretory vesicle assembly, apical secretion, and/or secretory vesicle membrane recycling in the lacrimal gland. (C) 1997 Academic Press Limited.