Hemidesmosome protein dynamics in live epithelial cells
Hemidesmosome protein dynamics in live epithelial cells
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DOI：
10.1002/cm.10089
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发表时间：
2003-02-01
影响因子：
--
通讯作者：
Jones, JCR
中科院分区：
文献类型：
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作者：
Tsuruta, D;Hopkinson, SB;Jones, JCR
半粒粒介导基底膜区上皮细胞对层粘连蛋白-5的稳定锚定，并被比作点焊。事实上，人们一直认为半半粒不是动态的，至少与其他基质粘附位点（包括局灶接触）相比是如此。我们通过监测绿色荧光蛋白（GFP）标记的人整合素β 4亚基（GFP- hβ 4）和GFP标记的180-kD人大疱性类天疱疮（BP）自身抗原（GFP- bp180）在804G细胞活培养物中的命运来验证这一观点，804G细胞聚集了许多成熟半粒。在亚融合的804G细胞中，gfp - hbet4和GFP-BP180蛋白簇都不稳定，而是以相对较快的速度组装和拆卸成猫爪状阵列。在804G细胞的融合群体中，虽然gfp - hbet4和GFP-BP180的一些猫爪状簇在100 - 60分钟内保持稳定，但其他gfp - hbet4和GFP-BP180蛋白阵列在同一时间段内形成和/或消失。此外，单个标记的颗粒在膜平面上表现出相当大的运动性。光漂白分析后的荧光恢复提供了半脂小体蛋白动力学的进一步指示。特别是，在融合细胞中，漂过的GFP-hbeta4蛋白团簇在大约30分钟内恢复信号，这表明沿细胞底物附着表面聚集的蛋白质阵列中的半粒酶组分的周转相对较快。回收率取决于一个完整的微丝系统。与此形成鲜明对比的是，在融合细胞中漂过的GFP-BP180蛋白簇即使观察超过60分钟也无法恢复信号。为了评估运动细胞中的半粒酶蛋白动力学，我们在体外监测了804G细胞中gfp - hbet4和GFP-BP180。在这些缺乏成熟半粒粒的迁移细胞中，整合素β 4亚基和BP180蛋白团逐渐组装和拆卸成线性和猫爪阵列。总之，半粒酶蛋白团簇，就像它们在局灶接触中的对应物一样，是动态的。我们讨论这些结果与半染色体功能的关系。
Hemidesmosomes mediate stable anchorage of epithelial cells to laminin-5 in the basement membrane zone and have been likened to spot-welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)-tagged human integrin beta4 subunit (GFP-hbeta4) and GFP-tagged 180-kD human bullous pemphigoid (BP) autoantigen (GFP-BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP-hbeta4 and GFP-BP180 protein clusters are not stable but assemble into and disassemble out of cat paw-like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw-like clusters of both GFP-hbeta4 and GFP-BP180 are stable over periods of >60 min, other GFP-hbeta4 and GFP-BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP-hbeta4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP-BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP-hbeta4 and GFP-BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin beta4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat-paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions.