Elucidating the principles of the molecular organization of heteropolymeric tight junction strands.

Elucidating the principles of the molecular organization of heteropolymeric tight junction strands.
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DOI:
10.1007/s00018-011-0680-z
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发表时间:
2011-12
影响因子:
8
通讯作者:
Blasig, Ingolf E.
Blasig, Ingolf E.
中科院分区:
生物学1区
文献类型:
--
作者:
Piontek, Joerg;Fritzsche, Susanne;Cording, Jimmi;Richter, Sandra;Hartwig, Jens;Walter, Maria;Yu, Dan;Turner, Jerrold R.;Gehring, Claudia;Rahn, Hans-Peter;Wolburg, Hartwig;Blasig, Ingolf E.

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组织的细胞旁屏障特性主要由克劳丁异聚体的组成所决定。为了分析紧密连接(TJ)的分子结构,我们研究了克劳丁(Cld)形成同聚体和异聚体的能力。对在脑屏障中表达的Cld1、-2、-3、-5和 -12进行了研究。通过对HEK293细胞进行克劳丁转染来重建TJ链。通过链内外的荧光共振能量转移分析顺式相互作用和/或空间邻近性,并排序为:Cld5/Cld5 > Cld5/Cld1 > Cld3/Cld1 > Cld3/Cld3 > Cld3/Cld5,无Cld3/Cld2。经典的Cld1、-3和 -5,但非经典的Cld12显示出同嗜性反式相互作用。冷冻断裂电子显微镜显示,与经典克劳丁不同,黄色荧光蛋白标记的Cld12不形成同聚体。在不同单转染细胞的共培养中分析了异嗜性反式相互作用。Cld3/Cld5的反式相互作用不如Cld3/Cld1、Cld5/Cld1、Cld5/Cld5或Cld3/Cld3明显。通过一种新的成像分析证明了重建的TJ链的屏障功能。生成了一个TJ分子结构的模型。
Paracellular barrier properties of tissues are mainly determined by the composition of claudin hetero-polymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis-Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5 > Cld5/Cld1 > Cld3/Cld1 > Cld3/Cld3 > Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans-interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans-interactions were analyzed in cocultures of differently monotransfected cells. trans-Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.
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