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成熟晶状体纤维细胞间大的岛状突起的形成依赖于肌动蛋白的原肌球蛋白1调节作用

Cheng C, Nowak RB, Biswas SK, et al.

期刊名称:Investigative ophthalmology & visual science

卷期:2016年第57卷第10期

摘要

目的:为了明确在晶状体纤维细胞的三细胞连接(顶点)中特殊的小的指状突起及大的岛状突起所必需的蛋白质。由于原肌球调节蛋白1 (Tmod1,一种F-肌动蛋白,尖锐的封端蛋白)缺失,我们建立一个新的方法进行免疫荧光染色单一晶状体纤维以及研究细胞形态学的变化。

方法:研究Tmod1+/+ 及Tmod1-/-单一成熟晶状体纤维的指状突起中F-肌动蛋白及F-肌动蛋白连接蛋白定位。

结果:F-肌动蛋白丰富的小的指状突起以及大的岛状突起与Tmod1+/+成熟纤维的细胞顶点一起出现。相反,Tmod1-/-纤维细胞缺乏正常的岛状区域,但是小的突起未受影响。在Tmod1+/+成熟纤维中,Tmod1、β2-膜收缩蛋白以及α-肌辅蛋白定位于岛间沟的大点中;但是在Tmod1-/-成熟纤维中,β2-膜收缩蛋白是分散的然而α-肌辅蛋白被重新分配到小突起及退化的岛状突起的基底。丝束蛋白及Arp3(肌动蛋白相关蛋白3)定位于小突起基底的点中,然而在Tmod1+/+ 及Tmod1-/-成熟纤维中,N-钙黏蛋白及埃兹蛋白均分布在细胞膜。

结论:与大的岛状突起相比,不同的F-肌动蛋白组织出现在小的突起。大的岛状区域的形成和/或维持依赖于通过Tmod1来稳定的β2膜收缩蛋白肌动蛋白网络。Tmod1的缺乏将增强α肌辅蛋白交叉连接F肌动蛋白束,这也提示细胞骨架组织的改变。Arp3分支及丝束蛋白连接的F-肌动蛋白网络可能辅助小突起的形成,而不依赖于Tmod1。这初步揭示了F肌动蛋白相关蛋白对于纤维间突起形成是必需的。

PURPOSE:To elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end-capping protein.METHODS:We investigated F-actin and F-actin-binding protein localization in interdigitations of Tmod1+/+ and Tmod1-/- single mature lens fibers.RESULTS:F-actin-rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1-/- mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1-/- mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1-/- mature fibers.CONCLUSIONS:These results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin-actin network stabilized by Tmod1. α-Actinin-crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin-associated proteins required for the formation of paddles between lens fibers.


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