Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules
Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules
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DOI：
10.1371/journal.pbio.3000355
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发表时间：
2019-08-01
期刊：
影响因子：
9.8
通讯作者：
Nabi, Ivan R.
中科院分区：
文献类型：
--
作者：
Gao, Guang;Zhu, Chengjia;Nabi, Ivan R.
内质网（ER）是一个扩张的，膜封闭的细胞器，由光滑的外周小管和粗糙的，核糖体镶嵌的中央ER片组成，其形态部分地由ER形成蛋白，reticulon（RTN）和细胞色素连接膜蛋白63（CLIMP-63）分别决定。在这里，受激发射耗尽（STED）超分辨率显微镜显示，reticulon 4a（RTN 4a）和CLIMP-63也调节外周ER小管纳米结构域的组织和动力学。STED成像显示，内腔ER单体氧化环境优化的绿色荧光蛋白（ERmoxGFP）、膜Sec 61 β GFP、敲入钙网蛋白-GFP和抗体标记的ER驻留蛋白calnexin和derlin-1都定位于沿沿着外周ER小管长度的周期性斑点，这些斑点不容易通过衍射限制的共聚焦显微镜观察到。RTN 4a与内腔团块分离并限制内腔团块长度，而CLIMP-63与内腔团块长度相关并增加内腔团块长度。RTN 4a和CLIMP-63还调节ER驻留蛋白的纳米结构域分布，这是钙连接蛋白和derlin-1斑点优先分离远离内腔ERmoxGFP斑点所需的。高速（40 ms/帧）活细胞STED成像显示RTN 4 a和CLIMP-63调节沿沿着外周ER小管的动态纳米级内腔区室化。RTN 4 a增强和CLIMP-63破坏内腔ERmoxGFP在沿ER小管沿着空间限定的位点的局部积累。因此，ER成形蛋白RTN和CLIMP-63调节内腔ER纳米结构域异质性、与ER驻留蛋白的相互作用以及外周ER小管中的动力学。
The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon (RTN) and cytoskeleton-linking membrane protein 63 (CLIMP-63), respectively. Here, stimulated emission depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), membrane Sec61 beta GFP, knock-in calreticulin-GFP, and antibody-labeled ER-resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER-resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER-shaping proteins RTN and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER-resident proteins, and dynamics in peripheral ER tubules.