Keppeke Gerson Dierley, Andrade Luís Eduardo C, Grieshaber Scott S, Chan Edward K L
Department of Oral Biology, University of Florida, 1395 Center Drive, Gainesville, FL 32610-0424 USA ; Rheumatology Division, Universidade Federal de São Paulo, Rua Botucatu 740, São Paulo, SP 04023-062 Brazil.
Rheumatology Division, Universidade Federal de São Paulo, Rua Botucatu 740, São Paulo, SP 04023-062 Brazil ; Immunology Division, Fleury Medicine and Health Laboratories, Avenida Gal Waldomiro Lima 508, São Paulo, SP 04102-050 Brazil.
Cell Biosci. 2015 Jan 5;5(1):1. doi: 10.1186/2045-3701-5-1. eCollection 2015.
Our laboratory previously reported interesting rods 3-10 μm long and rings 2-5 μm diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. In this study, we aimed to investigate the dynamic behavior of these RR in live cells.
RR were detected in >90% of COS-7 and HeLa cells treated with 1 mM ribavirin or 6-Diazo-5-oxo-L-norleucine (DON) for 24 h, and in 75% of COS-7 cells treated with 1 mM mycophenolic acid (MPA) for the same period of time. Microinjection of affinity-purified anti-IMPDH2 antibodies in live COS-7 cells treated with ribavirin, DON, or MPA showed mature forms of RR presented as stable and stationary structures in 71% of cells. In the remaining 29% of cells, RR acquired erratic movement and progressively disassembled into fragments and disappeared within 10 min. The specific stationary state and antibody-dependent disassembling of RR structures was independently confirmed in COS-7 and HeLa cells transfected with GFP-tagged IMPDH2.
This is the first demonstration of disassembly of RR structures upon microinjection of anti-IMPDH2 antibodies that led to the disappearance of the molecular aggregates. The disassembly of RR after microinjection of anti-IMPDH2 antibody further strengthens the notion that IMPDH2 are major building blocks of RR. Using two independent methods, this study demonstrated that the induced RR are primarily stationary structures in live cells and that IMPDH2 is a key component of RR.
我们实验室之前报道过在哺乳动物细胞的细胞质中存在长度为3 - 10微米的有趣杆状结构以及直径为2 - 5微米的环状结构（RR）。实验证据表明，肌苷 - 5'-单磷酸脱氢酶2（IMPDH2）和胞苷三磷酸合成酶（CTPS）都是RR结构的组成成分。包括小鼠胚胎干细胞在内的几种细胞类型，以及诸如小鼠3T3和大鼠NRK等细胞系，天然存在RR结构，而其他细胞在用干扰GTP/CTP生物合成途径的化合物处理时也可呈现RR结构。在本研究中，我们旨在研究这些RR在活细胞中的动态行为。
在用1 mM利巴韦林或6 - 重氮 - 5 - 氧代 - L - 正亮氨酸（DON）处理24小时的COS - 7和HeLa细胞中，超过90%检测到RR，在用1 mM霉酚酸（MPA）处理相同时间的COS - 7细胞中，75%检测到RR。在用利巴韦林、DON或MPA处理的活COS - 7细胞中显微注射亲和纯化的抗IMPDH2抗体，结果显示71%的细胞中成熟形式的RR呈现为稳定的静止结构。在其余29%的细胞中，RR出现不规则运动，并逐渐分解成片段，在10分钟内消失。在用绿色荧光蛋白标记的IMPDH2转染的COS - 7和HeLa细胞中，独立证实了RR结构的特定静止状态和抗体依赖性分解。
这是首次证明显微注射抗IMPDH2抗体后RR结构的分解导致分子聚集体消失。显微注射抗IMPDH2抗体后RR的分解进一步强化了IMPDH2是RR主要组成部分的观点。本研究使用两种独立方法证明，诱导产生的RR在活细胞中主要是静止结构，并且IMPDH2是RR的关键组成部分。
