Evidence that Proliferation of Golgi Apparatus Depends on Both De Novo Generation from the Endoplasmic Reticulum and Formation from Pre-Existing Stacks During the Growth of Tobacco BY-2 Cells
Evidence that Proliferation of Golgi Apparatus Depends on Both De Novo Generation from the Endoplasmic Reticulum and Formation from Pre-Existing Stacks During the Growth of Tobacco BY-2 Cells
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DOI：
10.1093/pcp/pct014
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发表时间：
2013-04-01
影响因子：
4.9
通讯作者：
Matsuoka, Ken
中科院分区：
文献类型：
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作者：
Abiodun, Moses Olabiyi;Matsuoka, Ken
在高等植物中，细胞质分布的高尔基堆积的数量因功能、年龄和细胞类型而异。目前尚不清楚高尔基数是如何控制的，细胞中所有高尔基体的功能是否相同，以及高尔基数的增加是内质网（ER）重新形成的结果还是原有堆栈的裂变的结果。烟草脯氨酰4-羟化酶（NtP4H1.1）是一种顺式高尔基定位型II型膜蛋白，用光敏荧光蛋白mKikGR（单体Kikume绿红）标记，在烟草亮黄2 （BY-2）细胞中表达。将转化后的细胞暴露在紫光下，使荧光从绿色变为红色。转换后的时间过程分析显示绿色点逐渐增加，红色点逐渐减少。3 ~ 6 h观察到高尔基体存在的红、黄、绿荧光点；高尔基体含有既存在又新合成的蛋白质；和新合成的高尔基体对细胞周期不同阶段高尔基体数量和荧光的分析表明，高尔基体数量的增加伴随着DNA的复制而发生分裂和重新合成。对不同抑制剂的研究表明，新的高尔基体的形成和高尔基体的产生都是由不同的机制介导的。这些结果和基于量化结果的建模表明，烟草BY-2细胞中的高尔基体并不均匀，并且表明内质网从头合成和高尔基分裂对增殖细胞的增加几乎相同。
In higher plants, the numbers of cytoplasmic-distributed Golgi stacks differ based on function, age and cell type. It has not been clarified how the numbers are controlled, whether all the Golgi apparatus in a cell function equally and whether the increase in Golgi number is a result of the de novo formation from the endoplasmic reticulum (ER) or fission of pre-existing stacks. A tobacco prolyl 4-hydroxylase (NtP4H1.1), which is a cis-Golgi-localizing type II membrane protein, was tagged with a photoconvertible fluorescent protein, mKikGR (monomeric Kikume green red), and expressed in tobacco bright yellow 2 (BY-2) cells. Transformed cells were exposed to purple light to convert the fluorescence from green to red. A time-course analysis after the conversion revealed a progressive increase in green puncta and a decrease in the red puncta. From 3 to 6 h, we observed red, yellow and green fluorescent puncta corresponding to pre-existing Golgi; Golgi containing both pre-existing and newly synthesized protein; and newly synthesized Golgi. Analysis of the number and fluorescence of Golgi at different phases of the cell cycle suggested that an increase in Golgi number with both division and de novo synthesis occurred concomitantly with DNA replication. Investigation with different inhibitors suggested that the formation of new Golgi and the generation of Golgi containing both pre-existing and newly synthesized protein are mediated by different machineries. These results and modeling based on quantified results indicate that the Golgi apparatuses in tobacco BY-2 cells are not uniform and suggest that both de novo synthesis from the ER and Golgi division contribute almost equally to the increase in proliferating cells.