Cellular localization of RNA degradation and processing components in Escherichia coli.
Cellular localization of RNA degradation and processing components in Escherichia coli.
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DOI：
10.1007/978-1-4939-2214-7_6
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发表时间：
2015-01-01
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影响因子：
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通讯作者：
Taghbalout, Aziz
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文献类型：
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作者：
Arluison, Veronique;Taghbalout, Aziz
研究细菌（如大肠杆菌）微小细胞内蛋白质的定位和组织的能力，为原核细胞生物学的一个新的令人兴奋的时代铺平了道路。在原核细胞中发现了以前未被认识到的蛋白质时空和超分子组织水平，而原核细胞一直被认为是一个“酶袋”。“免疫荧光（IF）显微镜，涉及用荧光标记的抗体对天然蛋白质进行细胞免疫染色，相对费力，需要细胞固定和高度特异性的抗体。然而，IF显微镜允许本地化研究的天然蛋白质表达到其正常的细胞水平，而不是标记蛋白质与大的荧光标签，可以改变蛋白质丰度依赖于mRNA和/或蛋白质的稳定性的变化，或其检测可能需要标记的蛋白质的过表达。此外，当针对天然蛋白质的抗体不可用或缺乏特异性时，表位标签如血凝素（HA）或Flag可用于标记染色体表达的蛋白质。短的Flag和HA标签，8或9个氨基酸，不太可能干扰蛋白质的定位或功能。我们描述和讨论在这里使用荧光显微镜测定的蛋白质组分的细胞组织的E。coli RNA加工和降解机制。我们目前的例子，细胞组织模式可视化的光显微镜，无论是通过IF显微镜的本地和表位标记的蛋白质在固定的细胞，或荧光标记的蛋白质在活细胞。
The ability to study the localization and organization of proteins within the tiny cells of bacteria, such as Escherichia coli, has paved the way for a new and exciting era of prokaryotic cellular biology. Previously unrecognized levels of spatiotemporal and supramolecular organization of proteins have been revealed within the prokaryotic cell that had long been assumed as a "bag of enzymes." Immunofluorescence (IF) microscopy, which involves cellular immunostaining of native proteins with fluorescently labeled antibodies, is relatively laborious and requires cell fixation and highly specific antibodies. However, IF microscopy allows localization studies of native proteins expressed to their normal cellular levels, as opposed to labeling proteins with large fluorescent tag that can alter protein abundance dependent on changes in mRNAs and/or proteins stability, or whose detection can require overexpression of labeled proteins. In addition, when antibodies against native proteins are not available or lack specificity, epitope tags such as hemagglutinin (HA) or Flag can be used to label chromosomally expressed proteins. The short Flag- and HA-tag, eight or nine amino acids, are unlikely to interfere with the localization or function of the proteins. We describe and discuss here the use of fluorescence microscopy for determination of cellular organization of protein components of the E. coli RNA processing and degradation machinery. We present examples of cellular organization patterns visualized by light microscopy, either by IF microscopy of native and epitope-tagged proteins in fixed cells, or by fluorescence labeling of the proteins in live cells.