An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria
An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria
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DOI：
10.1128/mbio.02117-14
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发表时间：
2015-01-01
期刊：
影响因子：
6.4
通讯作者：
Schueler, Dirk
中科院分区：
文献类型：
--
作者：
Borg, Sarah;Popp, Felix;Schueler, Dirk
由于它们的小尺寸和增强的稳定性，来源于骆驼科动物的纳米抗体先前已被用于构建细胞内“纳米陷阱”，其能够在活的植物和动物细胞内重定向和操纵绿色荧光蛋白（GFP）标记的靶标。通过利用磁性细菌Magnetoelllum gryphiswaldense的细胞内区室化，我们证明了蛋白质甚至整个细胞器也可以通过多功能纳米陷阱技术在原核细胞内重新定位。磁小体上多价GFP结合纳米抗体的表达异位地将趋化蛋白CheW（1）-GFP从极性化学感受器簇募集到中细胞，导致对需氧性的逐渐敲低。相反，整个磁小体链可以从中间细胞重新定向，并拴在细胞的一个极上。类似的方法可能用于构建合成的细胞结构和靶向蛋白敲除在其他bacteri.Importance Intrabodies通常用于真核系统的细胞内分析和不同的亚细胞区室内的蛋白质的操作。特别地，所谓的纳米抗体对于合成生物学方法具有巨大的潜力，因为它们可以容易地在异源宿主中表达，并且例如通过在活的动物和植物细胞中构建细胞内“纳米陷阱”而与细胞内靶标积极地相互作用。虽然原核细胞也表现出相当程度的细胞内组织，但几乎没有与真核生物中使用的成熟方法等同的工具。在这里，我们证明了异位重定向和耗尽极性膜蛋白和整个细胞器的不同隔室中的趋磁细菌，导致磁趋气性逐渐击倒。这种细胞内纳米陷阱方法有可能应用于其他细菌中，用于构建合成细胞结构，操纵蛋白质功能，并产生逐步的靶向敲除。我们的研究结果为普遍使用荧光标记蛋白质作为纳米陷阱的目标来完成这些任务提供了原则证明。
Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular "nanotraps," which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW(1)-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria.IMPORTANCE Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular " nanotraps" in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks.