Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins.
Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins.
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DOI：
10.1371/journal.pgen.1006116
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发表时间：
2016-06
期刊：
影响因子：
4.5
通讯作者：
Graumann PL
中科院分区：
文献类型：
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作者：
Dempwolff F;Schmidt FK;Hervás AB;Stroh A;Rösch TC;Riese CN;Dersch S;Heimerl T;Lucena D;Hülsbusch N;Stuermer CA;Takeshita N;Fischer R;Eckhardt B;Graumann PL
生物膜已被提议含有特定脂质成分的微域，其中聚集了不同的蛋白质组。 Flotillin 样蛋白在原核生物和真核生物之间是保守的，在几种真核细胞和细菌细胞中发挥重要功能，并在脊椎动物中定义了一种所谓的耐去污剂微结构域。使用 STED 显微镜，我们发现两种细菌 flotillins FloA 和 FloT 在模型细菌枯草芽孢杆菌中形成平均直径为 85 至 110 nm 的明确组件。有趣的是，flotillin 微域在真核细胞中具有相似的大小。 FloA 的可溶性结构域在体外形成高达数百 kDa 的高阶寡聚体，这表明与真核 Flotilins 一样，细菌组装部分基于其自我寡聚的能力。然而，枯草芽孢杆菌旁系同源物表现出显着不同的扩散速率，因此不会共定位到共同的微域中。对细菌中的弗洛替林蛋白和其他耐去垢剂蛋白质进行的双色延时实验表明，蛋白质共定位的时间不超过几百毫秒，并且不会一起移动。我们的数据表明，细菌膜包含确定大小的蛋白质结构域，而不是依赖于弗洛林素的功能性微结构域。基于其独特的动力学，FloA 和 FloT 赋予空间可区分的活性，但不充当分子支架。许多膜蛋白在生物膜内分布不均匀，并且可能更喜欢特定的脂质环境才能发挥最佳功能。使用超分辨率荧光显微镜，我们发现几种枯草芽孢杆菌膜蛋白确实聚集成 60 至 110 nm 的结构，验证了确定大小的蛋白质微结构域的存在。特定膜蛋白和flotillins（真核细胞和细菌细胞之间高度保守的蛋白质家族）的生化共分离表明，存在常见的“功能性”微结构域，其中包含所谓的“耐去垢剂”膜蛋白，以flotillins为中心。通过对枯草芽孢杆菌 FloA 和 FloT 的高速跟踪，我们发现这两种蛋白质并不存在于同一微域中，而是以不同的速度穿过膜。双色延时显微镜显示，与脊椎动物的flotillins相反，细菌flotillins不会与去污剂抗性蛋白质一起移动，从而排除了共簇的存在。两种flotillins的缺乏，而不是单一一种的缺乏，会导致细胞形状和细胞生长的显着缺陷，表明flotillin旁系同源物的重要重叠功能。我们的数据表明，基于 FloA 和 FloT 与 Sec 膜插入机制之间的密切联系，FloA 和 FloT 执行空间上不同的功能，可能是在插入需要特定脂质环境的膜蛋白时，但不充当洗涤剂抗性蛋白的支架。我们的跟踪分析为理解活细胞中膜蛋白之间的相互作用提供了重要基础。
Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds. Many membrane proteins are not uniformly distributed within biological membranes, and may prefer specific lipid environments to function optimally. Using super resolution fluorescence microscopy, we show that several Bacillus subtilis membrane proteins indeed cluster into structures of 60 to 110 nm, verifying the existence of defined-size protein microdomains. Biochemical co-isolation of specific membrane proteins and flotillins, a family of proteins highly conserved between eukaryotic and bacterial cells, suggested that common “functional” microdomains exist, containing so-called “detergent-resistant” membrane proteins, that are centered by flotillins. Through high speed tracking of Bacillus subtilis FloA and FloT we show that both proteins are not present in the same microdomain, but move through the membrane with different velocities. Dual colour time lapse microscopy showed that contrarily to vertebrate flotillins, bacterial flotillins do not move together with detergent-resistant proteins, ruling out the existence of coclusters. The lack of both flotillins, but not of a single one, leads to striking defects in cell shape and in cell growth, indicating important overlapping functions of flotillin paralogs. Our data show that FloA and FloT perform spatially distinct functions, possibly in the insertion of membrane proteins that require a specific lipid environment, based on a close connection between FloA and FloT with the Sec membrane insertion machinery, but do not act as scaffolds for detergent resistant proteins. Our tracking analyses provide an important basis for the understanding of interactions between membrane proteins in living cells.