Published: January 21st, 2013
We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.
Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with <20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.
1. Sample Preparation
Illustrated in Figure 3Aiv is a two-dimensional, super-resolution rendering of the Z-ring generated from the PALM imaging method described above. Below, we summarize qualitative and quantitative information that can be obtained from them.
Qualitatively, we observed that the Z-ring is an irregular structure that adopts multiple configurations (single band or helical arc) that are not distinguishable in conventional fluorescence images (compare Figure 3A-Dii
PALM images contain information about molecule counts and positions within a cell, allowing detailed analysis of the distribution and arrangement of target protein molecules that is difficult to achieve by other means. Below we outline precautions that should be taken to extract accurate quantitative information while maintaining the biological relevance of PALM images. We also explore the information that can be best obtained using live vs. fixed cells. Finally, we suggest avenues for obtaining additional super-.......
|Name of Reagent/Material
|50 x MEM Amino Acids
|100 x MEM Vitamins
|Electron Micrsocopy Sciences
|SeaPlaque GTG Agarose
|50 nm Gold Beads
|FCS2 Imaging Chamber
|1.45 NA, 60x Objective
|IXON EMCCD Camera
|488-nm Sapphire Laser
|561-nm Sapphire Laser
|405-nm CUBE Laser
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