Bacterial cytoskeleton filaments are shorter than expected

The self-assembly of the tubulin-homologue, FtsZ, is a critical step in bacterial cell division. Using dynamic light scattering (DLS), we measured the kinetics of polymerization and the length of Caulobacter crescentus FtsZ and found that this protein forms short linear polymers that are remarkably monodisperse in length. The assembly rate of FtsZ into polymers depends on the concentrations of GTP and GDP. The study demonstrates the insight that DLS provides for studying structure and dynamics of bacterial cytoskeletal proteins.

HFSP Young Investigator Grant holders Piotr Garstecki, Doug Weibel and Martin Thanbichler and colleagues
authored on Thu, 13 September 2012

The HFSP Young Investigator Grant team of Doug Weibel (University of Wisconsin-Madison), Martin Thanbichler (Philipps University, Germany) and Piotr Garstecki (Institute of Physical Chemistry of the Polish Academy of Sciences) demonstrated that bacterial tubulin polymers have a length scale that is significantly different than previously thought.  “Our studies reveal that FtsZ polymers are in fact very short, and are more than an order of magnitude shorter than previously believed”, says Dr. Sen Hou from the IPC PAS, the first author of the report that has just appeared in the Journal of Biological Chemistry.

Figure: Cartoon showing that DLS can be used for real-time measurement of the FtsZ polymerization without influencing the size of polymers.

In bacteria, the cytoskeleton plays a range of roles that are still being uncovered and include division, cell shape, and metabolism. FtsZ is an essential protein that assembles at the mid-cell and coordinates bacterial cell division. An understanding of the structure and mechanism of FtsZ may provide a starting point for the development of new antimicrobial agents and is a central step in understanding cell division.

The HFSP sponsored team studied the length of polymers formed by FtsZ in the model bacterium Caulobacter crescentus. DLS provided critical insight into the in vitro polymerization of FtsZ. DLS is also known as photon correlation spectroscopy and relies on recording light scattered due to the motion of molecules. The technique allows users to measure the structure and dynamics of molecules with dimensions below one micrometer and the study of assembly at time scales from microseconds to minutes.

Using DLS, we found that FtsZ polymers are short, consist of 9-18 monomers, and have a total length that is ~100 nanometers, which is at least one order of magnitude shorter than measurements with transmission electron microscopy (TEM).

By increasing and decreasing the polymerisation rate of samples, we demonstrated that FtsZ which comes into contact with the surface of the copper grid used for TEM stimulates the assembly of small linear polymers into large bundles within several seconds. This effect may cause the overestimation of the length of cytoskeletal polymers determined by TEM. “Our result is not only well documented, but it also better fits to the present knowledge of the chemical environment in the cell interior. The nanoviscosity of the cytoplasm in bacteria is up to a thousand times as high as that of water. Had the cytoskeleton polymers been long, their rate of diffusion in the cytoplasm would be remarkably slow”, says Prof. Robert Hołyst, IPC PAS.

Reference

Characterization of Caulobacter crescentus FtsZ Protein Using Dynamic Light Scattering. Hou S, Wieczorek SA, Kaminski TS, Ziebacz N, Tabaka M, Sorto NA, Foss MH, Shaw JT, Thanbichler M, Weibel DB, Nieznanski K, Holyst R, Garstecki P. (2012) J Biol Chem. 287(28), 23878-86. doi: 10.1074/jbc.M111.309492.

Pubmed link