Investigating the mechanism of Z-ring assembly and constriction in Bacillus subtilis
- Publication Type:
- Thesis
- Issue Date:
- 2014
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| Filename | Description | Size | |||
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 | 01front.pdf | contents and abstract | 207.75 kB | ||
| 02whole.pdf | thesis | 22.67 MB |
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NO FULL TEXT AVAILABLE. This thesis contains 3rd party copyright material. ----- Cell division is a fundamental process that is tightly regulated to occur in the right time and place in bacterial cells. However, despite decades of research the molecular mechanism which enables these seemingly simple unicellular and small organisms to divide is still not clear. At the core of the process of cell division is the assembly of a tubulin-like protein, FtsZ, into a scaffold structure known as the Z-ring. The main functions of the Z-ring are to identify the position in the cell where division will occur and to directly recruit other cell division proteins so that these proteins can aid in the formation of a division septum.
In recent years a third major function of the Z-ring has been recognized where this structure generates a contractile force to pull in the cell membrane during division. To understand how the Z-ring exerts a contractile force during cell division, the architecture of the Z-ring must be determined. This was achieved through the utilization of a super resolution microscopy technique known as 3D-structured illumination microscopy (3D-SIM). It was found that the Z-ring structure is heterogeneous, dynamic and possibly discontinuous, which has various implications for models that describe how the FtsZ protein exerts a contractile force. It was also shown that FtsZ encourages other cell division proteins to adopt a similar heterogeneous and dynamic localization pattern at the division site. Taken together these observations suggest that the invagination of the cell wall most likely occurs in discrete regions of the cell over time.
Next the role of an actin-like cell division protein, FtsA, during cell division was investigated. In Bacillus subtilis, it was originally thought that the FtsA promoted efficient Z-ring assembly by tethering FtsZ to the inner cell membrane. However, unpublished findings from this lab suggest that FtsA promotes the constriction of the Z-ring through an unknown mechanism. A series of experiments were performed to determine if FtsA directly promotes Z-ring constriction through its interaction with FtsZ. It was found that the absence of FtsA did not significantly alter the heterogeneous structure or dynamics of the Z-ring. Furthermore, the absence of FtsA did not alter the morphology of the division septum itself unlike other cell division mutants in B. subtilis. The examination of another cell division protein, DivIVA, revealed that its recruitment to the division site was temporally delayed and inefficient in the absence of FtsA. Therefore, it appears that FtsA indirectly promotes Z-ring constriction because it aids in the recruitment of additional cell division proteins to the division site which must occur before constriction.
Finally the work in this thesis focused on the assembly of the Z-ring. Prior studies in B. subtilis had demonstrated that the formation of the Z-ring is dependent on the remodeling of two distinct precursor FtsZ structures. These precursor structures have been visualized in a helical configuration within the cell and are recognized as the long and short FtsZ-helix. However, doubts have been recently raised about whether FtsZ truly forms a helical structure. Re-examination of the long and short FtsZ-helix was therefore performed and showed that the long FtsZ-helix might be an artifact of deconvolution image processing which will require additional work to validate. On the other hand, the short FtsZ-helix was shown to form a real helical structure, which assembles within the vicinity of the chromosome. It is regulated so that the short FtsZ-helix only assembles when the relative concentration of DNA located at the division site has decreased.
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