IST Austria Thesis
During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This so-called Z-ring acts as a scaffold recruiting several division-related proteins to mid-cell and plays a key role in distributing proteins at the division site, a feature driven by the treadmilling motion of FtsZ filaments around the septum. What regulates the architecture, dynamics and stability of the Z-ring is still poorly understood, but FtsZ-associated proteins (Zaps) are known to play an important role. Advances in fluorescence microscopy and in vitro reconstitution experiments have helped to shed light into some of the dynamic properties of these complex systems, but methods that allow to collect and analyze large quantitative data sets of the underlying polymer dynamics are still missing. Here, using an in vitro reconstitution approach, we studied how different Zaps affect FtsZ filament dynamics and organization into large-scale patterns, giving special emphasis to the role of the well-conserved protein ZapA. For this purpose, we use high-resolution fluorescence microscopy combined with novel image analysis workfows to study pattern organization and polymerization dynamics of active filaments. We quantified the influence of Zaps on FtsZ on three diferent spatial scales: the large-scale organization of the membrane-bound filament network, the underlying polymerization dynamics and the behavior of single molecules. We found that ZapA cooperatively increases the spatial order of the filament network, binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner, without compromising filament dynamics. Furthermore, we believe that our automated quantitative methods can be used to analyze a large variety of dynamic cytoskeletal systems, using standard time-lapse movies of homogeneously labeled proteins obtained from experiments in vitro or even inside the living cell.
I should also express my gratitude to the bioimaging facility at IST Austria, for their assistance with the TIRF setup over the years, and especially to Christoph Sommer, who gave me a lot of input when I was starting to dive into programming.
Dos Santos Caldas PR. Organization and dynamics of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinkers. 2020. doi:10.15479/AT:ISTA:8358
Dos Santos Caldas, P. R. (2020). Organization and dynamics of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinkers. IST Austria. https://doi.org/10.15479/AT:ISTA:8358
Dos Santos Caldas, Paulo R. “Organization and Dynamics of Treadmilling Filaments in Cytoskeletal Networks of FtsZ and Its Crosslinkers.” IST Austria, 2020. https://doi.org/10.15479/AT:ISTA:8358.
P. R. Dos Santos Caldas, “Organization and dynamics of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinkers,” IST Austria, 2020.
Dos Santos Caldas PR. 2020. Organization and dynamics of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinkers. IST Austria.
Dos Santos Caldas, Paulo R. Organization and Dynamics of Treadmilling Filaments in Cytoskeletal Networks of FtsZ and Its Crosslinkers. IST Austria, 2020, doi:10.15479/AT:ISTA:8358.
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