@article{11373, abstract = {The actin-homologue FtsA is essential for E. coli cell division, as it links FtsZ filaments in the Z-ring to transmembrane proteins. FtsA is thought to initiate cell constriction by switching from an inactive polymeric to an active monomeric conformation, which recruits downstream proteins and stabilizes the Z-ring. However, direct biochemical evidence for this mechanism is missing. Here, we use reconstitution experiments and quantitative fluorescence microscopy to study divisome activation in vitro. By comparing wild-type FtsA with FtsA R286W, we find that this hyperactive mutant outperforms FtsA WT in replicating FtsZ treadmilling dynamics, FtsZ filament stabilization and recruitment of FtsN. We could attribute these differences to a faster exchange and denser packing of FtsA R286W below FtsZ filaments. Using FRET microscopy, we also find that FtsN binding promotes FtsA self-interaction. We propose that in the active divisome FtsA and FtsN exist as a dynamic copolymer that follows treadmilling filaments of FtsZ.}, author = {Radler, Philipp and Baranova, Natalia S. and Dos Santos Caldas, Paulo R and Sommer, Christoph M and Lopez Pelegrin, Maria D and Michalik, David and Loose, Martin}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry}, publisher = {Springer Nature}, title = {{In vitro reconstitution of Escherichia coli divisome activation}}, doi = {10.1038/s41467-022-30301-y}, volume = {13}, year = {2022}, } @article{9414, abstract = {Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.}, author = {Ishihara, Keisuke and Decker, Franziska and Dos Santos Caldas, Paulo R and Pelletier, James F. and Loose, Martin and Brugués, Jan and Mitchison, Timothy J.}, issn = {1939-4586}, journal = {Molecular Biology of the Cell}, number = {9}, pages = {869--879}, publisher = {American Society for Cell Biology}, title = {{Spatial variation of microtubule depolymerization in large asters}}, doi = {10.1091/MBC.E20-11-0723}, volume = {32}, year = {2021}, } @phdthesis{8358, abstract = {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. }, author = {Dos Santos Caldas, Paulo R}, isbn = {978-3-99078-009-1}, issn = {2663-337X}, pages = {135}, publisher = {Institute of Science and Technology Austria}, title = {{Organization and dynamics of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinkers}}, doi = {10.15479/AT:ISTA:8358}, year = {2020}, } @inbook{7572, abstract = {The polymerization–depolymerization dynamics of cytoskeletal proteins play essential roles in the self-organization of cytoskeletal structures, in eukaryotic as well as prokaryotic cells. While advances in fluorescence microscopy and in vitro reconstitution experiments have helped to study the dynamic properties of these complex systems, methods that allow to collect and analyze large quantitative datasets of the underlying polymer dynamics are still missing. Here, we present a novel image analysis workflow to study polymerization dynamics of active filaments in a nonbiased, highly automated manner. Using treadmilling filaments of the bacterial tubulin FtsZ as an example, we demonstrate that our method is able to specifically detect, track and analyze growth and shrinkage of polymers, even in dense networks of filaments. We believe that this automated method can facilitate the analysis of a large variety of dynamic cytoskeletal systems, using standard time-lapse movies obtained from experiments in vitro as well as in the living cell. Moreover, we provide scripts implementing this method as supplementary material.}, author = {Dos Santos Caldas, Paulo R and Radler, Philipp and Sommer, Christoph M and Loose, Martin}, booktitle = {Methods in Cell Biology}, editor = {Tran, Phong }, issn = {0091679X}, pages = {145--161}, publisher = {Elsevier}, title = {{Computational analysis of filament polymerization dynamics in cytoskeletal networks}}, doi = {10.1016/bs.mcb.2020.01.006}, volume = {158}, year = {2020}, } @article{7197, abstract = {During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, 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.}, author = {Dos Santos Caldas, Paulo R and Lopez Pelegrin, Maria D and Pearce, Daniel J. G. and Budanur, Nazmi B and Brugués, Jan and Loose, Martin}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Cooperative ordering of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinker ZapA}}, doi = {10.1038/s41467-019-13702-4}, volume = {10}, year = {2019}, }