Bacterial cytoskeletal filaments

Bacterial cytoskeletal filaments: Towards a DNA segregation system for a synthetic cell

Amini Hounejani, R. (TU Delft BN/Bionanoscience)

Dogterom, A.M. (promotor)
Danelon, C.J.A. (copromotor)

Delft University of Technology

BN/Bionanoscience

2023-03-08

The past decades have seen the rapid development of many aspects of synthetic biology. For example, attempts to build synthetic cells under controlled conditions in the laboratory have led to significant achievements. Following a bottom-up approach, scientists aim at building a self-reproducing synthetic cell with a minimum number of biological modules. A functional synthetic cell should accomplish at least four processes during one cycle: growth,DNAreplication,DNA segregation, and division. DNA segregation, as a vital step in the life cycle of a synthetic cell, is in focus in this thesis. Segregation of replicated DNA in eukaryotes depends highly on microtubules. Microtubules are protein polymers which form cylindrical tubes, a dynamic structure with which they can exert both pulling and pushing forces. During cell division, microtubules form a spindle-like structure, named the mitotic spindle. Microtubules in the mitotic spindle apparatus attach to the replicated chromosomes through kinetochores and exert pulling forces to separate the sister chromosomes and place them in the newly born daughter cells. These hollow tubes can grow in the presence of GTPbound tubulin dimers. When all the GTP-tubulin subunits at the end of a filament turned into GDP-tubulin, themicrotubule exhibits a transition to shrinkage. While the addition of GTP-tubulin to the end of microtubulesmay cause pushing forces, shrinking microtubules are able to exert pulling forces with the help of microtubule adapter proteins. The random transitions between growth and shrinkage of a microtubule is called dynamic instability and has been studied throughout decades. DNA segregation components of a synthetic cell should preferably be fully expressible in a cell-free manner which employs reconstituted transcription-translation factors. Although mitosis in eukaryotes is the best-studied DNA segregation system to date, microtubules are unsuitable for cell-free expression due to their complex chaperondependent folding as well as essential post-translational modifications. Interesting alternatives for DNA segregation in synthetic cells are provided by bacterial segregation systems. An active bacterial segregation system is ideally composed of three elements: a cytomotive NTPase filament, a connecting or adapter protein, and a centromere-like DNA sequence. This thesis will examine various bacterial cytoskeletal filaments with a central focus on their suitability for building a DNA segregation systemfor synthetic cells...

Microtubules
bacterial microtubules
DNA segregation
PhuZ
TubZRC system
optogenetics
micro-fabrication
in vitro reconstitution
Monte Carlo simulations

https://doi.org/10.4233/uuid:79de9e6e-8201-499b-8d41-7c52ae450257

978-90-8593-553-7

Casimir PhD Series, Delft-Leiden, 2023 (05)

Institutional Repository

doctoral thesis

© 2023 R. Amini Hounejani