Abstracts for parallel session on Thursday May 19th
Cell division
5.1: Modeling area gain and loss during cytokinesis
Felix Frey, Timon Idema
Delft University of Technology, The Netherlands
The ability of cells to divide is fundamental for the self-replication of life. It is also one of the most challenging steps when building artificial cells. During cytokinesis of animal cells, the cell is divided into two equal parts. Since the cell’s volume is conserved, the projected area has to increase to allow for the change of shape. Here we aim to predict theoretically how membrane gain and loss processes adapt during cytokinesis. To address this question, we developed a kinetic model in which membrane gain and loss depend on membrane curvature and tension. We apply this model to a series of calculated vesicle shapes, which we take as a proxy for the shape of dividing cells. We find that the ratio of membrane gain and loss changes non-monotonically during cytokinesis due to the complex interplay between membrane area and shape. Our results imply that controlling membrane turnover will be crucial for the successful division of artificial cells.
5.2: Mimicking DNA segregation inside cell-sized confinement
Mai Tran¹, Rakesh Chatterjee², Yannik Dreher¹, Kevin Jahnke¹, Vasily Zaburdaev²*, Kerstin Göpfrich¹*
1: Max Planck Institute for Medical Research, Heidelberg University, Germany; 2: Friedrich-Alexander University Erlangen-Nurnberg, Germany
The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Even though significant progress has been made towards reliable compartment division, mechanisms to segregate the DNA-encoded informational content are still missing. Herein, DNA droplets are formed by liquid-liquid phase separation (LLPS) in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). DNA droplet fission is obtained by breaking down the linking component between two populations of DNA nanostars. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled segregation. Notably, the splitting process is slower in confinement than in bulk. This behavior was reproduced in a lattice-based simulation model which mimics the interactions between the DNA nanostar populations. The ionic strength of the solution and the nucleobase sequences were employed to regulate the segregation dynamics. In addition, local photoinduced segregation of DNA droplets within GUVs was achieved. Altogether, DNA droplet technology portrays a promising mechanism for information storage and segregation within bottom-up assembled artificial cells.
5.3: Module integrations in a synthetic cell: coupling DNA self-replication and phospholipid biosynthesis
Ana Restrepo Sierra, Federico Ramirez, Ilja Westerlaken, Christophe Danelon
Delft University of Technology, The Netherlands
Cellular life is supported by a set of interconnected biological modules. Efforts so far to build a synthetic cell have focused on the reconstitution of individual functions, such as DNA replication, expression of division protein complexes, and phospholipid biosynthesis for membrane growth. We here demonstrate the first integration of two functional modules in gene-expressing liposomes: DNA replication and phospholipid synthesis. The protein machinery for both heterologous modules was encoded on a single linear DNA template of 9.5 kb. The PURE system was used to express the replication proteins of the phage phi29 (the DNA polymerase and terminal protein)(1) and four enzymes of the E. coli Kennedy pathway for the production of phosphatidylserine (PS)(2). Using fluorescence microscopy and flow cytometry, we found that a subset of the liposome population exhibits dual activities, while a larger fraction displays either DNA replication or PS synthesis. To increase synergy between the two modules, we explored different incubation temperatures, initial molecular compositions and directed evolution approaches. This work marks the inception of integrated functionalities in a synthetic cell and unveils some of the challenges towards the implementation of more cellular modules.
References:
(1) Pauline van Nies, Ilja Westerlaken, Duco Blanken, Margarita Salas, Mario Mencia and Christophe Danelon. Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. Nature Communications, 2018.
(2) D. Blanken, D. Foschepoth, A. Calaça Serrão, and C. Danelon, Genetically controlled membrane synthesis in liposomes. Nat. Commun. 2020 Aug 28;11(1):4317.
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