biostudies-literatureKattan JDutch Research Council (NWO)2447-2455https://www.ebi.ac.uk/biostudies/studies/S-EPMC8524656biostudies-literatureUnknown10(10)Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus <i>Prosthecobacter</i>. They are formed by the tubulins BtubA and BtubB, which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, <i>de novo</i> synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by a light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.ACS synthetic biologyShaping Liposomes by Cell-Free Expressed Bacterial Microtubules.PMC8524656024.003.019Danelon CKattan JDoerr ADogterom MfalseShaping Liposomes by Cell-Free Expressed Bacterial Microtubules.Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus <i>Prosthecobacter</i>. They are formed by the tubulins BtubA and BtubB, which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, <i>de novo</i> synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by a light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.2021-01-01T00:00:00Z2021 Oct2025-04-20T02:02:26.705Z2025-04-20T02:02:26.705ZS-EPMC85246563458591810.1021/acssynbio.1c00278