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Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

César Díaz-Celis, Viviana I. Risca, Felipe Hurtado, Jessica K. Polka, Scott D. Hansen, Daniel Maturana, Rosalba Lagos, R. Dyche Mullins, Octavio Monasterio
doi: https://doi.org/10.1101/112987
César Díaz-Celis
aLaboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
bPrograma de Doctorado en Microbiología, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 33, Santiago, Chile
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  • For correspondence: cesardiazcelis@gmail.com monaster@uchile.cl
Viviana I. Risca
cBiophysics Graduate Group, University of California, Berkeley, CA 94720,USA
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Felipe Hurtado
aLaboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
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Jessica K. Polka
dDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
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Scott D. Hansen
dDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
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Daniel Maturana
aLaboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
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Rosalba Lagos
aLaboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
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R. Dyche Mullins
dDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
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Octavio Monasterio
aLaboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
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  • For correspondence: cesardiazcelis@gmail.com monaster@uchile.cl
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Abstract

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro bMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm of Prosthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.

Importance Microtubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

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Posted March 02, 2017.
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Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis
César Díaz-Celis, Viviana I. Risca, Felipe Hurtado, Jessica K. Polka, Scott D. Hansen, Daniel Maturana, Rosalba Lagos, R. Dyche Mullins, Octavio Monasterio
bioRxiv 112987; doi: https://doi.org/10.1101/112987
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Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis
César Díaz-Celis, Viviana I. Risca, Felipe Hurtado, Jessica K. Polka, Scott D. Hansen, Daniel Maturana, Rosalba Lagos, R. Dyche Mullins, Octavio Monasterio
bioRxiv 112987; doi: https://doi.org/10.1101/112987

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