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1. Microtubule dynamics at low temperature: evidence that tubulin recycling limits assembly

   https://doi.org/10.1091/mbc.E19-11-0634  

   Li, Gabriella ; Moore, Jeffrey K. ( May 2020 , Molecular Biology of the Cell)

   Théry, Manuel (Ed.)

   How temperature specifically affects microtubule dynamics and how these lead to changes in microtubule networks in cells have not been established. We investigated these questions in budding yeast, an organism found in diverse environments and therefore predicted to exhibit dynamic microtubules across a broad temperature range. We measured the dynamics of GFP-labeled microtubules in living cells and found that lowering temperature from 37°C to 10°C decreased the rates of both polymerization and depolymerization, decreased the amount of polymer assembled before catastrophes, and decreased the frequency of microtubule emergence from nucleation sites. Lowering to 4°C caused rapid loss of almost all microtubule polymer. We provide evidence that these effects on microtubule dynamics may be explained in part by changes in the cofactor-dependent conformational dynamics of tubulin proteins. Ablation of tubulin-binding cofactors (TBCs) further sensitizes cells and their microtubules to low temperatures, and we highlight a specific role for TBCB/Alf1 in microtubule maintenance at low temperatures. Finally, we show that inhibiting the maturation cycle of tubulin by using a point mutant in β-tubulin confers hyperstable microtubules at low temperatures and rescues the requirement for TBCB/Alf1 in maintaining microtubule polymer at low temperatures. Together, these results reveal an unappreciated step in the tubulin cycle. 

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2. Modeling microtubule-cytoplasm interaction in plant cells.

   Murshed M, Wei D ( September 2022 , bioRxiv)

   Although microtubules in plant cells have been extensively studied, the mechanisms that regulate the spatial organization of microtubules are poorly understood. We hypothesize that the interaction between microtubules and cytoplasmic flow plays an important role in the assembly and orientation of microtubules. To test this hypothesis, we developed a new computational modeling framework for microtubules based on theory and methods from the fluid-structure interaction. We employed the immersed boundary method to track the movement of microtubules in cytoplasmic flow. We also incorporated details of the encounter dynamics when two microtubules collide with each other. We verified our computational model through several numerical tests before applying it to the simulation of the microtubule-cytoplasm interaction in a growing plant cell. Our computational investigation demonstrated that microtubules are primarily oriented in the direction orthogonal to the axis of cell elongation. We validated the simulation results through a comparison with the measurement from laboratory experiments. We found that our computational model, with further calibration, was capable of generating microtubule orientation patterns that were qualitatively and quantitatively consistent with the experimental results. The computational model proposed in this study can be naturally extended to many other cellular systems that involve the interaction between microstructures and the intracellular fluid. 

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3. Simulation of microtubule–cytoplasm interaction revealed the importance of fluid dynamics in determining the organization of microtubules

   https://doi.org/10.1002/pld3.505  

   Murshed, Mohammad ; Wei, Donghui ; Gu, Ying ; Wang, Jin ( July 2023 , Plant Direct)

   Although microtubules in plant cells have been extensively studied, the mechanisms that regulate the spatial organization of microtubules are poorly understood. We hypothesize that the interaction between microtubules and cytoplasmic flow plays an important role in the assembly and orientation of microtubules. To test this hypothesis, we developed a new computational modeling framework for microtubules based on theory and methods from the fluid–structure interaction. We employed the immersed boundary method to track the movement of microtubules in cytoplasmic flow. We also incorporated details of the encounter dynamics when two microtubules collide with each other. We verified our computational model through several numerical tests before applying it to the simulation of the microtubule–cytoplasm interaction in a growing plant cell. Our computational investigation demonstrated that microtubules are primarily oriented in the direction orthogonal to the axis of cell elongation. We validated the simulation results through a comparison with the measurement from laboratory experiments. We found that our computational model, with further calibration, was capable of generating microtubule orientation patterns that were qualitatively and quantitatively consistent with the experimental results. The computational model proposed in this study can be naturally extended to many other cellular systems that involve the interaction between microstructures and the intracellular fluid.

    

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4. Membrane mediated motor kinetics in microtubule gliding assays

   https://doi.org/10.1038/s41598-019-45847-z  

   Lopes, Joseph ; Quint, David A. ; Chapman, Dail E. ; Xu, Melissa ; Gopinathan, Ajay ; Hirst, Linda S. ( July 2019 , Scientific Reports)

   Motor-based transport mechanisms are critical for a wide range of eukaryotic cell functions, including the transport of vesicle cargos over long distances. Our understanding of the factors that control and regulate motors when bound to a lipid substrate is however incomplete. We used microtubule gliding assays on a lipid bilayer substrate to investigate the role of membrane diffusion in kinesin-1 on/off binding kinetics and thereby transport velocity. Fluorescence imaging experiments demonstrate motor clustering on single microtubules due to membrane diffusion in the absence of ATP, followed by rapid ATP-induced dissociation during gliding. Our experimental data combined with analytical modeling show that the on/off binding kinetics of the motors are impacted by diffusion and, as a consequence, both the effective binding and unbinding rates for motors are much lower than the expected bare rates. Our results suggest that motor diffusion in the membrane can play a significant role in transport by impacting motor kinetics and can therefore function as a regulator of intracellular transport dynamics.

    

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5. Insights into allosteric control of microtubule dynamics from a buried β‐tubulin mutation that causes faster growth and slower shrinkage

   https://doi.org/10.1002/pro.3842  

   Ye, Xuecheng ; Kim, Tae ; Geyer, Elisabeth A. ; Rice, Luke M. ( March 2020 , Protein Science)

   αβ‐tubulin subunits cycle through a series of different conformations in the polymer lattice during microtubule growing and shrinking. How these allosteric responses to different tubulin:tubulin contacts contribute to microtubule dynamics, and whether the contributions are evolutionarily conserved, remains poorly understood. Here, we sought to determine whether the microtubule‐stabilizing effects (slower shrinking) of the β:T238A mutation we previously observed using yeast αβ‐tubulin would generalize to mammalian microtubules. Using recombinant human microtubules as a model, we found that the mutation caused slow microtubule shrinking, indicating that this effect of the mutation is indeed conserved. However, unlike in yeast, β:T238A human microtubules grew faster than wild‐type and the mutation did not appear to attenuate the conformational change associated with guanosine 5′‐triphosphate (GTP) hydrolysis in the lattice. We conclude that the assembly‐dependent conformational change in αβ‐tubulin can contribute to determine the rates of microtubule growing as well as shrinking. Our results also suggest that an allosteric perturbation like the β:T238A mutation can alter the behavior of terminal subunits without accompanying changes in the conformation of fully surrounded subunits in the body of the microtubule.

    

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