C-Terminal Tail Polyglycylation and Polyglutamylation Alter Microtubule Mechanical Properties.

Microtubules are biopolymers that perform diverse cellular functions. Microtubule behavior regulation occurs in part through post-translational modification of both the α- and ß-subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and/or glutamate residues to the disordered C-terminal tails (CTTs) of tubulin. Because of their prevalence in stable, high-stress cellular structures such as cilia, we sought to determine if these modifications alter microtubules' intrinsic stiffness. Here, we describe the purification and characterization of differentially modified pools of tubulin from Tetrahymena thermophila. We found that post-translational modifications do affect microtubule stiffness but do not affect the number of protofilaments incorporated into microtubules. We measured the spin dynamics of nuclei in the CTT backbone by NMR spectroscopy to explore the mechanism of this change. Our results show that the α-tubulin CTT does not protrude out from the microtubule surface, as is commonly depicted in models, but instead interacts with the dimer's surface. This suggests that the interactions of the α-tubulin CTT with the tubulin body contributes to the stiffness of the assembled microtubule, thus providing insight into the mechanism by which polyglycylation and polyglutamylation can alter microtubule mechanical properties.

Microtubule seams are not mechanically weak defects.

Microtubule rigidity is important for many cellular functions to support extended structures and rearrange materials within the cell. The arrangement of the tubulin dimers within the microtubule can be altered to affect the protofilament number and the lattice type. Prior electron microscopy measurements have shown that when polymerized in the presence of a high concentration of NaCl, microtubules were more likely to be ten protofilaments with altered intertubulin lattice types. Specifically, such high-salt microtubules have a higher percentage of seam defects. Such seams have long been speculated to be a mechanically weak location in the microtubule lattice, yet no experimental evidence supported this claim. We directly measured the persistence length of freely fluctuating filaments made either with high salt or without. We found that the microtubules made with high salt were more flexible, by a factor of 2, compared to those polymerized the same way without salt present. The reduced persistence length of the high-salt microtubules can be accounted for entirely by a smaller cross-sectional radius of these microtubules, implying that the mixed lattice interactions have little effect on the bending rigidity. Our results suggest that the microtubule seam is not weaker than the typical lattice structure as previously speculated from structural studies.

Control of molecular shuttles by designing electrical and mechanical properties of microtubules.

Kinesin-driven microtubules have been focused on to serve as molecular transporters, called "molecular shuttles," to replace micro/nanoscale molecular manipulations necessitated in micro total analysis systems. Although transport, concentration, and detection of target molecules have been demonstrated, controllability of the transport directions is still a major challenge. Toward broad applications of molecular shuttles by defining multiple moving directions for selective molecular transport, we integrated a bottom-up molecular design of microtubules and a top-down design of a microfluidic device. The surface charge density and stiffness of microtubules were controlled, allowing us to create three different types of microtubules, each with different gliding directions corresponding to their electrical and mechanical properties. The measured curvature of the gliding microtubules enabled us to optimize the size and design of the device for molecular sorting in a top-down approach. The integrated bottom-up and top-down design achieved separation of stiff microtubules from negatively charged, soft microtubules under an electric field. Our method guides multiple microtubules by integrating molecular control and microfluidic device design; it is not only limited to molecular sorters but is also applicable to various molecular shuttles with the high controllability in their movement directions.

Modern methods to interrogate microtubule dynamics.

Microtubules are essential protein filaments required to organize and rearrange the interior of the cell. They must be stiff with mechanical integrity to support the structure of the cell. Yet, they must also be dynamic to enable rearrangements of the cell during cell division and development. This dynamic nature is inherent to microtubules and comes about through the hydrolysis of chemical energy stored in guanosine triphosphate (GTP). Dynamic instability has been studied with a number of microscopy techniques both in cells and in reconstituted systems. In this article, we review the techniques used to examine microtubule dynamic instability and highlight future avenues and still open questions about this vital and fascinating activity.

Mechanical properties of doubly stabilized microtubule filaments.

Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5' [(α, ß)-methyleno] triphosphate, guanosine-5'-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules.