Manipulation and quantification of microtubule lattice integrity.

Taylor A Reid,Melissa K Gardner,Courtney Coombes

doi:10.1242/bio.025320

Abstract

ABSTRACTMicrotubules are structural polymers that participate in a wide range of cellular functions. The addition and loss of tubulin subunits allows the microtubule to grow and shorten, as well as to develop and repair defects and gaps in its cylindrical lattice. These lattice defects act to modulate the interactions of microtubules with molecular motors and other microtubule-associated proteins. Therefore, tools to control and measure microtubule lattice structure will be invaluable for developing a quantitative understanding of how the structural state of the microtubule lattice may regulate its interactions with other proteins. In this work, we manipulated the lattice integrity of in vitro microtubules to create pools of microtubules with common nucleotide states, but with variations in structural states. We then developed a series of novel semi-automated analysis tools for both fluorescence and electron microscopy experiments to quantify the type and severity of alterations in microtubule lattice integrity. These techniques will enable new investigations that explore the role of microtubule lattice structure in interactions with microtubule-associated proteins.

Highlights

Microtubules are long, hollow tubes that act as important structural and signaling components inside cells
We found that there was a significant increase in the Structure Metric for the 25°C storagecondition pool of GDP microtubules as compared to the 37°C storage-condition (Fig. 3C; P=2×10−6, t-test), and that this increase was due to shifts towards larger width variation and a larger curvature of the microtubules (Fig. 3D,E), suggesting that the lower temperature storage condition reduced the lattice structural integrity of the Taxol-stabilized GDP microtubules
After quantification using the Reporter Fraction (RF), we found that there was a 55% higher Reporter Fraction for the 25°C storage pool GDP microtubules as compared to the 37°C storage pool microtubules (Fig. 6C; P

Summary

Introduction

Microtubules are long, hollow tubes that act as important structural and signaling components inside cells. Microtubules are typically closed tubes that are formed by 13 laterally associated individual protofilaments, each of which is composed of αβ-tubulin heterodimers that are stacked end-to-end (Zhang et al, 2015; Wang and Nogales, 2005). While this regular, stacked αβ heterodimer arrangement of microtubules is widely conserved, electron microscopy studies have revealed the presence of a wide range of microtubule lattice structures and irregularities. Cryoelectron microscopy studies have revealed that the lattice structures near to growing microtubule ends are frequently characterized by flattened, open sheets, rather than closed tubes (Chrétien et al, 1995; Guesdon et al, 2016). It has been recently reported that hydrolysis of the β-tubulin subunit within the microtubule lattice leads to overall ‘compaction’ of the microtubule

Methods

Results

Conclusion