Analysis of microtubule dynamic instability using a plus-end growth marker

Regulation of microtubule dynamics is essential for many cell biological processes, and is likely to be variable between different subcellular regions. We describe a computational approach to analyze microtubule dynamics by detecting growing microtubule plus ends. Our algorithm tracks all EB1-EGFP comets visible in an image time-lapse sequence allowing the detection of spatial patterns of microtubule dynamics. We use spatiotemporal clustering of EB1-EGFP growth tracks to infer microtubule behaviors during phases of pause and shortening. The algorithm was validated by comparison to manually tracked, homogeneously labeled microtubules, and by analysis of the effects of well-characterized inhibitors of microtubule polymerization dynamics. We used our method to analyze spatial variations of intracellular microtubule dynamics in migrating epithelial cells

Insights into EB1 structure and the role of its C-terminal domain for discriminating microtubule tips from the lattice

End-binding proteins (EBs) comprise a conserved family of microtubule plus end–tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip–tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends