Abstract
Microtubules are highly dynamic structures that play an integral role in fundamental cellular functions. Different α- and β-tubulin isotypes are thought to confer unique dynamic properties to microtubules. The tubulin isotypes have highly conserved structures, differing mainly in their C-terminal tail sequences. However, little is known about the importance of the C-terminal tail in regulating and co-ordinating microtubule dynamics. We developed syngeneic human cell models using gene-editing to precisely modify the β-tubulin C-terminal tail region while preserving the endogenous microtubule network. Fluorescent microscopy of live cells, coupled with advanced image analysis revealed that the β-tubulin C-terminal tails differentially co-ordinate the collective and individual dynamic behaviour of microtubules by affecting microtubule growth rates and explorative microtubule assembly in an isotype-specific manner. Furthermore, βI- and βIII-tubulin C-terminal tails differentially regulate the sensitivity of microtubules to tubulin-binding agents and the microtubule depolymerising protein MCAK. The sequence of the β-tubulin tail encodes regulatory information that instructs and co-ordinates microtubule dynamics, thereby fine-tuning microtubule dynamics to support cellular functions.
Highlights
The microtubule cytoskeleton is a dynamic intracellular structure composed of α- and β-tubulin heterodimers
Through the development of novel syngeneic human cell models that preserve the endogenous microtubule network and eliminate the endogenous unmodified βIII-tubulin protein, we reveal that the βI- and βIII-tubulin C-terminal tails spatially regulate the coordination of microtubule dynamics in an isotype-specific manner
NCI-H460 cell lines, which endogenously express both βI- and βIII-tubulin isotypes, were gene-edited using zinc-finger nucleases targeted to the TUBB3 locus, which encodes the endogenous βIII-tubulin protein, to replace the endogenous βIII-tubulin protein with expression of either the full-length βIII-tubulin protein (ZB3), βIII-tubulin lacking the C-terminal tail (ZB3Δ), or a modified form of the protein where the βIII-tubulin C-terminal tail sequence from Thr429 was substituted for the βI-tubulin C-terminal tail sequence (ZB3/CB1) (Fig 1A)
Summary
The microtubule cytoskeleton is a dynamic intracellular structure composed of α- and β-tubulin heterodimers. The dynamic behavior of the microtubule cytoskeleton is critical in supporting cellular structure; in the transport of vesicles, proteins, and organelles; in enabling cell motility; and in ensuring correct segregation of the chromosomes during mitosis (Janke, 2014). The tubulin isotype composition forms a central component of the tubulin code, which together with posttranslational modifications and interactions with microtubule-associated proteins (MAPs), is hypothesized to form the regulatory mechanisms that specialize microtubule behavior (Gadadhar et al, 2017) but remains poorly defined. The C-terminal tail of the tubulin proteins extends outward from the wall of the microtubule, where it is a site for a wide range of posttranslational modifications and for interactions with proteins that regulate microtubule dynamics and other signaling effectors (Janke, 2014; Roll-Mecak, 2015). The C-terminal tails are the most divergent regions of the β-tubulin isotype sequence and serve to distinguish the tubulin isotypes from one another, making this region a prominent candidate in defining the isotypespecific function of the tubulin proteins
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