Abstract
Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. We present a protocol to produce [13C, 15N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Our results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.
Highlights
Microtubules are important components of the eukaryotic cytoskeleton
We incubated our prepared MTs with a fluorescently tagged microtubule-binding domain (MTBD) of MAP7 domain-containing protein 3 (MAP7D3, in the following abbreviated by MAP7), which served as a MT-binding probe, and found that the purified MTs were decorated by this protein fragment (Supplementary Fig. 1A, B, left)
Protein dynamics are critically involved in MT organization including the stochastic switching between growing and shrinking states as well as in the regulation of MTs by microtubule-associated proteins (MAPs) and other cellular factors
Summary
Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). We present a protocol to produce [13C, 15N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. The direct observation of dynamic regions of tubulin, including the unstructured C-terminal tails that are critical for binding of different MT-associated proteins such as tau[6] has remained challenging due to their intrinsic flexibility. We describe a solid-state NMR (ssNMR) approach to directly study the interaction of labeled MTs with their binding partners This method enables us to probe the dynamics of intact MTs. we examine the influence of two different MT-associated proteins upon the C-terminal tail dynamics. Our results suggest that MAP-tubulin tail interactions can involve both fast-time scale interactions between mobile chains as well as slowtime scale binding/unbinding events within stable MT–MAP complexes
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