Navigating microtubule diversity: Insights into microtubule stability and orientation

Abstract

Cells have diverse morphologies and correspondingly diverse microtubule networks crucial for their functions. The microtubules in these arrays can differ in terms of their dynamic behaviour or lifetime (i.e., their stability or lability), the post-translational modifications (PTMs) they bear, the microtubule-associated proteins (MAPs) bound to their surface, and their orientation (i.e., whether their fast-growing plus-end is pointing towards or away from the cell center). Because of these differences, some motor proteins prefer a certain type of microtubule. Specifically, kinesin-1 prefers to move on stable microtubules in cells, but the precise interplay of the aforementioned factors that dictates this remains poorly understood. In this thesis, we examine the orientation, reorganization, and unique properties of stable microtubules that allow them to serve as the preferred roads for kinesin-1-based transport. First, we develop and characterize a novel tool, StableMARK, to image long-lived, stable microtubules in live cells and find that it recognizes the expanded lattices of these microtubules. Next, we optimize motor-PAINT, a super-resolution method to map microtubule orientation, to study microtubule reorganization during neuronal development in cultured rat hippocampal neurons during the early stages of neurite outgrowth and axon specification. Using motor-PAINT and live-cell imaging of StableMARK, we find that stable microtubules are initially oriented plus-end-out in minor neurites, but later slide between and within neurites to reverse their orientation, adopting the minus-end-out organization seen in mature dendrites. Using another super-resolution method, expansion microscopy, we determine that these stable microtubules are first anchored at the centrioles and then released, helping to explain their reversal. We also study kinesin-1's preference for these stable microtubules, demonstrating that its subset specificity depends on the MAP7-binding domain in its stalk. Correspondingly, some MAP7 family members, which were previously demonstrated to be potent activators of kinesin-1 motility, are themselves selectively localized to stable microtubules, recruiting kinesin-1 to this subset. These MAP7s appear to recognize the expanded lattice of stable microtubules and not the PTMs that they bear. Thus, Taxol, which expands the microtubule lattice, redistributes these MAP7s and thus kinesin-1, to all microtubules. Exploiting this phenomenon, we record kinesin-1 steps in live cells for the first time using MINFLUX. We additionally combine MINFLUX with motor-PAINT, giving us impressive resolution that allows us to distinguish individual protofilaments. Finally, we integrate motor-PAINT with another microscopy technique, lattice light-sheet microscopy, to image microtubule orientation in full cell volumes, a crucial step towards determining microtubule orientation in tissue. Together, this thesis provides tools to study the orientation and stability of microtubules and helps us better understand why and how stable microtubules are distinct from dynamic microtubules and why kinesin-1 preferentially uses these microtubules as tracks in the cell. The techniques developed in this thesis can also contribute to the continued study of the unique properties and roles of stable microtubules, as well as how the misregulation of the balance of microtubule stability and dynamicity contributes to the pathology of neurodegenerative and neurodevelopmental disorders.