Exploring the role of Microtubule-Associated Proteins in organising microtubule bundles in cells using expansion microscopy

Abstract

Microtubules are an essential part of the cytoskeleton, involved in many cellular processes such as cell division, cell motility and intracellular transport. To execute their various functions, microtubules interact with a panoply of microtubule-associated proteins (MAPs), which by binding to microtubules can influence their individual as well as their collective behaviours. Most MAPs cause bundling of microtubules in cells when overexpressed, which aligns with the general view that MAPs are microtubule stabilisers. However, when inspected more closely, microtubule bundles can be very different depending on the MAP. Given that bundled microtubules are common in neurons, axonemes, or mitosis, understanding how those bundles are formed and how they contribute to the functioning of cells is essential for dissecting the underlying molecular mechanisms. How different MAPs bundle microtubules has so far only been studied for some examples. Given the different types of bundles observed with different MAPs, it is perceivable that they represent different microtubule arrangements, which are likely to be functionally important. We thus individually overexpressed MAPs in U2OS cells and analysed the ensuing microtubule bundles using expansion microscopy to increase resolution. We were able to pinpoint 4 principal types of bundle organization each induced by several MAPs. As in cells, MAPs are never present alone on microtubules, we investigated the interplay of MAPs on the microtubules by co-transfecting cells with two MAPs concomitantly. We could distinguish MAPs that are more dominant, can outcompete, or coexist with others. Together, this study has advanced our understanding of microtubule bundle formation and their critical functions in maintaining cellular viability.