A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

Abstract

The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.

Cellular cartography: Towards an atlas of the neuronal microtubule cytoskeleton

“…Interestingly, the rigor was observed to transiently associate with many microtubules in the cell, but had a high unbinding-rate from dynamic microtubules such that it only accumulated on stable microtubules. This marker was also shown to preferentially associate with (stable) microtubules with an expanded lattice in U2OS cells ( de Jager et al, 2022 ) and in vitro ( Jansen et al, 2023 ). This makes it interesting to consider how this compares with the track selectivity of wild-type (motile) kinesin-1.…”

“…Note how the KIF5B-rigor preferentially decorates the subset of microtubules that are acetylated, a commonly used marker for stable microtubules. Taken from ( Jansen et al, 2023 ).…”

“…Recently, a new tool has emerged to study stable microtubules, enabling their long-term tracking: a fluorescent rigor kinesin-1 construct (G234A) that cannot effectively hydrolyze ATP ( Rice et al, 1999 ) and selectively binds to stable microtubules in cells: Stable Microtubule-Associated Rigor Kinesin (StableMARK) ( Jansen et al, 2023 ) ( Figure 3F ). The stable microtubules marked by this construct, which are largely acetylated and detyrosinated, were observed to slide and curl in cells.…”

“…Kinesin-1/KIF5A,B,C Stable (expanded) Prefers acetylated microtubules in COS-7 cells and rat hippocampal neurons (Cai et al, 2009;Farías et al, 2015;Tas et al, 2017;Jansen et al, 2023); can expand the microtubule lattice (Peet et al, 2018); a kinesin-1 rigor localizes specifically to microtubules with an expanded lattice in U2OS cells (de Jager et al, 2022) Kinesin-4/KIF21B Stable Moves cargo with a retrograde bias in PEX assays in rat hippocampal neurons unless plus-end-out microtubules are stabilized (Masucci et al, 2021) Dynein Dynamic Cofactor p150 Glued binds preferentially to tyrosinated microtubules in fibroblasts and in vitro (Peris et al, 2006;McKenney et al, 2016) Kinesin-3/KIF1A Dynamic Rigor KIF1A is ~2-fold enriched on tyrosinated microtubules compared to acetylated microtubules in soma of neurons and COS-7 cells (Tas et al, 2017) Kinesin-13/MCAK/KIFA Dynamic Prefers tyrosinated microtubules in mouse embryonic fibroblasts and in vitro (Peris et al, 2009) MAP7 Stable Potently recruits and activates kinesin-1 in vitro and in HeLa cells (Monroy et al, 2018;Hooikaas et al, 2019;Ferro et al, 2022) MAP7D1 Stable MAP7D1 localizes to some sort of subset in HeLa cells; recruits and activates kinesin-1 (Hooikaas et al, 2019) MAP7D2 Stable Localizes to the axon initial segment and helps kinesin-1 traffic into the axon of rat hippocampal neurons (Pan et al, 2019) MAP7D3 Stable Recruits and activates kinesin-1 in vitro and in HeLa cells (Hooikaas et al, 2019) MAP6 Stable Stabilizes microtubules against cold and nocodazole-induced depolymerization in mouse (embryonic) fibroblasts, HeLa cells, and neurons (Bosc et al, 2003;Delphin et al, 2012;Cuveillier et al, 2020); resists tubulin compaction leading to coiled microtubules in vitro (Cuveillier et al, 2020) MAP4 Stable? Does not compact microtubule lattice in vitro (Siahaan et al, 2022); can bind microtubule at the same time as kinesin-1 (Shigematsu et al, 2018) MAP9 Dynamic?…”

Aurora B controls microtubule stability to regulate abscission dynamics in stem cells

“…Finally, we wondered whether this transient increase in stable microtubules at the bridge was a general feature of dividing cells. To test this, we imaged a previously established cell line stably expressing StableMARK (53) and found that contrary to ESCs, U2OS expressing low levels of StableMARK had stable microtubules at the point of contact between the bridge and the cell bodies of the daughter cells, but only few around the midbody throughout bridge maturation (Supplementary Movie 6, Supplementary Figure 3E,F). Thus, the transient stabilization of bridge microtubules along the length of the bridge is not conserved between cell lines and could be a specific feature of stem cells.…”