A live-cell marker to visualize the dynamics of stable microtubules
Posted on: 11 July 2021 , updated on: 12 July 2021
Preprint posted on 23 June 2021
Categories: cell biology, developmental biology, molecular biology
Background
The skeleton of a cell is its microtubule assembly, this underlies vital processes like cell division and intracellular transport. Various labeling studies have revealed that different microtubule types exist within a cell. Of these, the most widely studied are the dynamic microtubules. In this preprint, the authors instead aimed to facilitate the study of another microtubule subset: the stable microtubules.
These two types of microtubules differ in stability, distribution, and function: hence their names. The dynamic microtubules are prone to MT-destabilizing agents and are found uniformly distributed within the cell. In contrast, stable microtubules can withstand the effects of MT- destabilizing agents. This type is usually found in the leading edge of fibroblasts[1], and perinuclear areas in common cell lines like U2OS, HeLa, etc. The dynamic microtubule subset is well studied due to the wide availability of live-cell markers. However, stable microtubules have only been studied on fixed cells using immunocytochemistry, and the lack of a live-cell marker has limited the study of this subset of microtubules greatly.
To aid the direct visualization and study of stable microtubules, Jansen et.al. developed a live-cell marker, rigor-2xmNeongreen. This marker binds selectively to stable microtubules, facilitating studies on the dynamics and mechanisms of stable microtubules.
Key findings
Validation of rigor-2xmNeongreen as a potential live-cell marker for stable MTs:
The motor protein kinesin-1(hkif5b) moves specifically on stable microtubules[2,3,4]. A rigor mutant of this motor protein was developed by replacing glycine with alanine at the 234th position (G234A). This abolishes its ATPase activity and prevents its unbinding from the MTs[5]. This mutant can bind to acetylated stable MTs [4,6,7], a frequent post-translational modification observed in MTs.
A live-cell marker for stable microtubules was developed by fusing the G234A rigor mutant in tandem with a fluorophore, mNeongreen[8] (rigor-2xmNeongreen). Detection of rigor-2xmNeongreen expression in U2OS cells revealed that the marker could selectively bind to acetylated MTs, a proxy for stable MTs.
Fig 1:A and B – Strategy for the development of the live-cell marker and its binding to stable MTs within a cell. 1C shows the colocalizing of rigor-2xmNeongreen with acetylated stable MTs, near the perinuclear region of U2OS cells. (Adapted from Fig 1, Jansen et.al., 2021).
Next, the duration of rigor-2xmNeongreen binding with stable microtubules (labeled with rigor- 2xmNeongreen) was assessed in comparison to dynamic microtubules (labeled with mCherry-tubulin). TIRF(Total internal reflection fluorescence)-microscopy revealed that rigor-2xmNeongreen could be tracked on stable acetylated microtubules for at least thirty minutes, while dynamic microtubules (mCherry) show instability. Treatment with nocodazole (10uM), an MT destabilizing agent did not affect the bundle of rigor-2xmNeongreen stable MTs in U2OS cells. Further, serum starvation of Swiss 3T3 cells upon rigor expression, followed by 8h culture in serum added media, the rigor-2xmNeongreen MTs disassemble and resemble the stable MT network as in control cells.
Taken together, these experiments validate that rigor-2xmNeongreen could be used as a live-cell marker for stable MTs.
Rigor-2xmNeongreen can be used to study the dynamics of stable MTs:
Though stable microtubules are long-lived, they are dynamic and prone to mechanical forces. U2OS cells transfected with rigor-2xmNeongreen displayed distinct behaviors. Cytoplasmic microtubule transport, MT looping, undulation, and breakage were frequently observed in stable MTs. This indicates that stable microtubules may undergo fluctuations due to local pulling and pushing forces. The observation of such dynamic behaviors is restricted by immunocytochemistry on fixed cells, thus, demonstrating that rigor-2xmNeongreen may be a potential live-cell marker for exploring stable MTs dynamics.
A possible mechanism for the stability of stable microtubules using rigor-2xmNeongreen:
According to the authors, there are two possibilities for the stability of stable MTs: mechanisms that prevent its depolymerization, or the presence of stabilizing proteins/modifications along its length. To explore this, MTs expressing mCherry-tubulin and rigor-2xmNeongreen were photo-ablated in the perinuclear region of U2OS cells. Rigor-2xmNeongreen labeled MTs were stable on both sides of the cut. This suggests that the stability of stable MTs is uniform along the entire length. A detailed investigation is required to elucidate the exact mechanisms of the stability of the stable MTs.
Fig 2: A. Cartoon for the strategy of laser severing of microtubules. B. Yellow arrows marking rigor- 2xmNeongreen labeled microtubules retaining rigor expression on either side of the cut, time scale seconds: milliseconds. ( Adapted from Fig 7, Jansen et.al., 2021).
Importance of this preprint:
This work is the first to develop a live cell marker (rigor-2xmNeongreen) for the direct visualization of stable microtubules. This tool can be an asset for the study of this microtubule subset in the future. Live cell tracking of stable microtubules may help to decipher cell organization, origin, types, and post-translational modifications. In addition, it may be feasible to elucidate the mechanisms of intracellular transport that remain largely unexplored.
Questions to the authors:
- Are both stable and dynamic microtubules involved in cell division? If yes, can this marker be used to visualize and study stable microtubules during cell division?
- What are your thoughts on introducing this marker in model organisms(mouse/chick) to study microtubule organization and dynamics during early developmental processes?
- What do you speculate are the major differences between the surfaces of stable and dynamic MTs?
References
- Gundersen, G.G. & Bulinski, J.C., 1988. Selective stabilization of microtubules oriented toward the direction of cell Proceedings of the National Academy of Sciences of the United States of America, 5(16), pp.5946– 5950.
- Cai, et al., 2009. Single molecule imaging reveals differences in microtubule track selection between Kinesin motors. PLoS Biology, 7(10), e1000216.
- Dunn, et al., 2008. Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells.Journal of Cell Science, 121(Pt 7), pp.1085–1095.
- Guardia, C.M. et al., 2016. BORC Functions Upstream of Kinesins 1 and 3 to Coordinate Regional Movement of Lysosomes along Different Microtubule Tracks. Cell reports, 17(8), 1950–1961.
- Rice, et al., 1999. A structural change in the kinesin motor protein that drives motility. Nature, 402(6763), pp.778– 784.
- Tas, P. et al., 2017. Differentiation between Oppositely Oriented Microtubules Controls Polarized Neuronal Transport. Neuron, 96(6), pp.1264–1271.e5
- Farías, G.G. et al., 2017. BORC/kinesin-1 ensemble drives polarized transport of lysosomes into the Proceedings of the National Academy of Sciences of the United States of America, 114(14), pp.E2955–E2964.
- Shaner, C. et al., 2013. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature Methods, 10(5), pp.407–409.
doi: https://doi.org/10.1242/prelights.29988
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