A phospho-regulated ensemble signal motif of α-TAT1 drives dynamic microtubule acetylation

Background

Mechanical properties of microtubules can be altered by several post-translational modifications (PTMs). One such well-conserved PTM is acetylation. Acetylated microtubules are more stable, better able to resist bending forces, and therefore, are protected from mechanical stress^1,2. Tubulin acetylation contributes to the dynamic nature of microtubule organization and function in specialized processes such as intracellular protein trafficking, cilia motility, cell migration and cell division³.

Acetylation occurs on Lysine 40 position of a-tubulin in the cytoplasm and is catalyzed by α-TAT1, a highly conserved mammalian acetyltransferase². While the mechanistic insights into the catalytical role of α-TAT1 in promoting microtubule stability has been explored earlier, the regulation of intracellular distribution of the acetyltransferase itself has not been looked at in detail⁴. This preprint aims to understand the spatial regulation of acetyltransferase, α-TAT1, and its impact on the levels of microtubule acetylation in the cell.

                                   (A) 

                                   (B)

Figure (adapted from the preprint) shows the impact of change in α-TAT1 (acetyltransferase) localization on tubulin acetylation in HeLa cells. In control condition (A), majority of cells show cytoplasmic localization of α-TAT1 (left). Drug based inhibition of nuclear export (B) causes α-TAT1 to be sequestered in the nucleus (left). This leads to a reduction in the levels of acetylated microtubules in the cytoplasm (right).

Key Findings

While the N-terminus of α-TAT1 consists of the catalytic domain, its C -terminus was predicted to be intrinsically disordered and previously less well characterized. In this study, using a combination of sequence prediction and analysis software algorithms, α-TAT1 was predicted to comprise highly conserved regions of both NLS (nuclear localization signal) and NES (nuclear exclusion signal) domains. Upon expressing the fluorescently tagged α-TAT1 protein in HeLa cells, it was found to exhibit a range of localization patterns. Minor fraction of cells showed (a) a nuclear enrichment or (b) a diffuse localization of the protein. In majority of the cells, the protein was found to be exclusively localized to the cytoplasm. This dominant cytosolic distribution was expected since α-TAT1 is known to localize with the acetylated microtubules. Additionally, however, the intracellular fluorescence intensity of the protein was found to vary across time, indicative of a dynamic nature of a-TAT1 localization in cells. This categorical and dynamic localization pattern of a-TAT1 suggested that its function may be regulated by altering its distribution pattern in cells.

How is α-TAT1 localization regulated in the cell? The authors reported an interaction between α-TAT1 and exportin, a member of the nuclear export machinery, which suggested that α-TAT1 may be actively exported out of the nucleus, such that it can catalyze acetylation on microtubules in the cytoplasm. To test this, the authors used a generic drug to inhibit exportin-mediated transport and found that they could effectively trap a large pool of acetyltransferase in the nucleus (Major fraction of the cells showed a diffused localization of the protein). On doing so, expectedly, they were able to drastically reduce the levels of acetylated microtubules in the cytoplasm (animated video). Hence, the spatial switch of protein localization from the cytoplasm to the nucleus or vice versa is an efficient mechanism to regulate its function and alter the acetylation status of microtubules in the cells.

What controls the spatial shuttling of α-TAT1 between the nucleus and the cytoplasm? Upon expressing shorter fragments of the protein in the cells, it was found that the C-terminus of the protein, but not its N-terminus fragment, is preferentially in localized in the cytosol, suggesting that the export is mediated by the C-terminus. Truncation of putative NES domain abrogated its export from the nucleus. However, noticeably, as is the case with drug-mediated inhibition of nuclear export, this mutant protein too exhibited a dominant diffused localization pattern, suggesting that deletion of the NES alone is not sufficient to restrict the protein to the nucleus. Understandably, additional pathways are involved in the regulation of spatial distribution of α-TAT1.

Next, the authors set out to characterize the level of regulation on the putative NLS domain, which is also located on the C-terminus. The predicted NLS domain was found to be bind 14-3-3 proteins, which are reported to promote nuclear exclusion. Since interaction with 14-3-3 proteins conventionally requires phosphorylated serine and threonine residues, the authors subjected the cells to a battery of kinase inhibitors, and found that inhibition of kinases such as CDKs, CK2 and PKA increased the nuclear localization of the protein, such that major fraction of the cells showed diffused α-TAT1 protein localization. α-TAT1, therefore, must be in a phosphorylated state for its efficient nuclear export. Whether this phosphorylation occurs on the predicted NLS domain or not is still unresolved in the current study. [Of note, the current study does not comment on whether the nuclear enrichment observed upon kinase inhibition is because of a reduced affinity of 14-3-3 proteins for α-TAT1 or whether these kinases regulate α-TAT1 localization through an independent alternative mechanism. Further, the significance of interaction of α-TAT1 with 14-3-3 proteins on its spatial distribution pattern or on the regulation of tubulin acetylation has not been validated experimentally in the current version of the study.]

On further exploring the theme of phospho-regulation of α-TAT1 spatial distribution, the binding of the predicted NLS domain to importin, a member of the nuclear import machinery, was disrupted by mutating the basic residues flanking the NLS to neutral charged amino acid, alanine. This was done because importin has been reported to preferentially interact with basic residues enriched in the NLS domain. These mutations resulted in enhanced nuclear enrichment of α-TAT1, indicating that phosphorylation on these particular residues might be important for mediating nuclear export/import. [An important caveat of the experiment is that the whether these mutations indeed disrupt the interaction with importin or 14-3-3 proteins has not been tested in this study.]

In summary, this preprint explores the key processes by which the spatial distribution of acetyltransferase, α-TAT1, can be modulated in the cell. Nuclear export to the cytoplasm is independently carried out through the NES and NLS domains: – (a) direct binding of the NES domain to nuclear export machinery and (b) phosphorylation on the NLS domain.

What I like about this preprint

This preprint has exploited a combination of sequence prediction tools and bioinformatic analysis to experimentally characterize the unique spatial regulation of α-TAT1. Abnormal tubulin acetylation levels have been reported in several pathological conditions, such as neurodegenerative disorders and cancer³. The current study has provided a new outlook on exploring microtubule acetylation and associated mechanical properties of the cell with respect to the dynamic spatial distribution of the acetyltransferase, α-TAT1. While the precise mechanism of regulation of α-TAT1 distribution remains unclear from this study, it would be interesting to see how the delicate balance of kinases and phosphatases in the cell determines the spatial distribution of α-TAT1, and consequently microtubule acetylation and mechanical behaviour of the cell.

Future prospects and questions for the authors

1. Could the authors please describe or speculate on a scenario in which the change in microtubule acetylation, due to sequestration of α-TAT1 in the nucleus, directly modulates a cell biological process/ behaviour?
2. Could the authors comment on the time it would take for the spatial switch in distribution of α-TAT1, i.e., from the nucleus to the cytoplasm or from the cytoplasm to the nucleus, so as to significantly alter microtubule acetylation status of the cell? How much time would it take for cells to elicit any meaningful response to changes in microtubule acetylation?
3. The acetylation status of microtubules has been reported to change in certain neurodegenerative disorders and may also be important for biological processes such as migration of cancer cells. Could the authors predict the spatial distribution pattern of α-TAT1 in such conditions?
4. What do the authors have to say about the significance of kinases they found in their screen and their impact on microtubule acetylation and eventually, cell function? Additionally, the kinase inhibition screen in the study was done over several hours to overnight. Do the authors think that a shorter window of kinase inhibition could drive a meaningful, fast-acting response in the cell?
5. Here, 14-3-3 proteins have been shown to interact with α-TAT1. However, in the current study, it is not immediately clear that 14-3-3 proteins might directly affect the phosphorylation status of α-TAT1 or in fact, play any role in its intracellular spatial distribution. Can the authors explain briefly why they think 14-3-3 proteins would be an important future target for studying microtubule acetylation?
6. Microtubule acetylation may be important for the formation of specialized microtubule structures, such as the mitotic spindle. Further, α-TAT1 co-localizes with the microtubules at the time of formation of spindle. During mitosis, there is breakdown of the nuclear envelope, and therefore, should one not expect to see any important role of the NES and NLS domains and their potential phosphor-regulation identified in the study, during cell division?
7. This paper has helped characterize key processes and proteins that regulate the spatial distribution of α-TAT1 in microtubule acetylation. Could the authors briefly comment on whether microtubule de-acetyltransferases (say, histone de-acetylase) could be governed by a similar process, if at all?

 

References

1. Hammond, J. W., Cai, D. & Verhey, K. J. Tubulin modifications and their cellular functions. Curr. Opin. Cell Biol. 20, 71–76 (2008).
2. Janke, C. & Montagnac, G. Causes and Consequences of Microtubule Acetylation. Curr. Biol. 27, R1287–R1292 (2017).
3. Nekooki-Machida, Y. & Hagiwara, H. Role of tubulin acetylation in cellular functions and diseases. Med. Mol. Morphol. 53, 191–197 (2020).
4. Kalebic, N. et al. Tubulin Acetyltransferase αTAT1 Destabilizes Microtubules Independently of Its Acetylation Activity. Mol. Cell. Biol. 33, 1114–1123 (2013).

Tags: acetylation, acetyltransferase, tubulin ptm

Posted on: 2 December 2020

doi: https://doi.org/10.1242/prelights.26003

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