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Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics

Stephanie Torrino, Stephane Audebert, Ilyes Belhadj, Caroline Lacoux, Sabrina Pisano, Sophie Abélanet, Frederic Brau, Stephen Y. Chan, Bernard Mari, William M Oldham, Thomas Bertero

Preprint posted on 11 March 2020 https://doi.org/10.1101/2020.03.10.985036

Preprint from the Thomas Bertero lab shows how mechanical stress directly impacts microtubule glutamylation resulting in changes in cell elasticity, migration and proliferation

Selected by Sukriti Kapoor

Background

The ability of a cell to sense mechanical cues and elicit appropriate biological responses, such as changes in cell migration or proliferation, is termed mechanosenstation or mechanotranduction1. The mechanical cues that cells need to respond to include neighbouring cells, surrounding extracellular milieu, fluid flow, osmotic forces or internal cytoskeletal re- arrangements. In the context of mediating cellular responses, microtubule (MT) dynamics is a critical factor2.This preprint investigates the importance of MT glutamylation, one of the several MT post-translational modifications regulating MT dynamics, in eliciting changes in cell behaviour and physiology in response to mechanical stress.

Key findings

To understand the influence of extracellular matrix (ECM) stiffness on microtubule (MT) dynamics, MT diffusion ability and growth rate was measured using FRAP (fluorescence recovery after photobleaching) and GFP tagged MT tip end, respectively, in HeLa cells cultured on a gradient of increasing matrix stiffness (1kPa, 12kPa, 50kPa, Plastic). Increasing stiffness increased the diffusion rate, while simultaneously decreasing the growth rate. The effect was most pronounced on matrix stiffness of 50kPa, though there seems to be a large variability in data points.

What is the cause of underlying changes in MT dynamics? To answer this, the authors particularly focussed on MT glutamylation, which has been previously reported to regulate MT stability3. The authors found that different forms of mechanical stress, including ECM stiffening, osmotic shock and circular shear stress, led to increase in MT glutamylation, as observed in immunoblot analysis. Interestingly, immunofluorescence analysis revealed a shift in MT array organization, from net-like to straight cortical arrays (Figure). These results were consistent in cancer and primary cell lines.

The increase in MT glutamylation in the cells upon mechanical stress requires additional glutamate, which as the authors show, is due to increase in glutamine uptake, as indicated from metabolomic profiles, and also due to an increase in glutaminase (GLS) activity, an enzyme required for glutamine catabolism. Drug-based GLS inhibition (BPTES and CB839 – pharmacological and genetic inhibitors, respectively) suppressed the effect of increased matrix stiffness on MT array organization, which could be rescued by glutamate supplementation. These experiments conclusively suggest a direct impact of mechanical stress on glutamine metabolism and associated influence on MT dynamics.

The dynamic nature of MT glutamylation is maintained by antagonistic sets of enzymes, (a) glutamylase enzymes (TTLLs) and (b) deglutamylase enzymes (CCPs), which add and remove glutamate. siRNA mediated knockdown of individual enzymes from each set revealed primary enzymes (TTLL4, TTLL5, TTLL9 and CCP5) involved in MT glutamylation when cells are subjected to mechanical stress. Knockdown of TTL4 suppressed the effect of increased stiffness on MT array organization, while knockdown of CCP5, had an opposite effect on cells cultured on low stiffness. These experiments validate the sufficiency of primary enzymes associated with MT glutamylation in mediating a mechanical stress- induced cell response, in terms of MT array organization.

What is the relevant physiological relevance of MT glutamylation and associated changes in MT dynamics on cell physiology and function? Knockdown of TTLL and CCP, or overexpression of tubulin forms, either lacking the glutamylation region or with a glutamate mutation, significantly impacts cell elasticity, traction, contractility, circularity, migration and cell proliferation. Thus, MT glutamylation resulting from mechanical stress impacts the biophysical properties of the cell, mediated by enzymes directly associated with glutamate addition/removal.

 

Impact of mechanical stress on MT dynamics

Mechanical stress results in increase of MT glutamylation (red boxes) which is associated with decreased MT growth rate (represented by dashed lines) and re-arrangement of MT array from net-like to straight cortical parallel arrays. MT glutamylation proves to be sufficient for eliciting significant changes in mechanical properties of the cell.

What I like about this preprint

This study has been instrumental in demonstrating a very clear relationship between form and function, in that the mechanical stress results in changes in MT glutamylation and associated shifts in MT array organization and growth rate, which consequently results in very evident changes in cell structure, elasticity, proliferation and migration. It was informative to understand that mechanical stresses to the cell can rewire glutamate-glutamine biochemical metabolism within the cell, providing a concrete evidence for the extremely tight association of mechanical and biochemical inputs to cell physiology. I particularly liked how the authors used different forms of mechanical stress for their study (ECM stiffness, osmotic stress, shear stress). The rescue experiments which showed that the addition of glutamate completely reversed the effect of drug-targeted knockdown of GLS (reduced glutamylation) conclusively demonstrate the sufficiency of glutamate (MT glutamylation) for observed changes in stress- induced MT dynamics. The authors went on to pinpoint exactly which enzymes involved in MT glutamylation are actively employed when cells are subjected to stress. In summary, the results from the study highlight the importance of MT post-translation modification (PTMs), in particular, MT glutamylation, in mediating cellular response to mechanical stress.

Future prospective and questions for the authors

  1. It would be interesting to determine how mechanical stress affects MT What is the first-line sensor for stress which results in increased MT glutamylation?
  2. The authors show that MT glutamylation, due to mechanical stress, results in changes in alignment of the MT, i.e., from net-like to straight arrays, which show a slower growth rate. Based on current knowledge of MT PTMs, is there a scope for other PTMs apart from glutamylation, or in combination, to have the same or an additive impact?
  3. Could the authors comment on the importance of actin and associated cytoskeletal proteins with regards to the dramatic changes in mechanical properties of the cell (say, cell shape or cell migration)?
  4. What do the authors think is different in terms of response when cells are subjected to different types of mechanical stress? For instance, could the threshold of ECM stiffness required to observe apparent changes in glutamylation vary among different cell types? It would be very interesting to see if the results in the study can be applied to universal cell processes and 3-D settings, particularly in the context of cancer metastasis, lumen formation, cell sorting and gastrulation during the development of an embryo, which relies on specific mechanical cues.

 

  1. Petridou, N. I., Spiró, Z. & Heisenberg, C. P. Multiscale force sensing in development. Cell Biol.19, 581–588 (2017).
  2. Janke, C. The tubulin code: Molecular components, readout mechanisms, functions. Cell Biol. 206, 461–472 (2014).
  3. Valenstein, M. L. & Roll-Mecak, A. Graded Control of Microtubule Severing by Tubulin Glutamylation. Cell 164, 911–921 (2016).

 

Tags: cell mechanics, microtubule glutamylation, tubulin code

Posted on: 6 May 2020

doi: https://doi.org/10.1242/prelights.20162

Read preprint (1 votes)

Author's response

Stephanie Torrino and Thomas Bertero shared

Using a combination of super resolution microscopy, metabolomics, stable carbon isotope proteomic analysis and cell mechanics assays, we defined a crucial molecular connection between mechanotransduction, glutamine metabolism and microtubules (MTs) glutamylation. We found that mechano-activation of glutamine catabolism is able to induce MTs glutamylation to stabilize the MTs lattice which is required to balance external forces. Inhibiting either glutamine catabolism, MTs dynamics or MTs glutamylation reorganize the MT lattice and disrupt cell integrity, while forcing MTs glutamylation perturbs cell mechanics and several associated functions such as cell migration and proliferation.

Therefore, our findings shift multiple paradigms in cell biology, mechanobiology, cytoskeleton as well as cell metabolism by:

  1. Establishing mechanical forces as direct modulators of MTs glutamylation.
  2. Identifying mechano-activated glutamine catabolism as a necessary metabolic pathway for MTs glutamylation, connecting for the first time cell metabolism to MTs dynamics
  3. Establishing balanced MT glutamylation by TTL4 and CCP5 as a central modulator of MTs lattice which is required to maintain cell integrity.
  4. Establishing mechano-dependent metabolism as a lynchpin connecting mechanical environment, MTs dynamics and cell mechanics.
  5. Deciphering parts of the enigmatic tubulin code that coordinates the multiple functions of MTs in cells

 

Question: It would be interesting to determine how mechanical stress affects MT glutamylation. What is the first-line sensor for stress which results in increased MT glutamylation?

Response: You bring us a valid yet complex point. As you pointed out, we demonstrated that different mechanical cues increased MT glutamylation. Specifically, we demonstrated that 5mn osmotic shock is sufficient to significantly increase MT glutamylation (Fig.1d), indicating that is a pretty fast mechanism. In parallel, we also demonstrate that is a persistent mechanism, as we can observe increased MT glutamylation on cell cultivated on stiff matrix 48-72 hours after plaiting (Fig.1c).

Therefore, it seems that two distinct mechanisms (short-term vs long-term) control MT glutamylation. On short term, our metabolomics analyses indicated that the intracellular pool of glutamate and glutamine are rapidly (2mn) mobilized (Fig2a and extended Fig2b).  In contrast, at long term our metabolomics analyses indicated that the intracellular pool of glutamate is increased (Fig2a and extended Fig2a). Furthermore, our results clearly indicate that MT glutamylation rely on the concentration of the intracellular pool of glutamate (Fig2c). Therefore, while more in depth investigations will be necessary, it is tempting to speculate that the short term mechanism rely on the increased GLS activity and/or SCLC1A1/3 activity, which are regulated by the ADP/ATP ratio and the intracellular/extracellular gradient of glutamate/aspartate, respectively. The long term mechanism relies on the transcriptional increased of GLS and SLC1A1/3 expression levels, which have been shown to be YAP/TAZ dependent1,2.  In sum, our results indicate that MT glutamylation depend on the intracellular concentration of glutamate which can be regulated by a myriad of mechanisms including activation of YAP/TAZ-dependent pathway.

 

Question: The authors show that MT glutamylation, due to mechanical stress, results in changes in alignment of the MT, i.e., from net-like to straight arrays, which show a slower growth rate. Based on current knowledge of MT PTMs, is there a scope for other PTMs apart from glutamylation, or in combination, to have the same or an additive impact?

Response: PTMs of tubulin on MTs are emerging as crucial controllers of MT properties and functions in cells. The wide range of PTMs (such as acetylation, tyrosination, glutamylation, glycylation, methylation amination and phosphorylation) might, alone or in combination, generate chemical differences that are sufficient to confer specific cellular functions on MTs. Thus, many, if not all, functions of MTs are mediated by a highly complex and diverse set of PTMs, the so-called” tubulin code”3. This predestines PTMs to be a fine tuning mechanism of microtubule functions.

Previously, MT acetylation has been shown to be an important process to sustain MT persistence and induce MT resistance to depolymerization (nocodazole)4. It has also been proposed that MT acetylation, by controlling MT stability, plays an important role in mechanosensation5. In addition, tubulin detyrosination has been shown to modulate the cytoskeletal stiffness6,7. Therefore, while PTMs of tubulin have been shown to modulate the mechanical properties of MTs and thus affect MT mechanics8/ cell mechanics7, it has never been shown that mechanical cues affect MT PTMs. Indeed, both tensile forces and compressive forces fail to modulate tubulin acetylation/tyrosination4,6. Similarly, fluid shear stress fails to modulate tubulin tyrosination7.

Consistent with these previous reports, we do not observe MT acetylation/tyrosination by either ECM stiffening nor osmotic shock (data not shown). In contrast, mechanical forces increase MT glutamylation, thus reinforcing the importance of this PTM in the control of mechanotransduction.

 

Question: Could the authors comment on the importance of actin and associated cytoskeletal proteins with regards to the dramatic changes in mechanical properties of the cell (say, cell shape or cell migration)?

Response: We thank you for this question and would like to emphasize that while major emphasis in the mechanotransduction field has been placed on actin dynamics, the dynamics of MT in response to mechanical cues also suggests a central role for tubulin. MTs are the stiffest of all cytoskeletal filaments, which confers them the ability to organize and stabilize the cell structure. Yet, implications of MTs in the mechano-transduction processes are only emerging and the molecular mechanisms linking MT dynamics and mechanotransduction remain elusive. Here we demonstrate that mechanical cues sustain cell mechanics through glutaminolysis-dependent MT glutamylation, linking cell metabolism to MT dynamics and cell mechanics. Furthermore, our results decipher part of the enigmatic tubulin code that coordinates the fine tunable properties of MT mechanics, allowing cells to adjust the stiffness of their cytoskeleton to the mechanical loads of their environment.

Nevertheless, it appears that many of our observations could be explained by regulation of RhoA activity by GEF-H1. GEF-H1 is a Rho-GEF that associates with MTs. MT depolymerization frees GEF-H1, leading to an increase in RhoA activity and subsequent increases in actin polymerization, myosin contractility, and in at least some cancer models, increased proliferation. Alterations in MT glutamylation and hence stability could thus act to regulate cell circularity, contractility, migration, and proliferation, as we observe. Therefore, we investigated whether GEF-H1 and RhoA activity are involved in our mechanism. However, the mechanism seems more complex and not dependent of GEF-H1 (proliferation and migration) or partially dependent on GEF-H1 (cell contractility and cell spreading). Together these results indicate that while a tight crosstalk between tubulin and actin cytoskeleton exist, the mechanical property of MT by itself play an important role in cell mechanics.

 

Question: What do the authors think is different in terms of response when cells are subjected to different types of mechanical stress? For instance, could the threshold of ECM stiffness required to observe apparent changes in glutamylation vary among different cell types? It would be very interesting to see if the results in the study can be applied to universal cell processes and 3-D settings, particularly in the context of cancer metastasis, lumen formation, cell sorting and gastrulation during the development of an embryo, which relies on specific mechanical cues.

Response: Again you bring us a valid yet complex point. We agree with you that important discrepancy between 2D versus 3D migration could be observed. Therefore, investigating migration in 3D would increase the physiological relevance. However, monitoring migration in 3D is complex. Migration through 3D environments is challenging because it requires the cell to squeeze through complex or dense extracellular structures. Doing so requires specific cellular adaptations to mechanical features of the extracellular matrix (ECM) or its remodeling. In addition, besides navigating through diverse ECM environments and overcoming extracellular barriers, cells often interact with neighboring cells and tissues through physical and signaling interactions. Accordingly, cells need to call on an impressively wide diversity of mechanisms to meet these challenges.  Therefore, while it will be of major interest to investigate this cell mechanic-dependent read out in 3D context, we believe that is out of the scope of this study, and could be the basis for further study. We know acknowledge this limitation point in our discussion.

Yet, the true breadth of influence of MT PTMs on cell mechanics and mechanodependent cellular function may involve additional molecular components. While we demonstrated the importance of MT glutamylation on several mechanodependent cell functions such as proliferation and migration in 2 dimensional cell culture, further studies are necessary to decipher the role of MT glutamylation in 3 dimensional space, thereby unveiling novel hidden molecular codes embedded in this complex network of post-translationally modified tubulin isoforms.”

In addition, we are currently investigating the pathophysiological relevance of this mechanism in mechano-dependents diseases such as breast cancer and cardiovascular diseases.

 

  1. Bertero, T. et al. Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension. J. Clin. Invest. 126, 3313–3335 (2016).
  2. Bertero, T. et al. Tumor-Stroma Mechanics Coordinate Amino Acid Availability to Sustain Tumor Growth and Malignancy. Cell Metab. 29, 124-140.e10 (2019).
  3. Janke, C. & Magiera, M. M. The tubulin code and its role in controlling microtubule properties and functions. Nat. Rev. Mol. Cell Biol. (2020) doi:10.1038/s41580-020-0214-3.
  4. Xu, Z. et al. Microtubules acquire resistance from mechanical breakage through intralumenal acetylation. Science 356, 328–332 (2017).
  5. Janke, C. & Montagnac, G. Causes and Consequences of Microtubule Acetylation. Curr. Biol. 27, R1287–R1292 (2017).
  6. Robison, P. et al. Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes. Science 352, aaf0659 (2016).
  7. Lyons, J. S. et al. Microtubules tune mechanotransduction through NOX2 and TRPV4 to decrease sclerostin abundance in osteocytes. Sci Signal 10, (2017).
  8. Portran, D., Schaedel, L., Xu, Z., Théry, M. & Nachury, M. V. Tubulin acetylation protects long-lived microtubules against mechanical ageing. Nat. Cell Biol. 19, 391–398 (2017).

 

 

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