Biophysical forces rewire cell metabolism to guide microtubule-dependent cell mechanics
Posted on: 6 May 2020
Preprint posted on 11 March 2020
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 KapoorCategories: biochemistry, biophysics, cancer biology, cell biology
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
- 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?
- 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?
- 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)?
- 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.
- Petridou, N. I., Spiró, Z. & Heisenberg, C. P. Multiscale force sensing in development. Cell Biol.19, 581–588 (2017).
- Janke, C. The tubulin code: Molecular components, readout mechanisms, functions. Cell Biol. 206, 461–472 (2014).
- Valenstein, M. L. & Roll-Mecak, A. Graded Control of Microtubule Severing by Tubulin Glutamylation. Cell 164, 911–921 (2016).
doi: https://doi.org/10.1242/prelights.20162
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