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Mechanisms of Cellular Rigidity Sensing

Final Report Summary - RIGIDITY SENSING (Mechanisms of Cellular Rigidity Sensing)

The project “Rigidity Sensing” set out to address questions regarding the basic mechanisms by which cells are able to determine the rigidity of their outside environment. The funding provided for this project allowed the researcher, Dr. Haguy Wolfenson, to pursue this scientific goal and also provided essential support during a crucial period of his scientific career as a postdoctoral researcher. The plans for the project were mostly based on two technologies for generation of specialized surfaces to study cellular processes with extremely high precision – microfabrication of elastic pillar arrays and nanofabrication of surfaces with gold nano-dots. Unfortunately, we encountered numerous difficulties when dealing with the latter technology, and therefore were not able to obtain enough nanofabricated surfaces for a large-scale study. We thus focused mostly on the pillar array technology (see below). Despite this setback, the project was highly successful, yielding novel insights about rigidity sensing which were summarized in 5 research papers in top journals (Nature Cell Biology, Nature Materials, Nano Letters, molecular Biology of the Cell), plus one more research paper which detailed technical advances that we nevertheless made with gold nano-dot surfaces (ACS Nano). Based on this work, Dr. Wolfenson became a faculty member at the Technion – Israel Institute of Technology, where, together with his group, he is further studying processes related to rigidity sensing.
In recent years it has become evident that cells respond not only to biochemical signals such as hormones, but also to mechanical signals from their environment. In particular, the rigidity of the extracellular environment has proven to affect many cellular processes, including adhesion, survival, and proliferation [1-3]. Therefore, Dr. Wolfenson's goal during his time as a Marie Curie International Outgoing Fellow was to study the basic mechanisms by which cells test the rigidity. One of the major techniques that Dr. Wolfenson used during the outgoing phase of the fellowship at the lab of Prof. Mike Sheetz (Columbia University) is arrays of elastic PDMS pillars (“bed of needles”). These were used as substrates for cell spreading, since cells apply forces to the outside environment through specialized contact sites called ‘integrin adhesions’ to sense the rigidity, and these forces can be measured by tracking the movements of the pillar tops when the cells spread on them.
In the first study that we performed we showed that rigidity sensing involves actomyosin-based contractile units that resemble muscle sarcomeres. These units displace neighboring pillars by nanometer-level steps which are constant regardless of rigidity. Importantly, the steps and forces became significantly higher and depended on rigidity upon depletion of the actin-regulatory protein tropomyosin 2.1 (Tpm2.1 [4,5]). Surprisingly, Tpm2.1 depletion also allowed cells to grow under anchorage-independent conditions, whereas normal cells could not. These results, which were published in Nature Cell Biology [6], have important implications for our understanding of mechanosensing and provide a basis for future studies on how the mechanosensing machinery can affect cellular decisions.
In a follow-up study, published in Molecular Biology of the Cell [7], we explored the relationship between the local rigidity-sensing contractile units and the global cellular actin network. We showed that an essential component of these two cytoskeletal modules is the protein α-actinin, which initially localizes to the contractile units and then translocates into the larger actin network. When we depleted α-actinin from the cells, they were not able to sense rigidity properly since the forces were not properly regulated. This led the cells to be able to grow on soft matrices.
During his time as a postdoctoral fellow, Dr. Wolfenson also mentored several graduate students in the Sheetz lab. This experience not only prepared him for his current position as the head of his own lab, but also resulted in successful insights about the regulatory pathways that control rigidity sensing. The first of these studies focused on the role of the tyrosine kinases AXL and ROR2 in controlling rigidity sensing. These proteins came up in a previous screen as potential regulators of this process, and we found that both of them localize to the contractile units and associate there with Tpm2.1 myosin IIA, and filamin A. Similar to the case of α-actinin, depletion of either AXL or ROR2 allowed growth of cells on soft matrices by increasing the magnitude (AXL) or duration (ROR2) of the contraction events. This study was published in Nano Letters [8].
Another study that we performed focused on the role of epidermal growth factor receptor (EGFR, also known as ERBB1) and HER2 (also known as ERBB2) in the process. We showed that EGFR and HER2 activate rigidity sensing contractions on rigid, but not soft, substrates. At early time points when cells first make contact with the surface, rigidity sensing is activated without EGF (the ligand for EGFR); after a few hours, the cells enter a quiescent stage in which they are much less active, but addition of EGF at this point can re-activate rigidity sensing. Importantly, we also showed that EGFR and HER2 are activated through phosphorylation by Src family kinases (SFKs). On soft surfaces, neither EGFR inhibition nor EGF stimulation had any effects. This study was published in Nature Materials [9].
The third study which Dr. Wolfenson performed as a mentor for a graduate student in the lab focused on the role of one of the major adhesion-related proteins, talin. We found that it undergoes force-induced cleavage in early adhesions, and that in the absence of this cleavage event cell growth, adhesion maturation, and proper rigidity sensing are hampered. Based on these findings we suggest that an important function of talin is its control over cell cycle progression through its cleavage in early adhesions during the process of rigidity sensing. This study was also published in Nano Letters [10].
In addition, even though we did not use the nanodots surfaces as planned, we were able to define the conditions that allow functionalization of these surfaces with single molecules [11].
In Dr. Wolfenson's current lab at the Technion (http://wolfenson.net.technion.ac.il/) he is pursuing further questions regarding the long-term aspects and effects of rigidity sensing. In particular, the focus is on the link between rigidity sensing and cell growth as this aspect is closely linked to cancer. One of the major hallmarks of cancer cells is their ability to grow on very soft matrices, i.e. conditions that do not allow the formation of strong adhesions. Under such conditions, non-cancerous cells cannot grow and they typically die. This indicates that cancer cells do not properly sense the rigidity. Therefore, understanding the basics of rigidity sensing and the link to cell growth holds great promise for identifying novel mechano-biological avenues to attack cancer.

References:
1 Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265-275 (2006).
2 Wozniak, M. A. & Chen, C. S. Mechanotransduction in development: a growing role for contractility. Nat. Rev. Mol. Cell Biol. 10, 34-43, doi:nrm2592 [pii] 10.1038/nrm2592 (2009).
3 Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179-183 (2011).
4 Spudich, J. A., Huxley, H. E. & Finch, J. T. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632 (1972).
5 Perz-Edwards, R. J. et al. X-ray diffraction evidence for myosin-troponin connections and tropomyosin movement during stretch activation of insect flight muscle. Proc. Natl. Acad. Sci. USA 108, 120-125, doi:10.1073/pnas.1014599107 (2011).
6 Wolfenson, H. et al. Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices. Nat. Cell Biol. 18, 33-42, doi:10.1038/ncb3277 (2016).
7 Meacci, G. et al. alpha-Actinin links extracellular matrix rigidity-sensing contractile units with periodic cell-edge retractions. Mol. Biol. Cell 27, 3471-3479, doi:10.1091/mbc.E16-02-0107 (2016).
8 Yang, B. et al. Mechanosensing Controlled Directly by Tyrosine Kinases. Nano Lett 16, 5951-5961, doi:10.1021/acs.nanolett.6b02995 (2016).
9 Saxena, M. et al. EGFR and HER2 activate rigidity sensing only on rigid matrices. Nat Mater, doi:10.1038/nmat4893 (2017).
10 Saxena, M., Changede, R., Hone, J. C., Wolfenson, H. & Sheetz, M. P. Force induced calpain cleavage of talin is critical for growth, adhesion development and rigidity sensing. Nano Lett, doi:10.1021/acs.nanolett.7b02476 (2017).
11 Cai, H. et al. Molecular Occupancy of Nanodot Arrays. ACS Nano 10, 4173-4183, doi:10.1021/acsnano.5b07425 (2016).