---

Academic Profile: 

Dr. Yasuhiro Sawada received his MD from the University of Tokyo, Japan. After 6 years' clinical training as an orthopaedic surgeon, he joined the graduate program and received his PhD from the University of Tokyo. While he then returned to the clinical practice at the University of Tokyo Hospital, he resumed basic research in 2000 as a post-doctoral fellow at Prof. Michael Sheetz lab, Columbia University in the city of New York. Since then, he has been consistently working on the mechanism of how cells sense 'force' and exploring mechanotransduction, the emerging research field of life science. He joined NUS in November 2007 as an Assoc Prof at the Department of Biological Sciences and Division of Bioengineering (joint appointments).

Research Interests: 

• Signal transduction through mechanical modulation of signaling proteins
• Implications of mechanical factors in cell functions and biological phenomena including development, tissue regeneration, cardiovascular disorder, angiogenesis, neurogenesis, carcinogenesis, and bacteria/yeast physiology...'biological events are all mechanical?'

Exploration of Cell Mechano-Sensing 

A. Background and introduction

Mechanical factors are critical in defining both shape and function of the cells and organs of all living things from prokaryotes to eukaryotes. Diverse biological phenomena such as development, tissue regeneration, cardiac hypertrophy, atherosclerosis, neurogenesis, neuronal differentiation, as well as carcinogenesis are related to or modified by mechanical environments. Evidently, how mechanical forces are transduced as biological signals is a fundamental, yet significant question in biology.

Mechanically-initiated signal transduction, termed mechanotransduction, has been reported to involve numerous signaling systems including MAP kinase cascades, small GTPase signaling, non-receptor tyrosine kinase signaling, TGF-breceptor signaling, EGF receptor signaling, Wnt/b-catenin signaling, Hedgehog signaling, JAK/STAT signaling, NF-kB signaling, and immuno recognition receptor (T-cell and B-cell receptor) signaling.  Nearly all the signaling pathways appear more or less mechanically implicated.

To explore signal transduction induced by external stimuli, identification of specific receptors is crucial. However, specific force receptors, i.e. mechano-sensors that are directly modulated by mechanical stimuli and initiate intracellular signaling cascades (Fig. 1), were not identified at a molecular level, with the exception of mechano-sensitive ion channels. We postulated that direct mechano-sensors should be deformed by physical force and might not be totally diffusive in the cytoplasm. Thus, our recent work has sought to identify an ion channel-independent mechano-sensor in the cytoskeleton.  

Figure 1

B. p130Cas as a candidate of the cytoskeletal mechano-sensor

Since direct mechano-sensors were supposed to function at the most upstream of mechanotransduction, we began studying mechanotransduction with the relevant downstream signals such as MAP kinase activation and traced them upstream.

We first developed an original cell stretching system in which cells were cultured on stretchable substrate (silicone) and stretched (Fig. 2). Using this system, we found that all three major MAP kinase pathways (MEK1/2-ERK, MKK4(SEK1)/MKK7-JNK, and MKK3/6-p38) were activated by cell stretching and that a small GTPase Rap1 was involved in stretch-dependent activation of the MKK3/6-p38 pathway (Sawada et al., 2001).

We then found that stretch-dependent assembly of focal contact proteins in intact cells was reproduced in a detergent-insoluble cytoskeletal complex (Triton cytoskeleton) in which the role of ion movement was completely excluded (Sawada and Sheetz, 2002). These results suggested that cytoskeletons did involve mechano-sensors at focal contacts where cell-generated forces were supposed to be concentrated. Furthermore, we found that cytoskeletons could transmit a mechano-signal to Rap1 by binding Crk/C3G complex and that phosphorylation of the Src family kinase substrate p130Cas (Crk-associated substrate) in the cytoskeleton was required for the stretch-dependent Crk/C3G binding to cytoskeletons (Tamada et al., 2004). Thus, cytoskeletal mechano-sensing appeared to involve tyrosine phosphorylation of p130Cas that lead to activation of the Rap1-p38 pathway (Fig. 3).

C. How is p130Cas phosphorylated upon cell stretching?

Since p130Cas is a major substrate protein of Src family kinase, we postulated four mechanisms concerning stretch-dependent phosphorylation of p130Cas.

1. Kinase of p130Cas (i.e. Src family kinase) was activated by cell stretching
2. Phosphatase of p130Cas was inactivated by cell stretching
3. p130Cas and its kinase became physically associated with each other upon cell stretching.
4. Susceptibility of p130Cas to phosphorylation was enhanced by cell stretching.

Because our experimental results suggested that mechanisms 1) - 3) were not primarily responsible for the stretch-dependent phosphorylation of p130Cas, we tested the possibility 4). To eliminate any extraneous biochemical interactions or signaling pathways, we constructed an in vitro protein extension system in which bacterially expressed p130Cas substrate domain proteins (CasSD) were extended (Fig. 4). When we incubated CasSD with active Src kinase in vitro, we found that mechanical extension of CasSD enhanced its tyrosine phosphorylation with no apparent change in Src kinase activity. Physiological relevance of in vitro CasSD extension was confirmed by immunostaining using an extension-specific anti-p130Cas antibody. These findings indicated that p130Cas converted stretching forces into a biochemical signal through enhancement of susceptibility to phosphorylation caused by the mechanical extension of its substrate domain (Fig. 5), which we designated 'substrate priming.' We concluded that p130Cas acts as an ion channel-independent, cytoskeletal mechano-sensor. 

D. Mechano-sensors other than p130Cas: Substrate priming may be a general mechanism that regulates Src family kinase signaling

We found that a number of proteins other than p130Cas were tyrosine phosphorylated by Src family kinase (SFK) upon stretching of cytoskeletons from fibroblastic cells (Tamada et al., 2004). Since SFK activation appeared not to be primarily responsible for the stretch-response in the tyrosine phosphorylation of those cytoskeletal proteins (Sawada et al., 2006), they most likely act as direct mechano-sensors by a mechanism similar to p130Cas phosphorylation, i.e. substrate priming.

A variety of signaling pathways have been reported to involve SFKs, including growth factor (receptor) signaling, and immune recognition receptor signaling. In addition, the regulating mechanisms of SFKs have been extensively documented. Csk (C-terminal Src kinase) phosphorylates C-terminal tyrosine of SFKs, causing them to form 'closed' inactive configuration, while RPTP-a dephosphorylates that inhibitory tyrosine and thereby activates SFKs. Furthermore, SFKs themselves have been shown to be activated by mechanical stimulation.  However, the physiological stimulation that enhances SFK activity remains undefined in several 'SFK-dependent' pathways.  We speculate that substrate priming is a fairly general mechanism that regulates signaling events involving SFKs and even other tyrosine kinases.

E. More mechano-sensors.

Different sets of cytoplasmic proteins bound to cytoskeletons depending on the extension status (i.e. stretched or relaxed) of cytoskeletons (Sawada and Sheetz, 2002), suggesting the existence of cytoskeletal proteins that serve as mechano-sensors by altering their conformation (extension status)-dependent binding affinities to cytoplasmic proteins (Fig. 6). Identification of those mechano-sensors is under way in our lab.

F. Significance of our research.

Considering the versatile roles of mechanical factors, exploration of cell mechano-sensing will contribute to better understanding of the mechanisms of how tissues and organs develop and execute their normal functions, how tissues regenerate and wounds are healed, and how pathological conditions are caused. Thus, our research will provide scientific foundations needed for the development of new treatment methods particularly in regenerative medicine and cancer care or prevention. 

E. References 

1. Sawada, Y., K. Nakamura, K. Doi, K. Takeda, K. Tobiume, M. Saitoh, K. Morita, I. Komuro, K. De Vos, M. Sheetz, and H. Ichijo. 2001. Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase. J Cell Sci. 114:1221-7. 

2. Sawada, Y., and M.P. Sheetz. 2002. Force transduction by Triton cytoskeletons. J Cell Biol. 156:609-15. 

3. Sawada, Y., M. Tamada, B.J. Dubin-Thaler, O. Cherniavskaya, R. Sakai, S. Tanaka, and M.P. Sheetz. 2006. Force Sensing by Mechanical Extension of the Src Family Kinase Substrate p130Cas. Cell. 127:1015-26. 

4. Tamada, M., M.P. Sheetz, and Y. Sawada. 2004. Activation of a signaling cascade by cytoskeleton stretch. Dev Cell. 7:709-18.