The regulation of traction force in relation to cell shape and focal adhesions
Introduction
The shape of adherent cells is known to have profound effects on a number of important properties including cytoskeletal structure, growth, and differentiation [1], [2], [3], [4]. When allowed to adhere and spread without constraints, most adhesive cells reach an extended polygonal shape and form large cables of actin filaments, accompanied by active DNA synthesis and growth [1]. Using patterned substrates to control cell shape, it was discovered that the rate of fibroblast growth increases with the spreading area [1], [5]. More importantly, spreading area appears to control the differentiation of stem cells. Human mesenchymal stem cells allowed to spread over a large area differentiate into osteocytes, whereas those confined within small areas differentiate into adipocytes [6]. In addition to spreading area, aspect ratio has also been found to affect the differentiation of mesenchymal stem cells [7].
Despite the significance, how shape information is transduced into intracellular chemical signals remains largely unclear. An important clue is that shape-dictated differentiation requires RhoA-dependent contractility of the actin cytoskeleton, which is known to generate “traction forces” – mechanical forces exerted by adherent cells on the underlying substrate [8], [9], [10]. Therefore, an attractive possibility is that traction forces are involved in reading out cell shape. Consistent with this hypothesis, responses of adherent cells to cell geometry share similarities to those elicited by applied mechanical forces, including the reinforcement of the actin cytoskeleton and focal adhesions [11], [12] and the activation of similar signaling pathways [13], [44]. In addition, traction forces were found to increase with the cell spreading area [15], [16], [17], [18], [19]. Since traction forces are actively generated near the leading edge [20], [21], [22], [23], their distribution and magnitude may respond to cell shape and serve as the readout as cells actively change their geometry.
The goal of this study is to identify the aspect of cell shape that determines the responses and the structural basis of the readout mechanism. Previous studies have been limited by technical challenges, specifically difficulties in micro-patterning soft gels for traction force measurements under controlled geometry [17]. To address the challenge we have developed a novel method that allows easy and reproducible generation of adhesive micropatterns on hydrogels at a high resolution. We then applied this approach to systematically investigate the role of cellular geometry in traction force regulation by varying the area, aspect ratio, and adhesion pattern of cells in various rectangular shapes. The knowledge of how cells sense and respond to their own shape should provide the field of tissue engineering with a diverse toolbox for controlling cellular fate when combined with other chemical and mechanical manipulations.
Section snippets
Preparation of patterned polyacrylamide hydrogels
A novel patterning method was developed for patterning polyacrylamide hydrogels. Initially, 50 bloom gelatin at a concentration of 0.1% was activated by incubation with 3.6 mg/ml sodium m-periodate (Sigma, St Louis, MO) at room temperature for 30 min, as previously described [24]. Polydimethylsiloxane (PDMS) stamps were fabricated by standard soft lithography procedures. Briefly, a positive photoresist, SPR-220.3 (Microchem, Newton, MA), was spun on a glass coverslip. It was then exposed to UV
Developing a new patterning technique for polyacrylamide hydrogels
To address the question of how cell geometry regulates the generation of traction stress, it is essential to constrain cells geometrically on adhesive islands of defined shape cast on elastic hydrogels. Among cell culture suitable materials, protein-conjugated polyacrylamide hydrogels have emerged as an ideal, tunable substrate for many purposes. However, it has been technically challenging to create high-quality adhesive patterns on these non-adhesive gels for controlling cell migration or
Discussion
Traction forces, generated by the actin-myosin II cytoskeleton [10], are transmitted to the extracellular matrix through the associated focal adhesion complexes [30]. Active traction forces are concentrated at the frontal periphery and are oriented toward the cell center [20], [21]. Their characteristics are consistent with a propulsive role during cell migration, which has been the focus of previous analyses [21], [22], [23]. However, the present observation, that traction forces are sensitive
Conclusion
A systematic study of the effect of adherent cell shape on traction stresses using a new hydrogel micropatterning method led to the conclusion that traction stress correlates linearly with the center-to-corner distance of rectangular cells. Shape control of traction stress is in turn mediated by the regulation of focal adhesion size, which dominates over the overall cell size and shape. Importantly, this same mechanism is capable of detecting a wide range of mechanical and geometrical signals.
Acknowledgements
We thank Prof. Micah Dembo, Boston University, for providing the LIBTRC program package for computing traction forces. This work was supported by grant GM-32476 from the National Institutes of Health to Y.L.W.
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