Wei-hui Guo

The regulation of traction force in relation to cell shape and focal adhesions.

Mechanical forces provide critical inputs for proper cellular functions. The interplay between the generation of, and response to, mechanical forces regulate such cellular processes as differentiation, proliferation, and migration. We postulate that adherent cells respond to a number of physical and topographical factors, including cell size and shape, by detecting the magnitude and/or distribution of traction forces under different conditions. To address this possibility we introduce a new simple method for precise micropatterning of hydrogels, and then apply the technique to systematically investigate the relationship between cell geometry, focal adhesions, and traction forces in cells with a series of spread areas and aspect ratios. Contrary to previous findings, we find that traction force is not determined primarily by the cell spreading area but by the distance from cell center to the perimeter. This distance in turn controls traction forces by regulating the size of focal adhesions, such that constraining the size of focal adhesions by micropatterning can override the effect of geometry. We propose that the responses of traction forces to center-periphery distance, possibly through a positive feedback mechanism that regulates focal adhesions, provide the cell with the information on its own shape and size. A similar positive feedback control may allow cells to respond to a variety of physical or topographical signals via a unified mechanism.

Guidance of Cell Migration by Substrate Dimension

There is increasing evidence to suggest that physical parameters, including substrate rigidity, topography, and cell geometry, play an important role in cell migration. As there are significant differences in cell behavior when cultured in 1D, 2D, or 3D environments, we hypothesize that migrating cells are also able to sense the dimension of the environment as a guidance cue. NIH 3T3 fibroblasts were cultured on micropatterned substrates where the path of migration alternates between 1D lines and 2D rectangles. We found that 3T3 cells had a clear preference to stay on 2D rather than 1D substrates. Cells on 2D surfaces generated stronger traction stress than did those on 1D surfaces, but inhibition of myosin II caused cells to lose their sensitivity to substrate dimension, suggesting that myosin-II-dependent traction forces are the determining factor for dimension sensing. Furthermore, oncogene-transformed fibroblasts are defective in mechanosensing while generating similar traction forces on 1D and 2D surfaces. Dimension sensing may be involved in guiding cell migration for both physiological functions and tissue engineering, and for maintaining normal cells in their home tissue.

Microtubule depolymerization induces traction force increase through two distinct pathways

Traction forces increase after microtubule depolymerization; however, the signaling mechanisms underlying this, in particular the dependence upon myosin II, remain unclear. We investigated the mechanism of traction force increase after nocodazole-induced microtubule depolymerization by applying traction force microscopy to cells cultured on micropatterned polyacrylamide hydrogels to obtain samples of homogeneous shape and size. Control cells and cells treated with a focal adhesion kinase (FAK) inhibitor showed similar increases in traction forces, indicating that the response is independent of FAK. Surprisingly, pharmacological inhibition of myosin II did not prevent the increase of residual traction forces upon nocodazole treatment. This increase was abolished upon pharmacological inhibition of FAK. These results suggest two distinct pathways for the regulation of traction forces. First, microtubule depolymerization activates a myosin-II-dependent mechanism through a FAK-independent pathway. Second, microtubule depolymerization also enhances traction forces through a myosin-II-independent, FAK-regulated pathway. Traction forces are therefore regulated by a complex network of complementary signals and force-generating mechanisms.

Fibroblasts probe substrate rigidity with filopodia extensions before occupying an area

Significance Mechanical properties of the extracellular environment provide important cues that regulate cell behavior. Understanding this mechanical signaling has become important in disease treatment as well as tissue engineering. To efficiently study cellular responses to rigidity signals, we have created a model system of micropatterned composite material based on the “cell-on-a-chip” concept. We demonstrate that a migrating fibroblast uses filopodia to probe substrate rigidity, such that it “feels” its way based on the deformability of a material before occupying an area. Myosin II plays a key role in rigidity sensing and responses. This mechanism allows cells to migrate efficiently by avoiding mechanically unfavorable areas without backtracking. Rigidity sensing and durotaxis are thought to be important elements in wound healing, tissue formation, and cancer treatment. It has been challenging, however, to study the underlying mechanism due to difficulties in capturing cells during the transient response to a rigidity interface. We have addressed this problem by developing a model experimental system that confines cells to a micropatterned area with a rigidity border. The system consists of a rigid domain of one large adhesive island, adjacent to a soft domain of small adhesive islands grafted on a nonadhesive soft gel. This configuration allowed us to test rigidity sensing away from the cell body during probing and spreading. NIH 3T3 cells responded to the micropatterned rigidity border similarly to cells at a conventional rigidity border, by showing a strong preference for staying on the rigid side. Furthermore, cells used filopodia extensions to probe substrate rigidity at a distance in front of the leading edge and regulated their responses based on the strain of the intervening substrate. Soft substrates inhibited focal adhesion maturation and promoted cell retraction, whereas rigid substrates allowed stable adhesions and cell spreading. Myosin II was required for not only the generation of probing forces but also the retraction in response to soft substrates. We suggest that a myosin II-driven, filopodia-based probing mechanism ahead of the leading edge allows cells to migrate efficiently, by sensing physical characteristics before moving over a substrate to avoid backtracking.

Microtubules stabilize cell polarity by localizing rear signals

Significance We discovered that cells on micropatterned strips change from highly persistent migration into striking oscillations upon the disassembly of microtubules. The oscillation phenomenon then allowed us to apply computer modeling to understand how the positive feedback in the local-excitation–global-inhibition (LEGI) mechanism, responsible for the persistence of migration, might be converted into negative feedback to drive oscillations upon the disassembly of microtubules. Our analyses led to the conclusion that microtubules facilitate the transport of inhibitory signals and their global distribution. Depending on the relative position of excitation and inhibitory signals, the resulting feedback in the integrated control circuit may be either positive or negative. Our finding therefore provides important insights into the role of microtubules in the control circuit of cell migration. Microtubules are known to play an important role in cell polarity; however, the mechanism remains unclear. Using cells migrating persistently on micropatterned strips, we found that depolymerization of microtubules caused cells to change from persistent to oscillatory migration. Mathematical modeling in the context of a local-excitation–global-inhibition control mechanism indicated that this mechanism can account for microtubule-dependent oscillation, assuming that microtubules remove inhibitory signals from the front after a delayed generation. Experiments further supported model predictions that the period of oscillation positively correlates with cell length and that oscillation may be induced by inhibiting retrograde motors. We suggest that microtubules are required not for the generation but for the maintenance of cell polarity, by mediating the global distribution of inhibitory signals. Disassembly of microtubules induces cell oscillation by allowing inhibitory signals to accumulate at the front, which stops frontal protrusion and allows the polarity to reverse.

A three-component mechanism for fibroblast migration with a contractile cell body that couples a myosin II–independent propulsive anterior to a myosin II–dependent resistive tail

Frontal, cell body, and rear regions perform distinct functions in the complex process of cell migration. A low-capacity, directional mechanism in the front coupled to a high-capacity, nondirectional mechanism in the middle represents a highly appealing model for driving cell migration under high mechanical load.

Micropatterning Cell Adhesion on Polyacrylamide Hydrogels

Cell shape and substrate rigidity play critical roles in regulating cell behaviors and fate. Controlling cell shape on elastic adhesive materials holds great promise for creating a physiologically relevant culture environment for basic and translational research and clinical applications. However, it has been technically challenging to create high-quality adhesive patterns on compliant substrates. We have developed an efficient and economical method to create precise micron-scaled adhesive patterns on the surface of a hydrogel (Rape et al., Biomaterials 32:2043-2051, 2011). This method will facilitate the research on traction force generation, cellular mechanotransduction, and tissue engineering, where precise controls of both materials rigidity and adhesive patterns are important.

Responses of Cells to Adhesion-Mediated Signals: A Universal Mechanism

Cells are exposed to a plethora of signals that typically coerce them to function properly, but aberrant signaling can lead to pathological conditions. In the treatment of diseases and the rational design of functioning tissues, it is vital to understand and be able to manipulate these inputs. In the past, much of the interest has been on chemical signaling but recently, there has been an explosion of research into a diverse array of mechanical signals. Mechanical signals have been shown to influence cellular growth, survival, migration, and differentiation. Despite its obvious importance, relatively little is known about the mechanism of mechanosensing. In this chapter, we describe what is currently known about potential mechanosensing molecules and then describe a model by which a wide array of mechanical signals can be interpreted by a common mechanism. By understanding this mechanism, one may be able to develop new therapeutic interventions for devastating diseases such as cancer and break through critical barriers facing the field of tissue engineering. We expect the knowledge gained from the study of basic biology to greatly impact the treatment of many patients in the clinical setting in the coming years.

Preparation of a Micropatterned Rigid-Soft Composite Substrate for Probing Cellular Rigidity Sensing

Substrate rigidity has been recognized as an important property that affects cellular physiology and functions. While the phenomenon has been well recognized, understanding the underlying mechanism may be greatly facilitated by creating a microenvironment with designed rigidity patterns. This chapter describes in detail an optimized method for preparing substrates with micropatterned rigidity, taking advantage of the ability to dehydrate polyacrylamide gels for micropatterning with photolithography, and subsequently rehydrate the gel to regain the original elastic state. While a wide range of micropatterns may be prepared, typical composite substrates consist of micron-sized islands of rigid photoresist grafted on the surface of polyacrylamide hydrogels of defined rigidity. These islands are displaced by cellular traction forces, for a distance determined by the size of the island, the rigidity of the underlying hydrogel, and the magnitude of traction forces. Domains of rigidity may be created using this composite material to allow systematic investigations of rigidity sensing and durotaxis.