Migration regulates cellular mechanical states.

Recent studies indicate that adherent cells are keenly sensitive to external physical environment, such as substrate rigidity and topography, and internal physical states, such as cell shape and spreading area. Many of these responses are believed to involve coupled output and input of mechanical forces, which may constitute the key sensing mechanism to generate downstream regulatory signals for cell growth and differentiation. Here, we show that the state of cell migration also plays a regulatory role. Compared with migrating cells, stationary cells generate stronger, less dynamic, and more peripherally localized traction forces. These changes are coupled to reduced focal adhesion turnover and enhanced paxillin phosphorylation. Further, using cells migrating along checkerboard micropatterns, we show that the appearance of new focal adhesions directly in front of existing focal adhesions is associated with the down-regulation of existing focal adhesions and associated traction forces. Together, our results imply a mechanism where cell migration regulates traction forces by promoting dynamic turnover of focal adhesions, which may then regulate processes such as wound healing and embryogenesis where cell differentiation must coordinate with migration state and proper localization.

Microtubules stabilize cell polarity by localizing rear signals.

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.

Fibroblasts probe substrate rigidity with filopodia extensions before occupying an area.

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.

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.

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.

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.

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.

To understand the mechanism of cell migration, we cultured fibroblasts on micropatterned tracks to induce persistent migration with a highly elongated morphology and well-defined polarity, which allows microfluidic pharmacological manipulations of regional functions. The function of myosin II was probed by applying inhibitors either globally or locally. Of interest, although global inhibition of myosin II inhibited tail retraction and caused dramatic elongation of the posterior region, localized inhibition of the cell body inhibited nuclear translocation and caused elongation of the anterior region. In addition, local application of cytochalasin D at the tip inhibited frontal extension without inhibiting forward movement of the cell nucleus, whereas local treatment posterior to the nucleus caused reversal of nuclear movement. Imaging of cortical dynamics indicated that the region around the nucleus is a distinct compression zone where activities of anterior and posterior regions converge. These observations suggest a three-component model of cell migration in which a contractile middle section is responsible for the movement of a bulky cell body and the detachment/retraction of a resistive tail, thereby allowing these regions to undergo coordinated movement with a moving anterior region that carries little load.

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.

Assessing the spatial resolution of cellular rigidity sensing using a micropatterned hydrogel-photoresist composite.

The biophysical machinery that permits a cell to sense substrate rigidity is poorly understood. Rigidity sensing of adherent cells likely involves traction forces applied through focal adhesions and measurement of resulting deformation. However, it is unclear if this measurement takes place underneath single focal adhesions, over local clusters of focal adhesions, or across the length of the entire cell. To address this question, we developed a composite, chip-based material containing many arrays of 6.5 µm × 6.5 µm rigid adhesive islands, with an edge-edge distance of 8 µm, grafted onto the surface of a non-adhesive polyacrylamide hydrogel. This material is thus rigid within single islands while long-range rigidity is determined by the hydrogel. On soft gels, most NIH 3T3 cells spread only across two islands in a given dimension forming small stress fibers and focal adhesions. On stiff gels, cell spreading, stress fibers, and focal adhesions were indistinguishable from those on regular culture surfaces. We conclude that rigidity sensing is dictated by material compliance across the cell length and that responses to rigidity may be inhibited at any point when large substrate strain is encountered during spreading. Our finding may serve as a guideline for the design of biomaterials for tissue engineering.

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.

Insulin-like growth factor 2 and the insulin receptor, but not insulin, regulate fetal hepatic glycogen synthesis.

Whether insulin or IGFs regulate glycogen synthesis in the fetal liver remains to be determined. In this study, we used several knockout mouse strains, including those lacking Pdx-1 (pancreatic duodenal homeobox-1), Insr (insulin receptor), and Igf2 (IGF-II) to determine the role of these genes in the regulation of fetal hepatic glycogen synthesis. Our data show that insulin deficiency does not alter hepatic glycogen stores, whereas Insr and Igf2 deficiency do. We found that both insulin receptor isoforms (IR-A and IR-B) are present in the fetal liver, and their expression is gestationally regulated. IR-B is highly expressed in the fetal liver; nonetheless, the percentage of hepatic IR-A isoform, which binds Igf2, was significantly higher in the fetus than the adult. In vitro experiments demonstrate that Igf2 increases phosphorylation of hepatic Insr, insulin receptor substrate-2, and Akt proteins and also the activity of glycogen synthase. Igf2 ultimately increased glycogen synthesis in fetal hepatocytes. This increase could be blocked by the phosphoinositide 3-kinase inhibitor LY294008. Taken together, we propose Igf2 as a major regulator of fetal hepatic glycogen metabolism, the insulin receptor as its target receptor, and phosphoinositide 3-kinase as the signaling pathway leading to glycogen formation in the fetal liver.

IGF2 knockout mice are resistant to kainic acid-induced seizures and neurodegeneration.

Insulin-like growth factor 2 (Igf2), a member of the insulin gene family, is important for brain development and has known neurotrophic properties. Though Igf2, its receptors, and binding proteins, are expressed in the adult CNS, their role in the adult brain is less well-understood. Here we studied how Igf2 deficiency affects brains of adult Igf2 knockout (Igf2(-/-)) mice following neurotoxic insult produced by the glutamate analog kainic acid (KA). Igf2(-/-) mice exhibited attenuated epileptiform activity in response to KA and were less susceptible to hippocampal neurodegeneration compared with Igf2(+/+) mice. Other brain areas protected by the lack of Igf2 included the amygdala complex, septal nuclei, and thalamic region. Apoptosis, as determined by TUNEL and Hoechst 33342 staining, was accordingly less for Igf2(-/-) mice. Hippocampal slices from Igf2(-/-) mice also were protected against the effects epileptogenic effects of KA compared to Igf2(+/+) mice suggesting that neuroprotection afforded by a lack of Igf2 may be developmental in origin and experiments demonstrating enhanced synaptic inhibition in slices taken from Igf2(-/-) mice support this hypothesis. Taken together, these results suggest that Igf2 may be important for mechanisms and circuits that contribute to neurodegeneration and epilepsy.

Retrograde fluxes of focal adhesion proteins in response to cell migration and mechanical signals.

Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell.

Igf2 deficiency results in delayed lung development at the end of gestation.

IGF-II is a polypeptide hormone with structural homology to insulin and IGF-I. IGF-II plays an important role in fetal growth as mice with targeted disruption of the IGF-II gene (Igf2) exhibit severe growth retardation. The role of IGFs in the fetal lung has been suggested by several studies, including those that have identified IGF mRNA expression, and that of their receptors and binding proteins in the lungs at different stages of development. In this study, we used mice carrying a null mutation of Igf2 (Igf2-/- mice) to determine whether the absence of IGF-II had any effect in fetal lung maturation. Our results showed that the lungs of Igf2-/- fetuses had thicker alveolar septae and poorly organized alveoli when compared with those of Igf2+/+ on d 17.5 and 18.5 of gestation. These morphological alterations may be the result of exposure to lower levels of glucocorticoids because plasma corticosterone levels were significantly lower in Igf2-/- mothers compared with wild-type controls. In support of this, fetuses from homozygous knockout matings, where mothers were treated with 15 microg/ml corticosterone, and Igf2-/- fetuses obtained from heterozygous matings had similar lung histology to those of wild-type fetuses. Finally, we found that IGF-I and SP-B mRNA levels were up-regulated in the lungs of Igf2-/- fetuses at the end of gestation. This study suggests that Igf2 plays an important role in the development of the fetal lung and may affect fetal lung maturation by regulating maternal factors, such as corticosterone levels, during pregnancy.

Substrate rigidity regulates the formation and maintenance of tissues.

The ability of cells to form tissues represents one of the most fundamental issues in biology. However, it is unclear what triggers cells to adhere to one another in tissues and to migrate once a piece of tissue is planted on culture surfaces. Using substrates of identical chemical composition but different flexibility, we show that this process is controlled by substrate rigidity: on stiff substrates, cells migrate away from one another and spread on surfaces, whereas on soft substrates they merge to form tissue-like structures. Similar behavior was observed not only with fibroblastic and epithelial cell lines but also explants from neonatal rat hearts. Cell compaction on soft substrates involves a combination of weakened adhesions to the substrate and myosin II-dependent contractile forces that drive cells toward one another. Our results suggest that tissue formation and maintenance is regulated by differential mechanical signals between cell-cell and cell-substrate interactions, which in turn elicit differential contractile forces and adhesions to determine the preferred direction of cell migration and association.

Inhibition of growth of mouse gastric cancer cells by Runx3, a novel tumor suppressor.

We reported recently that the silencing of RUNX3 is causally related to gastric cancer in humans. Here we report that in three of four cell lines derived from N-methyl-N-nitrosourea-induced mouse glandular stomach carcinomas, Runx3 is silenced due to hypermethylation of CpG islands in the promoter region, as we also observed for human gastric cancer cells. Although two of the sites we tested in the promoter of the fourth line were not methylated, in all four cases the silencing of Runx3 could be reversed by treatment of the cells with 5'-azacytidine and trichostatin A. Interestingly, the exogenous expression of RUNX3 in cell lines that do not express the endogenous gene caused an inhibition of growth in soft agar, suggesting that anchorage-independent growth could be used as an assay of RUNX3 activity in vitro. These observations suggest that the mouse system described here may be useful as a model for the study of human gastric carcinogenesis.