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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.