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.

A synthetic hydrogel for the high-throughput study of cell-ECM interactions.

It remains extremely challenging to dissect the cooperative influence of multiple extracellular matrix (ECM) parameters on cell behaviour. This stems in part from a lack of easily deployable strategies for the combinatorial variation of matrix biochemical and biophysical properties. Here we describe a simple, high-throughput platform based on light-modulated hyaluronic acid hydrogels that enables imposition of mutually independent and spatially continuous gradients of ligand density and substrate stiffness. We validate this system by showing that it can support mechanosensitive differentiation of mesenchymal stem cells. We also use it to show that the oncogenic microRNA, miR18a, is nonlinearly regulated by matrix stiffness and fibronectin density in glioma cells. The parallelization of experiments enabled by this platform allows condensation of studies that would normally require hundreds of independent hydrogels to a single substrate. This system is a highly accessible, high-throughput technique to study the combinatorial variation of biophysical and biochemical signals in a single experimental paradigm.

A composite hydrogel platform for the dissection of tumor cell migration at tissue interfaces.

Glioblastoma multiforme (GBM), the most prevalent primary brain cancer, is characterized by diffuse infiltration of tumor cells into brain tissue, which severely complicates surgical resection and contributes to tumor recurrence. The most rapid mode of tissue infiltration occurs along blood vessels or white matter tracts, which represent topological interfaces thought to serve as "tracks" that speed cell migration. Despite this observation, the field lacks experimental paradigms that capture key features of these tissue interfaces and allow reductionist dissection of mechanisms of this interfacial motility. To address this need, we developed a culture system in which tumor cells are sandwiched between a fibronectin-coated ventral surface representing vascular basement membrane and a dorsal hyaluronic acid (HA) surface representing brain parenchyma. We find that inclusion of the dorsal HA surface induces formation of adhesive complexes and significantly slows cell migration relative to a free fibronectin-coated surface. This retardation is amplified by inclusion of integrin binding peptides in the dorsal layer and expression of CD44, suggesting that the dorsal surface slows migration through biochemically specific mechanisms rather than simple steric hindrance. Moreover, both the reduction in migration speed and assembly of dorsal adhesions depend on myosin activation and the stiffness of the ventral layer, implying that mechanochemical feedback directed by the ventral layer can influence adhesive signaling at the dorsal surface.

Microenvironmental stiffness enhances glioma cell proliferation by stimulating epidermal growth factor receptor signaling.

The aggressive and rapidly lethal brain tumor glioblastoma (GBM) is associated with profound tissue stiffening and genomic lesions in key members of the epidermal growth factor receptor (EGFR) pathway. Previous studies from our laboratory have shown that increasing microenvironmental stiffness in culture can strongly enhance glioma cell behaviors relevant to tumor progression, including proliferation, yet it has remained unclear whether stiffness and EGFR regulate proliferation through common or independent signaling mechanisms. Here we test the hypothesis that microenvironmental stiffness regulates cell cycle progression and proliferation in GBM tumor cells by altering EGFR-dependent signaling. We began by performing an unbiased reverse phase protein array screen, which revealed that stiffness modulates expression and phosphorylation of a broad range of signals relevant to proliferation, including members of the EGFR pathway. We subsequently found that culturing human GBM tumor cells on progressively stiffer culture substrates both dramatically increases proliferation and facilitates passage through the G1/S checkpoint of the cell cycle, consistent with an EGFR-dependent process. Western Blots showed that increasing microenvironmental stiffness enhances the expression and phosphorylation of EGFR and its downstream effector Akt. Pharmacological loss-of-function studies revealed that the stiffness-sensitivity of proliferation is strongly blunted by inhibition of EGFR, Akt, or PI3 kinase. Finally, we observed that stiffness strongly regulates EGFR clustering, with phosphorylated EGFR condensing into vinculin-positive focal adhesions on stiff substrates and dispersing as microenvironmental stiffness falls to physiological levels. Our findings collectively support a model in which tissue stiffening promotes GBM proliferation by spatially and biochemically amplifying EGFR signaling.

Hutchinson-Gilford progeria syndrome alters nuclear shape and reduces cell motility in three dimensional model substrates.

Cell migration through tight interstitial spaces in three dimensional (3D) environments impacts development, wound healing and cancer metastasis and is altered by the aging process. The stiffness of the extracellular matrix (ECM) increases with aging and affects the cells and cytoskeletal processes involved in cell migration. However, the nucleus, which is the largest and densest organelle, has not been widely studied during cell migration through the ECM. Additionally, the nucleus is stiffened during the aging process through the accumulation of a mutant nucleoskeleton protein lamin A, progerin. By using microfabricated substrates to mimic the confined environment of surrounding tissues, we characterized nuclear movements and deformation during cell migration into micropillars where interspacing can be tuned to vary nuclear confinement. Cell motility decreased with decreased micropillar (µP) spacing and correlated with increased dysmorphic shapes of nuclei. We examined the effects of increased nuclear stiffness which correlates with cellular aging by studying Hutchinson-Gilford progeria syndrome cells which are known to accumulate progerin. With the expression of progerin, cells showed a threshold response to decreased µP spacing. Cells became trapped in the close spacing, possibly from visible micro-defects in the nucleoskeleton induced by cell crawling through the µP and from reduced force generation, measured independently. We suggest that ECM changes during aging could be compounded by the increasing stiffness of the nucleus and thus changes in cell migration through 3D tissues.

Altered cell mechanics from the inside: dispersed single wall carbon nanotubes integrate with and restructure actin.

With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs) are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

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.

Carbon nanotubes reorganize actin structures in cells and ex vivo.

The ability of globular actin to form filaments and higher-order network structures of the cytoskeleton is essential for cells to maintain their shape and perform essential functions such as force generation, motility, and division. Alterations of actin structures can dramatically change a cell's ability to function. We found that purified and dispersed single wall carbon nanotubes (SWCNTs) can induce actin bundling in cells and in purified model actin systems. SWCNTs do not induce acute cell death, but cell proliferation is greatly reduced in SWCNT-treated cells with an increase in actin-related division defects. Actin, normally present in basal stress fibers in control cells, is located in heterogeneous structures throughout the SWCNT-treated cell. These SWCNT-induced changes in actin structures are seen functionally in multinucleated cells and with reduced force generation. Ex vivo, purified actin filaments cross-linked with alpha-actinin and formed isotropic networks, whereas SWCNTs caused purified actin filaments to assemble into bundles. While purified, isolated SWCNTs do not appear acutely toxic, this subcellular reorganization may cause chronic changes to cellular functions.