No abstract

Migrating fibroblasts reorient directionality by PI3K-dependent branching and pivoting of protrusions, a mechanism that allows fibroblasts to align with an external chemotactic gradient.

During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3Ј-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3Ј-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts. Journal of Cell Science 314 is largely PI3K dependent. This is seen most dramatically when microtubules are depolymerized using nocodazole, which induces rapid protrusion-retraction events as seen by TIRF. In the context of random fibroblast migration, formation of branched lamellipodia and turning events were found to coincide with localized enrichment of 3Ј-phosphoinositides. Results PI3K lipid products accumulate in a dynamic fashion during fibroblast spreadingTo assess the activation of PI3K signaling and its possible relation to adhesion-based motility, we established stable expression of the 3Ј-phosphoinositide-specific Akt pleckstrin-homology domain, fused with enhanced green fluorescent protein (EGFP-AktPH) in NIH3T3 fibroblasts and monitored these cells by TIRF microscopy as they attached and spread on glass coated with fibronectin or poly-D-lysine (Fig. 1). Glass coated with bovine serum albumin, which was also present in our imaging buffer, does not promote adhesion of these cells (results not shown). After allowing the cells to spread for 30-50 minutes, a saturating dose of PDGF was added to evaluate the maximal activation of PI3K in each cell, followed by a large dose of PI3K inhibitor to evaluate the fluorescence intensity associated with EGFP-AktPH in the cytosol; the latter is used to normalize the PI3K-dependent response (Schneider and Haugh, 2004).Cells spreading on fibronectin consistently showed dynamic enrichment of 3Ј-phosphoinositides, with transient bursts of signaling that were often localized in actively protruding regions at the cell periphery and other times showed a more global pattern ( Fig. 1A; supplementary material Movie 1). These patterns of PI3K signaling are distinct from the more stable, ring-like patterns seen in response to PDGF stimulation, which we have previously explained in quantitative detail (Schneider and Haugh, 2004). Analysis of a...

There is significant interest in developing new approaches for culturing mammalian cells in a three-dimensional (3D) environment due to the fact that it better recapitulates the in vivo environment. The goal of this work was to develop thiol-acrylate, biodegradable hydrogels that possess highly tunable properties to support in vitro 3D culture. Six different hydrogel formulations were synthesized using two readily available monomers, a trithiol (ETTMP 1300 [ethoxylated trimethylolpropane tri (3-mercaptopropionate) 1300]) and a diacrylate (PEGDA 700 [polyethylene glycol diacrylate 700]), polymerized by a base-catalyzed Michael addition reaction. The resultant hydrogels were homogeneous, hydrophilic, and biodegradable. Different mechanical properties such as gelation time, storage modulus (or the elasticity G'), swelling ratio, and rate of degradation were tuned by varying the weight percentage of polymer, the molar ratio of thiol-to-acrylate groups, and the pH of the solution.Cytocompatibility was assessed using two model breast cancer cell lines by both 2D and 3D cell culturing approaches. The hydrogel formulations with a thiol-to-acrylate molar ratio of 1.05 were found to be optimal for both 2D and 3D cultures with MDA-MB-231 cellular aggregates found to be viable after 17 days of 3D continuous culture. Finally, MCF7 cells were observed to form 3D spheroids up to 600 μm in diameter as proof of principle for the thiol-acrylate hydrogel to function as a scaffold for in vitro 3D cell culture. A comparison of the different mechanical properties of the six hydrogel formulations coupled with in vitro cell culture results and findings from previously published hydrogels conclude that the thiol-acrylate hydrogels have significant potential as a scaffold for 3D cell culture.

Cell movement biased by a chemical gradient, or chemotaxis, coordinates the recruitment of cells and collective migration of cell populations. During wound healing, chemotaxis of fibroblasts is stimulated by platelet-derived growth factor (PDGF) and certain other chemoattractants. Whereas the immediate PDGF gradient sensing response has been characterized previously at the level of phosphoinositide 3-kinase (PI3K) signaling, the sensitivity of the response at the level of cell migration bias has not yet been studied quantitatively. In this work, we used live-cell total internal reflection fluorescence microscopy to monitor PI3K signaling dynamics and cell movements for extended periods. We show that persistent and properly aligned (i.e., high-fidelity) fibroblast migration does indeed correlate with polarized PI3K signaling; accordingly, this behavior is seen only under conditions of high gradient steepness (>10% across a typical cell length of 50 μm) and a certain range of PDGF concentrations. Under suboptimal conditions, cells execute a random or biased random walk, but nonetheless move in a predictable fashion according to the changing pattern of PI3K signaling. Inhibition of PI3K during chemotaxis is accompanied by loss of both cell-substratum contact and morphological polarity, but after a recovery period, PI3K-inhibited fibroblasts often regain the ability to orient toward the PDGF gradient.