In Chemotaxing Fibroblasts, Both High-Fidelity and Weakly Biased Cell Movements Track the Localization of PI3K Signaling

Abstract

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.

Migration of cells in a social context

“…Contrary to previous work (22,(24)(25)(26)29), these cells form neither 2D sheets nor 3D structures, nor are they highly polarized, and their single-cell migratory behavior is established (13,14,16). The microfluidic cell culture platform hosts independent and isolated culture conditions in each of the isolated 96 polymethylsiloxane (PDMS) chambers (34-nL volume) that mimic physiological conditions more plausibly than traditional cell-culture environments in which concentrations of, for instance, secreted signaling molecules are diluted into large volumes of surrounding fluid.…”

“…In addition to its aesthetic qualities, social cell migration is involved in embryonic development (7), wound healing (8), and immune response (9), and unregulated migration leads to disease, including cancer metastasis (10). Previous work on single-cell migration has focused on isolated (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) or strongly polarized and aligning (21,22) cell types, mostly using population-averaged bulk assays (23) or simple observations in a social context (2,3). However, strongly cross-correlated cell motion and collective substrate deformation has been found to arise in mechanically interlinked cells transmitting forces through both cell-cell linkages and the substrate (24)(25)(26)(27)(28)(29).…”

“…Furthermore, striking social effects such as highly sensitive collective responses in a number of sensing systems [e.g., quorum sensing (31,32) and onset of collective behavior in Dictyostelium discoideum (33)] mediated by increased levels of cell-secreted signals in higher cell density indicate that mechanical links are not necessary for collective behavior. At the subcellular level, many types of nonswimming motile cells involved in multicellular biology [e.g., fibroblasts, Dictyostelium, and neutrophils (13,14,(16)(17)(18)] have been found to transmit traction force to the substrate by intracellularly polymerizing their cytoskeletons in dynamically formed membrane protrusions known as pseudopodia. However, whether the social context changes this, mechanisms by which the social context manifests itself, and the implications of being close to neighboring cells all remain unexplored.…”

Mechanical Regulation of Fibroblast Migration and Collagen Remodeling in Healing Myocardial Infarcts

“…However, factors regulating infarct anisotropy are not well understood. Mechanical, structural, and chemical environmental cues have all been shown to regulate alignment of fibroblasts and collagen in vitro Melvin et al, 2011), but understanding of fibroblast behavior in the complex environment of a healing infarct is lacking. A better understanding of how fibroblasts integrate these cues as they deposit and remodel extracellular matrix in a healing infarct is needed in order to develop interventions that modify infarct scar anisotropy for therapeutic benefit.…”

“…According to this model, any cue for fibroblast alignment may subsequently cause collagen alignment. In vitro studies have shown that fibroblasts will align in the direction of uniaxial mechanical stretch (Eastwood et al, 1998;Wang & Grood, 2000;Neidlinger-Wilke et al, 2001;Raeber et al, 2008;Hsu et al, 2009;Steward et al, 2010;, in the direction of aligned matrix fibers or micropatterned ridges (Guido & Tranquillo, 1993;Sutherland et al, 2005;Doyle et al, 2009;Sun et al, 2010), and in the direction of chemokine gradients (Knapp et al, 1999;Grinnell et al, 2006;Melvin et al, 2011). Mechanical, structural, and chemical cues are all present during infarct healing, which makes all of them potential determinants of infarct collagen alignment.…”