Supplementary Materials01

Supplementary Materials01. phenomenon that underlies diverse physiological Goat monoclonal antibody to Goat antiRabbit IgG HRP. and pathological processes such as tissue morphogenesis, immune response, and cancer metastasis. Much of what we know about the mechanisms of cell migration stems from in vitro studies with 2D substrates (Friedl and Alexander, 2011; Mogilner and Oster, 1996; Pollard and Borisy, 2003). The classical model of cell migration along 2D planar surfaces is characterized by cycles of actin polymerization-driven lamellipodial protrusion, integrin-dependent adhesion, myosin II-mediated contraction, and de-adhesion at the trailing edge. Although 2D migration is relevant in certain processes, such as neutrophil migration along the endothelium or epithelial cell wound healing, most 2D assays fail to recapitulate the physiological tissue environment encountered in vivo (Wirtz et al., 2011). Cells often migrate in vivo within 3D extracellular matrices (ECMs). Cells also migrate through 3D longitudinal tracks with bordering 2D interfaces (i.e., channels). These channels are formed between the connective tissue and the basement membrane of muscle, nerve, and BAY-850 epithelium (Friedl and Alexander, 2011). 3D longitudinal channels are also formed between adjacent bundled collagen fibers in fibrillar interstitial tissues. Importantly, cells have been reported to migrate through such 3D channels in vivo (Alexander et al., 2008). The cross-sectional areas (Wolf et al., 2009) of pores/channels encountered in vivo range from 10 to 300 m2, suggesting that cells migrating in vivo experience varying degrees of physical confinement. Mounting evidence suggests that physical confinement alters cell migration mechanisms (Balzer et BAY-850 al., 2012; Konstantopoulos et al., 2013; Pathak and Kumar, 2012; Stroka et al., 2013). To isolate the effect of physical confinement that tumor cells experience as they migrate through the ECM microtracks in vivo, we have developed a chemotaxis-based microfluidic device containing microchannels of varying cross-sectional areas (Balzer et al., 2012; Tong et al., 2012). Migration of cells through wide microchannels (width by height 50 10 m2) recapitulates the earmarks of 2D cell motility and BAY-850 depends on actin polymerization and myosin II-mediated contractility. However, metastatic breast cancer cells migrate through narrow (3 10 m2) microchannels even when actin polymerization, Rho/ROCK- or myosin II-dependent contractility, or 1-integrin function are inhibited (Balzer et al., 2012). Here, we present an actin- and myosin-independent mechanism of cell migration that is based on water permeation and active and passive ion transport in confined spaces. Ion channels and aquaporins (AQPs) have previously been implicated in 2D cell migration (Papadopoulos et al., 2008; Schwab et al., 2007). However, their specific molecular roles during migration are not well understood. Cytoskeletal components regulate the activity of ion channels (Dreval et al., 2005; Grunnet et al., 2002; Mazzochi et al., 2006), and as a result, volume regulation via these ion pumps requires an intact cytoskeleton. For example, the sodium hydrogen exchanger-1 (NHE-1) is known to physically interact with the actin cytoskeleton (Goss et al., 1994; Grinstein et al., 1993; Wakabayashi et al., 1992). Pharmacological inhibition of NHE-1 restrains leukocyte chemotaxis (Ritter et al., 1998) and the migration speeds of endothelial and epithelial cells (Klein et al., 2000). AQPs, transmembrane proteins that allow transport of water molecules across the cell membrane, are also involved in cell migration. Specifically, aquaporin 5 (AQP5) is overexpressed in lung and breast tumor cells and facilitates 2D migration of these cells (Chae et al., 2008; Jung et al., 2011), presumably by regulating water influx to facilitate protrusions by actin polymerization (Papadopoulos et al., 2008) and/or by stabilizing microtubules (Sidhaye et al., 2012). AQPs have been identified as potential targets for cancer therapeutic development, but like ion channels, their contribution to 2D versus confined migration is not well understood. Here, we present an integrated experimental and theoretical approach showing that water BAY-850 permeation is a major mechanism of cell migration in confined microenvironments. We have termed this mode of migration the Osmotic Engine Model, which is dependent on cell-volume regulation and the fluxes of ions and water into and out of the cell. Specifically, the polarized cell inside a narrow channel establishes a.