(584e) Role of Water Permeation in Confined Cell Migration and Polarization
Introduction: Cell homeostasis and diverse processes, including migration, are tightly regulated by cell volume. During migration through tissues, metastatic cancer cells experience varying degrees of physical confinement as they migrate through longitudinal tracks within the matrix (1). Intriguingly, cell migration persists in narrow channels even when typical hallmarks of 2D planar migration, such as actin polymerization and myosin II-mediated contractility, are inhibited (2). We therefore hypothesized that an alternate mechanism based on cell volume regulation via ion channels and aquaporins drives cell migration in these confined microenvironments, where cells must deform in order to squeeze through physically restrictive spaces. We used an integrated experimental and theoretical approach, which we have termed the “osmotic engine model”, to show that directed water permeation regulates migration in confined microenvironments. Importantly, the theoretical model predicts all key experimental results. Here, our objective was to extend the model by examining the temporal and spatial aspects of cell repolarization in response to an osmotic shock.
Materials and Methods: We engineered a novel microfluidic-based chemotactic device with narrow (3 μm-wide, 10 μm-high, 200 μm-long) extracellular matrix-coated channels. Using timelapse microscopy, we imaged live sarcoma cell migration before and after an osmotic shock at the leading or trailing edge. Pharmacological drugs or siRNA treatments were used to block or knock down proteins relevant to our model, including Na+/H+ion channels, aquaporins, myosin II, actin, or microtubules. Cell velocity, volume, and ion channel or aquaporin distribution were quantified and correlated in order to understand cell repolarization and volume changes in response to osmotic shock in confinement.
Results and Discussion: Inhibition of Na+/H+ ion channels or knockdown of Aquaporin 5 (AQP5) reduced confined cell migration velocity. These results indicate the importance of ion and water flux across the cell membrane during confined migration and motivated our model. To test the model, osmotic shocks were applied to cells’ leading and/or trailing edges within the microfluidic device. In accord with our model, cells reversed migration direction within 5 minutes of applying a hypotonic shock at the leading edge or hypertonic shock at the trailing edge, followed by a two-phase (fast, then slower) migration pattern (Fig. 1). The fast phase correlated with the time during which cell volume was reduced by 40%. Blockage of Na+/H+ ion channels, inhibition of actin polymerization, or knockdown of AQP5 interfered with this response. To explore the repolarization mechanism, we quantified the distribution of NHE-1 (a specific Na+/H+ion channel) and AQP5 within the cells. As predicted by our model, both NHE-1 and AQP5 polarized to the leading edge of cells migrating in narrow channels. However, they did not begin redistributing to the new leading edge until approximately 30 minutes after the osmotic shock. Thus, cell migration initiated in the opposite direction even before NHE-1 repolarized, indicating that confined cell migration driven by an osmotic shock does not require NHE-1 to be localized at the leading edge, which is a key aspect of our model. Inhibition of actin polymerization completely prevented repolarization of NHE-1.
Conclusions: Together, the results support our “osmotic engine” model of confined cell migration that is driven water permeation via activity of ion channels and aquaporins. This study presents an alternate mechanism of migration in confined spaces that cells exploit in the absence of actin polymerization in confining microchannels. Our osmotic engine model is especially applicable during cell repolarization, where migration in the opposing direction precedes ion channel redistribution. Future work will include live imaging of microtubule, actin, aquaporin, and ion channel dynamics during migration and repolarization in confined channels.
References: (1) Alexander S. et al, Histochemistry and Cell Biology 130: 1147-1154 (2008). (2) Balzer E.M. et al, FASEB J 26: 4045-4056 (2012).