(591b) Polarized NHE1 and SWELL1 Regulate Migration Direction, Ef?ciency and Metastasis

Introduction: Cell migration regulates diverse (patho) physiological processes, including cancer metastasis. According to the Osmotic Engine Model, polarization of NHE1 at the leading edge of confined cells facilitates water uptake, cell protrusion and motility. However, the role of NHE1 in promoting isosmotic swelling has yet to be established. Furthermore, it is currently unknown which ion channel(s) preferentially localize at the cell rear, and mediate isosmotic shrinkage of the cell trailing edge. Also, does the spatial polarization of these molecules confer migration directionality? To address these fundamental and translational questions, we combined microfluidics with live-cell imaging, novel optogenetic tools to demonstrate that in MDA-MB-231 cells, NHE1 and SWELL1 preferentially polarize at the cell leading and trailing edges, respectively, mediate cell volume regulation, and confined migration. Optogenetic RhoA activation at the cell front triggers SWELL1 re-distribution and migration direction reversal, while Akt1 activation promotes NHE1 membrane localization and impairs cell motility when it is enriched at the cell rear.

Materials and methods: We generated NHE1, SWELL1 and dual knock down cell lines with shRNA lentivirus. Knock down efficiency was confirmed by Western blot. polydimethylsiloxane (PDMS)-based microfluidic devices containing an array of parallel microchannels of prescribed height (10 µm), width (3 µm or 10 µm), and length (200 µm) were fabricated for immunofluorescence, migration and optogenetic experiments. Optogenetic tools were utilized to control the subcellular activation of SWELL1, RhoA or Akt1 with high spatiotemporal accuracy, using the cryptochrome 2 (Cry2)-CIBN light-gated dimerizer system. This system relies on the fusion of the corresponding protein to Cry2-mCherry (optoSWELL1, optoGEF, or optoAkt1) and its GFP-labeled dimerization partner, CIBN, engineered to bind to the plasma via the CAAX anchor (CAAX-CIBN-GFP). To this end, cells migrating inside confining channels were monitored in real-time by imaging the mCherry channel to identify the leading and trailing edges. Light stimulation was performed with a 488 nm laser at 1% power on a rectangular area placed either at the cell leading or trailing edge. Cell velocity were quantified using a custom MATLAB script.

Results: According to OEM, cell migration inside confining channels involves regulatory volume increase (RVI) at the front and regulatory volume decrease (RVD) at the rear. As reported, NHE1 is polarized at the cell front of cells migrating inside PDMS-based confining channels (Fig. a). The RVD-mediating SWELL1 chloride channel is revealed to polarize at the trailing edge by live-cell imaging using ectopically expressed SWELL1-GFP (Fig. a). In line with the role of NHE1 and SWELL1 in isosmotic swelling and shrinkage, respectively, their individual knockdown changed cell volume accordingly and impaired cell migration in confining channels. Importantly, dual silencing results in a cooperative inhibition of confined migration (Fig. b, c, d).

To establish the role of SWELL1 polarization pattern in the direction and efficiency of confined migration, we developed optoSWELL1. Before light stimulation, SWELL1 localizes primarily at the trailing edge. Light stimulation at the cell leading edge gradually promotes local SWELL1 enrichment, accompanied by a reduction at the opposite pole. During this process, cell migration velocity decreases as the front-to-rear ratio of SWELL1 expression increases. When SWELL1 is equally distributed at the cell poles at t = t₁, cell motility halts. Further light stimulation induces preferential SWELL1 enrichment at the cell front along with the reversal of migration direction, as evidenced by the negative velocity values (Fig. e, f).

Because volume sensitive chloride channels are modulated by RhoA, we examined how optogenetic regulation of RhoA activity alters the spatial localization of SWELL1 using SWELL1-iRFP-expressing MDA-MB-231 breast cancer cells. Light-induced upregulation of RhoA activity via optoGEF-RhoA/CAAX-CIBN-GFP at the leading edge of cells inside unconfined channels enriched SWELL1 localization, followed by a reversal of migration direction (Fig. g). It suggests that RhoA activity regulates the polarization of SWELL1 and modulates cell migration direction.

We next aimed to delineate the underlying mechanism of NHE1 polarization and its contribution to the migration efficiency. As PI3K/Akt axis initiate signals that stimulate cell movement, we tested how the spatiotemporal regulation of Akt1 affects NHE1. Light-induced upregulation of Akt1 activity in cells on 2D greatly promotes NHE1 membrane localization (Fig. h). Furthermore, upregulation of Akt1 activity at the trailing edge of cells inside confined channels impaired cell motility (Fig. i). It indicates the critical role of PI3K/Akt signaling pathway in controlling the spatiotemporal NHE1 localization and migration efficiency.

Discussion: Cell migration is a pivotal step in the metastatic dissemination of cancer cells from a primary tumor to distant organs in the body. Cell motility is governed by cell-matrix interactions, the actomyosin cytoskeleton, and cell volume regulation as proposed by OEM. According to OEM, a cell migrating in confinement establishes a spatial gradient of ion transporters and ion channels in the cell membrane so that local swelling at the leading edge and shrinkage at the trailing edge, respectively, facilitate net cell movement. We show that the coordinated action of NHE1 and SWELL1, respectively, due to their distinct polarization patterns, supports efficient confined migration. Using optogenetic tools, we further demonstrate that SWELL1 localization is regulated by RhoA activity, while NHE1 is modulated by PI3K/Akt signaling pathway.