NHE3 phosphorylation via PKCη marks the polarity and orientation of directionally migrating cells

Abstract

Endogenous electric fields (EF) may provide an overriding cue for directional cell migration during wound closure. Perceiving a constant direction requires active sodium-hydrogen exchanger (pNHE3) at the leading edge of HEK 293 cells but its activation mechanism is not yet fully understood. Because protein kinase C (PKC) is required in electrotaxis, we asked whether NHE3 is activated by PKC during wound healing. Using pharmacological (pseudosubstrate and edelfosine) inhibition, we showed that inhibition of PKCη isoform impairs directional cell migration in HEK 293 cells in the presence of a persistent directional cue (0.25–0.3 V/mm of EF for 2 h). Further, we found that pNHE3 forms complexes with both PKCη and ɣ-tubulin, suggesting that these molecules may regulate the microtubule-organizing center. In addition, cellular pNHE3 content was reduced significantly when PKCη was inhibited during directional cell migration. Taken together, these data suggest that PKCη-dependent phosphorylation of NHE3 and the formation of pNHE3/PKCη/ɣ-tubulin complexes at the leading edge of the cell are required for directional cell migration in an EF.

Endogenous electric fields as guiding cue for cell migration

“…What is more, we found in mammalian cells that pNHE3 forms complexes with both PKCη and γ-tubulin at filopodia, suggesting that these molecules may regulate the microtubule-organizing center. Our data further suggest that PKCη-dependent phosphorylation of NHE3 and the formation of pNHE3/PKCη/ γ-tubulin complexes at the leading edge of the cell are required for directional cell migration in an EF (see Figure 1 ) (Ozkucur et al, 2014 ; Perike et al, 2014 ). Thus, the molecular details of the translational force of EF that induce transporters, cytoskeletal elements and migration were unraveled.…”

Na⁺/H⁺ Exchangers Are Required for the Development and Function of Vertebrate Mucociliary Epithelia

“…The family of transmembrane Na + /H + exchangers (NHEs) is encoded by solute carrier genes SLC9s , which are subdivided into three groups SLC9A-C [Fuster, and Alexander, 2014], NHEs contribute to pH regulation of the cytoplasm and organelle content, and act in concert with other ion transporters to maintain cellular homeostasis by extruding protons in exchange for sodium ions [Slepkov et al, 2007; Souza et al, 1998; Schreiber, 2005], NHE1-5 ( SLC9A1-5 ) predominantly localize to the cell membrane, while NHA1-2 (Na + /H + antiporters, SLC9B1-2 ) localize mostly to intracellular sites, and SLC9C1-2 genes encode one sperm-specific and one putative NHE form, respectively [Fuster, and Alexander, 2014], Additionally, multiple NHEs were shown to influence cell signaling and specific cell behavior in vitro and in vivo , including Wnt/planar cell polarity signaling, directional cell migration and embryonic development [Siyanov, and Baltz, 2013; Schneider et al, 2009; Ozkucur et al, 2014; Slepkov et al, 2007; Simons et al, 2009], Consequently, NHE function or dysregulation were implicated in human diseases, such as tumor formation and metastasis, and a higher susceptibility to airway infections in cystic fibrosis patients [Sera et al, 2012; Harguindey et al, 2005; Slepkov et al, 2007; Pereira et al, 2017a; Dorfman et al, 2011; Corvol et al, 2015; Li et al, 2014].…”

Environmental Factors That Influence Stem Cell Migration: An “Electric Field”

“…This increase in calcium level could activate the protein kinase C (PKC) pathway either directly or through phosphatidylinositol 4,5-bisphosphate (PIP2), whose expression was also modified during EF exposure, and second messenger signalling lipid diacylglycerol [ 60 – 62 ]. PKC was shown to contribute to the patchy localization of NHE3 which colocalized with β -actin at the leading edge membrane protrusions [ 61 , 62 ]. Furthermore, γ -tubulin complexes interacted with phosphorylated NHE3 in patches in the leading edge of the cell [ 61 ].…”

“…PKC was shown to contribute to the patchy localization of NHE3 which colocalized with β -actin at the leading edge membrane protrusions [ 61 , 62 ]. Furthermore, γ -tubulin complexes interacted with phosphorylated NHE3 in patches in the leading edge of the cell [ 61 ]. Indeed, the γ -tubulin was thought to aid in establishing the microtubule polarity within the cell as they were observed at the microtubule-organizing centres which could help orient the cell in the presence of persistent cues such as EFs [ 63 ].…”