Albrecht Schwab

Role of Ion Channels and Transporters in Cell Migration

Cell motility is central to tissue homeostasis in health and disease, and there is hardly any cell in the body that is not motile at a given point in its life cycle. Important physiological processes intimately related to the ability of the respective cells to migrate include embryogenesis, immune defense, angiogenesis, and wound healing. On the other side, migration is associated with life-threatening pathologies such as tumor metastases and atherosclerosis. Research from the last ≈ 15 years revealed that ion channels and transporters are indispensable components of the cellular migration apparatus. After presenting general principles by which transport proteins affect cell migration, we will discuss systematically the role of channels and transporters involved in cell migration.

Directional Cell Migration and Chemotaxis in Wound Healing Response to PDGF-AA are Coordinated by the Primary Cilium in Fibroblasts

Cell motility and migration play pivotal roles in numerous physiological and pathophysiological processes including development and tissue repair. Cell migration is regulated through external stimuli such as platelet-derived growth factor-AA (PDGF-AA), a key regulator in directional cell migration during embryonic development and a chemoattractant during postnatal migratory responses including wound healing. We previously showed that PDGFRα signaling is coordinated by the primary cilium in quiescent cells. However, little is known about the function of the primary cilium in cell migration. Here we used micropipette analysis to show that a normal chemosensory response to PDGF-AA in fibroblasts requires the primary cilium. In vitro and in vivo wound healing assays revealed that in ORPK mouse (IFT88Tg737Rpw) fibroblasts, where ciliary assembly is defective, chemotaxis towards PDGF-AA is absent, leading to unregulated high speed and uncontrolled directional cell displacement during wound closure, with subsequent defects in wound healing. These data suggest that in coordination with cytoskeletal reorganization, the fibroblast primary cilium functions via ciliary PDGFRα signaling to monitor directional movement during wound healing.

Protons make tumor cells move like clockwork

Cancer accounts for 13% of the yearly total mortality worldwide. Most cancer deaths are the sequel of metastatic diseases rather than of primary tumor growth. Thus, the major challenge in tumor therapy is the tumor cells’ ability to metastasize. The extent to which a tumor metastasizes correlates with the tumor cells’ migratory activity. Tumor cell migration requires a coordinated formation and release of cell adhesion contacts, a controlled cytoskeletal dynamics, the digestion and reorganization of the extracellular matrix, and local ion and water transport across the plasma membrane. All of these operations depend on intracellular pH (pHi) and extracellular pH (pHe). Numerous H+, HCO3−, and monocarboxylate transporters as well as different carbonic anhydrase isozymes have considerable impact on pHi and pHe which spotlights them as possible, potential targets for anticancer therapeutics. Especially in solid tumors whose vascularization is often not sufficient, tumor cells cope with hypoxia and the resulting glycolysis by overexpressing the Na+/H+ exchanger NHE1, monocarboxylate transporters MCT1 and/or MCT4, and the carbonic anhydrase CA IX. NHE1, MCT, and CA IX activity lead to an acidification of the extracellular space in order to maintain the cytosolic pH homeostasis stable. The present article gives a review on how this characteristic, acidic tumor micro- and nanoenvironment controls tumor cell migration.

Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange.

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell–matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin α2β1. Cells reached their maximum motility at pHe∼7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell–matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin α2β1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell–matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition).

Cells move when ions and water flow.

Cell migration is a process that plays an important role throughout the entire life span. It starts early on during embryogenesis and contributes to shaping our body. Migrating cells are involved in maintaining the integrity of our body, for instance, by defending it against invading pathogens. On the other side, migration of tumor cells may have lethal consequences when tumors spread metastatically. Thus, there is a strong interest in unraveling the cellular mechanisms underlying cell migration. The purpose of this review is to illustrate the functional importance of ion and water channels as part of the cellular migration machinery. Ion and water flow is required for optimal migration, and the inhibition or genetic ablation of channels leads to a marked impairment of migration. We briefly touch cytoskeletal mechanisms of migration as well as cell–matrix interactions. We then present some general principles by which channels can affect cell migration before we discuss each channel group separately.

Autocrine Purinergic Receptor Signaling Is Essential for Macrophage Chemotaxis

Amplification of outside-in chemotactic signaling by inside-out purinergic signaling drives macrophage migration. Self-Help Migration Immune cells such as neutrophils and macrophages migrate to sites of infection or inflammation by following gradients of chemoattractants. These include chemokines, which can be released by other cells at the target site; components of the complement system, such as C5a; and bacterial products, such as the formylated peptide, fMLP. While they navigate along the chemoattractant gradient toward their destination, cells are also exposed to other signals, some of which may compete with the chemoattractant that the cells were already following. Another level of complexity in the regulation of cell migration came from the discovery that migrating neutrophils release adenosine triphosphate (ATP), which then functions in an autocrine fashion through the purinergic receptor P2Y2 to enhance migration; cells deficient in P2Y2 have impaired gradient sensing. Kronlage et al. provide evidence that autocrine ATP signaling is also required for the migration of macrophages in vitro and in an in vivo model. The authors found that more than one type of ATP receptor type as well as metabolites of ATP contributed to the migratory responses of macrophages; furthermore, ATP was not released through pannexin-1 proteins, as has been suggested for neutrophils. Together, these data suggest that autocrine purinergic receptor signaling may play a general role in regulating the chemotactic responses of immune cells. Chemotaxis, the movement of cells along chemical gradients, is critical for the recruitment of immune cells to sites of inflammation; however, how cells navigate in chemotactic gradients is poorly understood. Here, we show that macrophages navigate in a gradient of the chemoattractant C5a through the release of adenosine triphosphate (ATP) and autocrine “purinergic feedback loops” that involve receptors for ATP (P2Y2), adenosine diphosphate (ADP) (P2Y12), and adenosine (A2a, A2b, and A3). Whereas macrophages from mice deficient in pannexin-1 (which is part of a putative ATP release pathway), P2Y2, or P2Y12 exhibited efficient chemotactic navigation, chemotaxis was blocked by apyrase, which degrades ATP and ADP, and by the inhibition of multiple purinergic receptors. Furthermore, apyrase impaired the recruitment of monocytes in a mouse model of C5a-induced peritonitis. In addition, we found that stimulation of P2Y2, P2Y12, or adenosine receptors induced the formation of lamellipodial membrane protrusions, causing cell spreading. We propose a model in which autocrine purinergic receptor signaling amplifies and translates chemotactic cues into directional motility.

Functional importance of Ca2+-activated K+ channels for lysophosphatidic acid-induced microglial migration

Migration of microglial cells towards damaged tissue plays a key role in central nervous system regeneration under pathological conditions. Using time lapse video microscopy we show that lysophosphatidic acid (LPA) enhances chemokinetic migration of murine microglial cells. In the presence of 1 µm LPA, the mean migration rate of microglial cells was increased 3.8‐fold. In patch‐clamp studies we demonstrate that LPA induces activation of a Ca2+‐activated K+ current. Microglial Ca2+‐activated K+ currents were abolished by either 50 nm charybdotoxin or 10 µm clotrimazole. In contrast, 5 µm paxilline did not have any significant effects on Ca2+‐activated K+ currents. The LPA‐stimulated migration of microglial cells was inhibited by blockers of IKCa1 Ca2+‐activated K+ channels. The mean migration rate of LPA‐stimulated cells was decreased by 61% in the presence of 50 nm charybdotoxin or by 51% during exposure to 10 µm clotrimazole. Microglial migration was not inhibited by 5 µm paxilline. It is concluded that IKCa1 Ca2+‐activated K+ channels are required for LPA‐stimulated migration of microglial cells.

Mechanisms of angiotensin II signaling on cytoskeleton of podocytes

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin–angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of α actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of α actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis.

pH Nanoenvironment at the Surface of Single Melanoma Cells

Extracellular pH and the Na+/H+ exchanger (NHE1) modulate tumor cell migration. Yet, the pH nanoenvironment at the outer surface of the cell membrane (pHem) where cell/matrix interaction occurs and matrix metalloproteinases work was never measured. We present a method to measure this pH nanoenvironment using proton-sensitive dyes to label the outer leaflet of the plasma membrane or the glycocalyx of human melanoma cells. Polarized cells generate an extracellular proton gradient at their surface that increases from the rear end to the leading edge of the lamellipodium along the direction of movement. This gradient collapses upon NHE1 inhibition by HOE642. NHE1 stimulation by intracellular acidification increases the difference in pHem between the tips of lamellipodia and the cell body in a Na+ dependent way. Thus, cells create a pH nanoenvironment that promotes cell migration by facilitating cell adhesion at their front and the release of cell/matrix contacts at their rear part.

Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume.

Abstract Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca2+-sensitive K+ channel (IK channel; Pflügers Arch 432:R87–R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca2+ concentration ([Ca2+]i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca2+]i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca2+]i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca2+]i. We therefore suggest that cell volume – possibly via [Ca2+]i– is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells.