Peter Dieterich

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).

Anomalous dynamics of cell migration

Cell movement—for example, during embryogenesis or tumor metastasis—is a complex dynamical process resulting from an intricate interplay of multiple components of the cellular migration machinery. At first sight, the paths of migrating cells resemble those of thermally driven Brownian particles. However, cell migration is an active biological process putting a characterization in terms of normal Brownian motion into question. By analyzing the trajectories of wild-type and mutated epithelial (transformed Madin–Darby canine kidney) cells, we show experimentally that anomalous dynamics characterizes cell migration. A superdiffusive increase of the mean squared displacement, non-Gaussian spatial probability distributions, and power-law decays of the velocity autocorrelations is the basis for this interpretation. Almost all results can be explained with a fractional Klein–Kramers equation allowing the quantitative classification of cell migration by a few parameters. Thereby, it discloses the influence and relative importance of individual components of the cellular migration apparatus to the behavior of the cell as a whole.

Endothelial barrier function under laminar fluid shear stress

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress–induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 Ωcm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of α-, β-, and γ-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress–induced junction rearrangement. Additionally, catenins were partly colocalized with β-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress–dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.

The Na+/H+ exchanger NHE1 is required for directional migration stimulated via PDGFR-α in the primary cilium

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) α–mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-α and the Na+/H+ exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in Tg737orpk MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-α ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5′-(N-ethyl-N-isopropyl) amiloride potently reduces PDGF-AA–mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in Tg737orpk MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-α signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-α stimulation.

Subcellular distribution of calcium-sensitive potassium channels (IK1) in migrating cells.

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular “migration machinery.” We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK‐F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK‐F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK‐F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA‐tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK‐F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK‐F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK‐F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells.

TRPC1 channels regulate directionality of migrating cells.

Cell migration depends on the generation of structural asymmetry and on different steps: protrusion and adhesion at the front and traction and detachment at the rear part of the cell. The activity of Ca2+ channels coordinate these steps by arranging intracellular Ca2+ signals along the axis of movement. Here, we investigated the role of the putative mechanosensitive canonical transient receptor potential channel 1 (TRPC1) in cell migration. We analyzed its function in transformed renal epithelial (Madin–Darby canine kidney-focus) cells with variation of TRPC1 expression. As shown by time lapse video microscopy, TRPC1 knockdown cells have partially lost their polarity and the ability to persistently migrate into a given direction. This failure is linked to the suppression of a local Ca2+ gradient at the front of migrating TRPC1 knockdown cells, whereas TRPC1 overexpression leads to steeper Ca2+ gradients. We propose that the Ca2+ signaling events regulated by TRPC1 within the lamellipodium determine polarity and directed cell migration.

Quantitative Morphodynamics of Endothelial Cells within Confluent Cultures in Response to Fluid Shear Stress

To evaluate shear stress-induced effects on cultured cells we have extended the mechanical setup of a multichannel in vitro rheological system and developed software allowing entire processing control and image data analysis. The values of cell motility, degree of orientation (alignment), and cell elongation were correlated as a function of time (morphodynamics). Collective and individual endothelial cells within confluent cultures displayed a shear stress-dependent characteristic phase behavior of the following time course: resting conditions (phase I), change of motility (phase II), onset of alignment (phase III), and finally cell elongation (phase IV). Especially cell motility was characterized by a randomized zigzag movement around mean trajectories (fluctuations) together with mean cell locomotion. Onset of shear stress caused a down-regulation of fluctuations of 30% within <10 min and simultaneously increased locomotion velocities preferring the flow direction (phase II). After a lag period of 10 to 20 min cells orientated in the direction of flow (phase III) without significant cell elongation, which finally occurs within hours (phase IV). These data provide first evidence that cells within confluent endothelial monolayers respond to shear stress with a characteristic phase behavior.

The role of Ca2+ transport across the plasma membrane for cell migration.

Cell migration plays a central role in many physiological and pathophysiological processes. On a cellular level it is based on a highly coordinated restructuring of the cytoskeleton, a continuous cycle of adhesion and de-adhesion as well as on the activity of ion channels and transporters. The cytoplasmic Ca²⁺ ([Ca²⁺]_i) concentration is an important coordinator of these intracellular processes. Thus, [Ca²⁺]_i must be tightly controlled in migrating cells. This is among other things achieved by the activity of Ca²⁺ permeable channels, the plasma membrane Ca²⁺-ATPase (PMCA) and the Na⁺/Ca²⁺ exchanger (NCX) in the plasma membrane. Here, we wanted to determine the functional role of these transport proteins in cell migration. We therefore quantified the acute effect of inhibitors of these transport proteins (Gd³⁺, vanadate, KB-R7943) on migration, [Ca²⁺]_i, and intracellular pH (pH_i) of MDCK-F cells. Migration was monitored with computer-assisted time-lapse video microscopy. [Ca²⁺]_i and pH_i were measured with the fluorescent indicators fura-2 and BCECF. NCX expression in MDCK-F cells was verified with ion substitution experiments, and expression of PMCA was tested with RT-PCR. All blockers lead to a rapid impairment of cell migration. However, the most prominent effect is elicited by NCX-inhibition with KB-R7943. NCX-blockade leads to an almost complete inhibition of migration which is accompanied by a dose-dependent increase of [Ca²⁺]_i and an intracellular alkalinisation. We show that inhibition of NCX and PMCA strongly affects lamellipodial dynamics of migrating MDCK-F cells. Taken together, our results show that PMCA and in particular NCX are of critical importance for cell migration.

Dynamics of single potassium channel proteins in the plasma membrane of migrating cells

Cell migration is an important physiological process among others controlled by ion channel activity. Calcium-activated potassium channels (K(Ca)3.1) are required for optimal cell migration. Previously, we identified single human (h)K(Ca)3.1 channel proteins in the plasma membrane by means of quantum dot (QD) labeling. In the present study, we tracked single-channel proteins during migration to classify their dynamics in the plasma membrane of MDCK-F cells. Single hK(Ca)3.1 channels were visualized with QD- or Alexa488-conjugated antibodies and tracked at the basal cell membrane using time-lapse total internal reflection fluorescence (TIRF) microscopy. Analysis of the trajectories allowed the classification of channel dynamics. Channel tracks were compared with those of free QD-conjugated antibodies. The size of the label has a pronounced effect on hK(Ca)3.1 channel diffusion. QD-labeled channels have a (sub)diffusion coefficient D(QDbound) = 0.067 microm(2)/s(alpha), whereas that of Alexa488-labeled channels is D(Alexa) = 0.139 microm(2)/s. Free QD-conjugated antibodies move much faster: D(QDfree) = 2.163 microm(2)/s(alpha). Plotting the mean squared distances (msd) covered by hK(Ca)3.1 channels as a function of time points to the mode of diffusion. Alexa488-labeled channels diffuse normally, whereas the QD-label renders hK(Ca)3.1 channel diffusion anomalous. Free QD-labeled antibodies also diffuse anomalously. Hence, QDs slow down diffusion of hK(Ca)3.1 channels and change the mode of diffusion. These results, referring to the role of label size and properties of the extracellular environment, suggest that the pericellular glycocalyx has an important impact on labels used for single molecule tracking. Thus tracking fluorescent particles within the glycocalyx opens up a possibility to characterize the pericellular nanoenvironment.

Improved vessel preservation after 4 days of cold storage: Experimental study in rat arteries

BACKGROUND Cold storage of arteries for reconstructive and bypass surgery may result in injury of endothelial cells which may promote low perfusion and graft vasculopathy. METHODS A recently developed N-acetyl histidine-buffered, potassium-chloride enriched, and amino acid-fortified vascular storage solution augmented with iron chelators deferoxamine (100 micromol/L) and LK 614 (20 micromol/L) was studied in the rat superior mesenteric artery and aorta with respect to: (1) potassium-induced vessel tone, (2) endothelium-dependent and -independent relaxation, and (3) endothelial nitric oxide synthase (eNOS) protein expression over 4-days cold storage (4 degrees C).This solution was compared with traditional storage solutions, histidine-tryptophan-ketoglutarate (HTK) and physiological saline solution (PSS). RESULTS Vessels stored for 4 days in the new solution were significantly better protected than those stored in traditional HTK or PSS. The protective effects comprised: (1) vessel tone development after stimulation with potassium-chloride solution, (2) endothelium-dependent and -independent vessel relaxation, and (3) eNOS expression. With iron chelators (deferoxamine 100 microM, LK 614 20 microM) present in the storage solution, endothelium-dependent relaxations (eNOS-dependent and K(Ca)-channel-dependent) were fully maintained after 96 hours of cold storage. Endothelial cell structure was significantly better maintained after 96 hours in the new solution than in HTK or PSS solutions. Already, 2 hours of cold storage in HTK resulted in a significant loss of structurally intact endothelium. The structural changes correlated significantly with the diminished vessel relaxation capacity. Furthermore, tissue reductive capacity was only preserved after 96 hours storage if the new solution was used. CONCLUSION The new storage solution is superior to traditional HTK and PSS cold storage with respect to: (1) preservation of vessel structure and function; (2) the presence of iron chelators significantly improved protection of endothelial function; and (3) the new solution permits cold vessel storage for a minimum of 4 days with full maintenance of endothelial function and its coupling to smooth muscle.