Intercellular crosstalk shapes purinergic metabolism and signaling in cancer cells.

CD73-derived adenosine suppresses anti-cancer immunity, and CD73 inhibitors are currently evaluated in several clinical trials. Here, we have assessed enzyme kinetics of all key purinergic ectoenzymes in five cancer cell lines (Hodgkin lymphoma, multiple myeloma, pancreas adenocarcinoma, urinary bladder carcinoma, and glioblastoma) under normoxia and hypoxia. We found that adenosine metabolism varied considerably between individual cancer types. All cell lines investigated exhibited high ecto-adenosine deaminase (ADA) activity, which critically influenced the kinetics of adenosine accumulation. Combining kinetics data with single-cell RNA sequencing data on myeloma and glioblastoma cancerous tissue revealed that purine metabolism is not homogeneously organized, but it differs in a cancer type-specific fashion between malignant cells, stromal cells, and immune cells. Since purine metabolism in cancerous tissue is most likely spatially heterogeneous and differs between the various cell types, diffusion distances in the microenvironment as well as ADA activity may be important variables that influence the level of bioactive adenosine.

Quantifying and mathematical modelling of the influence of soluble adenylate cyclase on cell cycle in human endothelial cells with Bayesian inference.

Adenosine-3', 5'-cyclic monophosphate (cAMP) produced by adenylate cyclases (ADCYs) is an established key regulator of cell homoeostasis. However, its role in cell cycle control is still controversially discussed. This study focussed on the impact of soluble HCO3 - -activated ADCY10 on cell cycle progression. Effects are quantified with Bayesian inference integrating a mathematical model and experimental data. The activity of ADCY10 in human umbilical vein endothelial cells (HUVECs) was either pharmacologically inhibited by KH7 or endogenously activated by HCO3 - . Cell numbers of individual cell cycle phases were assessed over time using flow cytometry. Based on these numbers, cell cycle dynamics were analysed using a mathematical model. This allowed precise quantification of cell cycle dynamics with model parameters that describe the durations of individual cell cycle phases. Endogenous inactivation of ADCY10 resulted in prolongation of mean cell cycle times (38.7 ± 8.3 h at 0 mM HCO3 - vs 30.3 ± 2.7 h at 24 mM HCO3 - ), while pharmacological inhibition resulted in functional arrest of cell cycle by increasing mean cell cycle time after G0 /G1 synchronization to 221.0 ± 96.3 h. All cell cycle phases progressed slower due to ADCY10 inactivation. In particular, the G1 -S transition was quantitatively the most influenced by ADCY10. In conclusion, the data of the present study show that ADCY10 is a key regulator in cell cycle progression linked specifically to the G1 -S transition.

Anomalous diffusion and asymmetric tempering memory in neutrophil chemotaxis.

The motility of neutrophils and their ability to sense and to react to chemoattractants in their environment are of central importance for the innate immunity. Neutrophils are guided towards sites of inflammation following the activation of G-protein coupled chemoattractant receptors such as CXCR2 whose signaling strongly depends on the activity of Ca2+ permeable TRPC6 channels. It is the aim of this study to analyze data sets obtained in vitro (murine neutrophils) and in vivo (zebrafish neutrophils) with a stochastic mathematical model to gain deeper insight into the underlying mechanisms. The model is based on the analysis of trajectories of individual neutrophils. Bayesian data analysis, including the covariances of positions for fractional Brownian motion as well as for exponentially and power-law tempered model variants, allows the estimation of parameters and model selection. Our model-based analysis reveals that wildtype neutrophils show pure superdiffusive fractional Brownian motion. This so-called anomalous dynamics is characterized by temporal long-range correlations for the movement into the direction of the chemotactic CXCL1 gradient. Pure superdiffusion is absent vertically to this gradient. This points to an asymmetric 'memory' of the migratory machinery, which is found both in vitro and in vivo. CXCR2 blockade and TRPC6-knockout cause tempering of temporal correlations in the chemotactic gradient. This can be interpreted as a progressive loss of memory, which leads to a marked reduction of chemotaxis and search efficiency of neutrophils. In summary, our findings indicate that spatially differential regulation of anomalous dynamics appears to play a central role in guiding efficient chemotactic behavior.

HIF2α supports pro-metastatic behavior in pheochromocytomas/paragangliomas.

Mutations that drive the stabilization of hypoxia inducible factor 2α (HIF2α) and downstream pseudohypoxic signaling are known to predispose to the development of pheochromocytomas and paragangliomas (PPGLs). However, any role of HIF2α in predisposition to metastatic disease remains unclear. To assess such a role we combined gene-manipulations in pheochromocytoma cell lines with retrospective analyses of patient data and gene expression profiling in tumor specimens. Among 425 patients with PPGLs identified with mutations in tumor-susceptibility genes, those with tumors due to activation of pseudohypoxic pathways had a higher frequency of metastatic disease than those with tumors due to activation of kinase-signaling pathways, even without inclusion of patients with mutations in SDHB (18.6% vs 4.3% in, P < 0.0001). Three out of nine (33%) patients with gain-of-function mutations in HIF2α had metastatic disease. In cell line studies, elevated expression of HIF2α enhanced cell proliferation and led to increased migration and invasion capacity. Moreover, HIF2α expression in HIF2α-deficient cells resulted in increased cell motility, diffuse cluster formation and emergence of pseudopodia indicating changes in cell adhesion and cytoskeletal remodeling. In a mouse liver metastasis model, Hif2a enhanced the metastatic load. Transcriptomics data revealed alterations in focal adhesion and extracellular matrix-receptor interactions in HIF2α-mutated PPGLs. Our translational findings demonstrate that HIF2α supports pro-metastatic behavior in PPGLs, though other factors remain critical for subsequent transition to metastasis. We identified LAMB1 and COL4A2 as new potential therapeutic targets for HIF2α-driven PPGLs. Identified HIF2α downstream targets might open a new therapeutic window for aggressive HIF2α-expressing tumors.

Docosahexaenoic acid reduces adenosine triphosphate-induced calcium influx via inhibition of store-operated calcium channels and enhances baseline endothelial nitric oxide synthase phosphorylation in human endothelial cells.

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, 100 µM) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to 50 µM. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated Ca2+-transient. This effect was preserved in the presence of BAPTA (10 and 20 µM) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane (75 µM) to inhibit store-operated calcium channel or thapsigargin (2 µM) to delete calcium store. In addition, DHA (12 µM) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a Ca2+ activated mode to a preferentially controlled phosphorylation mode.

Invasive and non-invasive point-of-care testing and point-of-care monitoring of the hemoglobin concentration in human blood - how accurate are the data?

In this review, scientific investigations of point-of-care testing (POCT) and point-of-care monitoring (POCM) devices are summarized with regard to the measurement accuracy of the hemoglobin concentration. As a common basis, information according to the Bland and Altman principle [bias, limits of agreement (LOA)] as well as the measurement accuracy and precision are considered, so that the comparability can be mapped. These collected data are subdivided according to the manufacturers, devices and procedures (invasive and non-invasive). A total of 31 devices were identified. A comparability of the scientific investigations in particular was given for 23 devices (18 invasive and five non-invasive measuring devices). In terms of measurement accuracy, there is a clear leap between invasive and non-invasive procedures, while no discernible improvement can be derived in the considered time frame from 2010 to 2018. According to the intended use, strict specifications result from the clinical standards, which are insufficiently met by the systems. More stringent requirements can be derived both in the area of blood donation and in the treatment of patients.

A steady state system for in vitro evaluation of steroidogenic pathway dynamics: Application for CYP11B1, CYP11B2 and CYP17 inhibitors.

Disorders featuring dysregulated adrenal steroidogenesis, such as primary aldosteronism, can benefit from targeted therapies. The aldosterone and cortisol producing enzymes, aldosterone synthase (CYP11B2) and 11-beta-hydroxylase (CYP11B1), share 93% homology requiring selective drugs for pharmacological treatment. Herein, we introduce an effective in vitro assay for evaluation of steroidogenic enzyme kinetics based on intracellular flux calculations. H295RA cells were cultured in chambers under constant medium flow. Four hourly samples were collected (control samples), followed by collections over an additional four hours after treatment with fadrozole (10 nM), metyrapone (10 µM), SI_191 (5 nM), a novel CYP11B2 inhibitor or SI_254 (100 nM), a newly synthesized 17-alpha-hydroxylase/17,20-lyase inhibitor. Mass spectrometric measurements of multiple steroids combined with linear system computational modeling facilitated calculation of intracellular fluxes and changes in rate constants at different steroidogenic pathway steps, enabling selectivity of drugs for those steps to be evaluated. While treatment with fadrozole, metyrapone and SI_191 all reduced fluxes of aldosterone, corticosterone and cortisol production, treatment with SI_254 led to increased flux through the mineralocorticoid pathway and reduced production of steroids downstream of 17-alpha-hydroxylase/17,20-lyase. Drug-induced decreases in rate constants revealed higher selectivity of SI_191 compared to other drugs for CYP11B2 over CYP11B1, this reflecting additional inhibitory actions of SI_191 on catalytic steps of CYP11B2 downstream from the initial 11-beta-hydroxlase step. By culturing cells under perfusion the described system provides a realistic model for simple and rapid calculations of intracellular fluxes and changes in rate constants, thereby offering a robust procedure for investigating drug or other effects at specific steps of steroidogenesis.

Biotechnological production of the angiotensin-converting enzyme inhibitory dipeptide isoleucine-tryptophan.

Peptides with angiotensin-converting enzyme (ACE)-inhibitory and antihypertensive effects are suggested as innovative food additives to prevent or treat hypertension. Currently, these substances are isolated from food proteins following nonselective hydrolysis as a mixture of ACE-inhibitory peptides and other protein fragments. This study presents an innovative biotechnological method, based on recombinant DNA technology that was established to specifically produce the ACE-inhibitory dipeptide isoleucine-tryptophan. In a first step, a repetitive isoleucine-tryptophan construct fused to the maltose-binding protein was generated and expressed in Escherichia coli BL21 cells. The chromatographically purified recombinant fusion protein was enzymatically hydrolyzed using α-chymotrypsin to liberate the dipeptide isoleucine-tryptophan. The identity of the liberated isoleucine-tryptophan was confirmed by MS and derivatization of its N-terminus. The ACE-inhibitory effect of the recombinant dipeptide on soluble and membrane bound ACE was found to be indistinguishable from the inhibitory potential of the chemically produced commercially available dipeptide. The established experimental strategy represents a promising approach to the biotechnical production of sufficient amounts of recombinant peptide-based ACE-inhibitory and antihypertensive substances that are applicable as functional food additives to delay or even prevent hypertension.

CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Epicardium-derived cells (EPDCs) play a fundamental role in embryonic cardiac development and are reactivated in the adult heart in response to myocardial infarction (MI). In this study, EPDCs from post-MI rat hearts highly expressed the ectoenzyme CD73 and secreted the profibrotic matricellular protein tenascin-C (TNC). CD73 on EPDCs extensively generated adenosine from both extracellular ATP and NAD. This in turn stimulated the release of additional nucleotides from a Brefeldin A-sensitive intracellular pool via adenosine-A2BR signaling, forming a positive-feedback loop. A2BR activation, in addition, strongly promoted the release of major regulatory cytokines, such as IL-6, IL-11, and VEGF. TNC was found to stimulate EPDC migration and, together with ATP-P2X7R signaling, to activate inflammasomes in EPDCs via TLR4. Our results demonstrate that EPDCs are an important source of various proinflammatory factors in the post-MI heart controlled by purinergic and TNC signaling.-Hesse, J., Leberling, S., Boden, E., Friebe, D., Schmidt, T., Ding, Z., Dieterich, P., Deussen, A., Roderigo, C., Rose, C. R., Floss, D. M., Scheller, J., Schrader, J. CD73-derived adenosine and tenascin-C control cytokine production by epicardium-derived cells formed after myocardial infarction.

Computational analysis of liquid chromatography-tandem mass spectrometric steroid profiling in NCI H295R cells following angiotensin II, forskolin and abiraterone treatment.

Adrenal steroid hormones, which regulate a plethora of physiological functions, are produced via tightly controlled pathways. Investigations of these pathways, based on experimental data, can be facilitated by computational modeling for calculations of metabolic rate alterations. We therefore used a model system, based on mass balance and mass reaction equations, to kinetically evaluate adrenal steroidogenesis in human adrenal cortex-derived NCI H295R cells. For this purpose a panel of 10 steroids was measured by liquid chromatographic-tandem mass spectrometry. Time-dependent changes in cell incubate concentrations of steroids - including cortisol, aldosterone, dehydroepiandrosterone and their precursors - were measured after incubation with angiotensin II, forskolin and abiraterone. Model parameters were estimated based on experimental data using weighted least square fitting. Time-dependent angiotensin II- and forskolin-induced changes were observed for incubate concentrations of precursor steroids with peaks that preceded maximal increases in aldosterone and cortisol. Inhibition of 17-alpha-hydroxylase/17,20-lyase with abiraterone resulted in increases in upstream precursor steroids and decreases in downstream products. Derived model parameters, including rate constants of enzymatic processes, appropriately quantified observed and expected changes in metabolic pathways at multiple conversion steps. Our data demonstrate limitations of single time point measurements and the importance of assessing pathway dynamics in studies of adrenal cortical cell line steroidogenesis. Our analysis provides a framework for evaluation of steroidogenesis in adrenal cortical cell culture systems and demonstrates that computational modeling-derived estimates of kinetic parameters are an effective tool for describing perturbations in associated metabolic pathways.

Early Staphylococcus aureus-induced changes in endothelial barrier function are strain-specific and unrelated to bacterial translocation.

The vascular endothelium provides the critical barrier during hematogenous spreading of bacteria, a phenomenon that might contribute to severe diseases in humans including endocarditis and sepsis as known from infections by Staphylococcus aureus. Here we aimed to uncover early responses of the endothelium to S. aureus infection with respect to (a) inflammatory reactions such as paracellular endothelial barrier function and expression of cell adhesion molecule-1 (ICAM-1) and (b) translocation through the endothelium. After infection of the cultured endothelium with 22 different clinical isolates of S. aureus and two well-characterized lab strains a diverse and strain-specific change in para- and transcellular endothelial barrier function was observed. Bayesian data analysis revealed positive correlation of paracellular barrier function decrease followed by expression of ICAM-1 while these parameters negatively correlated with transcellular bacterial translocation. Translocating bacteria largely blocked TNFα-induced ICAM-1 expression indicating an active anti-inflammatory effect mediated by those strains probably due to intracellularly released virulence factors. Furthermore, the underlying background of barrier function decrease was investigated in more detail using two well-characterized lab strains, ls 8325-4 and ls 6850 and respective mutants. Barrier function decrease was found to be independent of early cell death and early release of virulence factors into the medium, but require internalization of live bacteria. The data show for the first time that endothelial cells respond diversely to infection with different strains of S. aureus and that translocating strains downregulate inflammatory response of the endothelium. Furthermore, data indicate that S. aureus-mediated activation of the endothelium reduces bacterial translocation.

Preservation of human artery function following prolonged cold storage with a new solution.

OBJECTIVE: Blood vessels are an important tissue for allogenic vessel replacement surgery, which is needed for example following infection of artificial grafts. For tissue banking, European legislation requires evidence of tissue sterility with assays performed over 1 week. Currently, used cold storage solutions do not protect vascular function longer than 2 days. This does not allow completion of microbiological testing. This discrepancy has almost completely stopped vessel banking in Europe. METHODS: We compared the recently developed storage solution TiProtec (Dr F Köhler Chemie, Bensheim, Germany) with traditionally used histidine-tryptophan-ketoglutarate (HTK) solution, 0.9% NaCl, and phosphate-buffered saline (physiological saline solution [PSS]) solution for extended cold (4°C) storage up to 25 days. Isolated rings of human internal mammary artery were studied with respect to several parameters of vessel function, including vessel tone development, endothelium-dependent and endothelium-independent relaxation, and tissue reductive capacity. RESULTS: Vessels stored in NaCl or PSS for ≥10 hours failed to develop tone after rewarming. Mammary arteries stored in HTK for 4 hours at 4°C initially showed a well-preserved vessel function with respect to vessel tone development, as well as endothelial and smooth muscle dilatative function. However, following 4 days of cold storage, vessel tone development and dilatative responses were significantly impaired. In contrast, arteries stored in TiProtec showed full preservation of vessel tone as well as endothelial and smooth muscle function after 4 days of cold storage. Even after 10 days of cold storage, endothelium-dependent relaxation was approximately 50% of control, and smooth muscle function was fully preserved. Over 2 weeks, tissue reductive capacity was significantly better maintained after cold storage in TiProtec compared with vessels stored in NaCl. CONCLUSIONS: In contrast to traditional HTK, NaCl, or PSS storage, TiProtec solution offers an excellent potential for prolonged cold storage of human arteries, which may close the existing gap between legal requirements for tissue banking and current cold preservation methods.

Validation of (99m)Tc-labeled "4+1" fatty acids for myocardial metabolism and flow imaging: Part 1: myocardial extraction and biodistribution.

INTRODUCTION: (13)C, (18)F and (123)I fatty acids (FA) are used for myocardial imaging. Recently, our group showed that [(99m)Tc]-labeled "4+1" FA are extracted into the rat and guinea pig myocardium. The present study evaluates determinants of myocardial uptake and whole body biodistribution of these FA derivatives. METHODS: Studies were performed with isolated perfused hearts of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) with a FAT/CD36 deficiency, as well as with heart type FA binding protein knockout mice (H-FABP)(-/-) and H-FABP(+/+). Eight 4+1-(99m)Tc-FA were applied for 3 min followed by 1-min washout. A mathematical model was used to analyze FA dynamics and binding to proteins. Whole-body distribution was studied in rats with and without Tween 80. In vitro fractionation studies with [(99m)Tc]-FA assessed red blood cell uptake as well as association with plasma lipoproteins very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). RESULTS: Myocardial extraction was 19.0-33.0% of the infused dose in isolated WKY and 15.2-26.4% in SHR hearts. However, H-FABP(-/-) showed a marked reduction of tracer extraction [2.8+/-0.6%ID (percent injected dose) vs. 17+/-2%ID P<.001]. Uptake in red blood cells (<1.2%ID) and incorporation into lipoproteins were negligible. Incubation of (99m)Tc-FA with albumin reduced ventricular extraction (P<.001) into the range of established iodinated FA tracers. polyoxyethylene(20) sorbitan monooleate improved the heart-to-liver ratio in the biodistribution studies. CONCLUSIONS: Myocardial uptake of [(99m)Tc]-FA 4+1 derivatives is dependent on H-FABP. These substances may therefore provide a new tool to specifically assess regional myocardial changes of H-FABP.

Validation of (99m)Tc-labeled "4+1" fatty acids for myocardial metabolism and flow imaging: Part 2. Subcellular distribution.

INTRODUCTION: Our group has synthesized technetium-labeled fatty acids (FA) that are extracted into the myocardium and sequestered due to heart-type fatty acid binding protein (H-FABP) binding. In this article, we further address the detailed subcellular distribution and potential myocardial metabolism of [(99m)Tc]"4+1" FA. METHODS: Experiments were conducted using isolated hearts of Wistar rats, as well as of wild-type and H-FABP(-/-) mice. Myocardium samples underwent subcellular fractionation [subsarcolemmal mitochondria (SM), intermyofibrillar mitochondria (IM), cytosol with microsomes, and nuclei and crude membranes] and analysis by thin-layer chromatography and high-performance liquid chromatography. RESULTS: The largest fraction of tissue radioactivity was associated with cytosol [79.69+/-8.88% of infused dose]. About 9.07+/-0.95% and 3.43+/-1.38% of the infused dose were associated with SM and IM fractions, respectively. In the rat heart, etomoxir, an inhibitor of carnitin-palmitoyl transferase I, did not significantly decrease radioactivity associated with mitochondrial fractions, whereas myocardial extraction of [(123)I]-labeled 15-(p-iodophenyl)-pentadecanoic acid (13.26% vs. 49.49% in controls) and the radioactivity associated with the SM and IM fractions were blunted. The percentage of the infused dose in the mitochondrial and crude fractions increased with the number of NH-amide groups of the FA derivative. Absence of H-FABP significantly decreased radioactivity count in the cytosolic fraction (P<.001). No metabolic product of [(99m)Tc]"4+1" FA could be detected in any isolated heart. CONCLUSIONS: Myocardial [(99m)Tc]"4+1" FA extraction reflects binding to H-FABP and membrane structures (including the mitochondrial membrane). However, the compounds do not undergo mitochondrial metabolism because they do not reach the mitochondrial matrix.

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.

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

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) alpha-mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-alpha 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 Tg737(orpk) MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-alpha 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 Tg737(orpk) MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-alpha signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-alpha stimulation.

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.

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.

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.