Stephan Paschke

Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8–18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.

The regulatory role of cell mechanics for migration of differentiating myeloid cells

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytokeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.

Viscoelastic Properties of Differentiating Blood Cells Are Fate- and Function-Dependent

Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured using an optical stretcher, to suit their ultimate fate and function. Myeloid cells circulating in blood have to be advected through constrictions in blood vessels, engendering the need for compliance at short time-scales (<seconds). Intriguingly, only the two circulating myeloid cell types have increased short time scale compliance and flow better through microfluidic constrictions. Moreover, all three differentiated cell types reduce their steady-state viscosity by more than 50% and show over 140% relative increase in their ability to migrate through tissue-like pores at long time-scales (>minutes), compared to undifferentiated cells. These findings suggest that reduction in steady-state viscosity is a physiological adaptation for enhanced migration through tissues. Our results indicate that the material properties of cells define their function, can be used as a cell differentiation marker and could serve as target for novel therapies.

Keratins: markers and modulators of liver disease.

Purpose of review Keratins are a subgroup of intermediate filaments expressed in the epithelia. Keratins emerged as important tissue-protecting genes and keratin variants cause/predispose to development of more than 50 human disorders. Our review focuses on the importance of keratins in context of liver disease. Recent findings K8/K18 variants are found in approximately 4% of white population and predispose to development and adverse outcome of multiple liver diseases. K8/K18 are major constituents of Mallory–Denk bodies, that is inclusions found in alcoholic and nonalcoholic steatohepatitis (NASH) and dysregulated keratin expression, K8 hyperphosphorylation, misfolding and crosslinking via transglutaminase 2 facilitate aggregate formation. Necrosis-generated and apoptosis-generated keratin serum fragments are emerging as important noninvasive markers of multiple liver diseases, particularly NASH. Keratins are established markers of tumor origin and in hepatocellular carcinoma, K19 expression is associated with poor prognosis. Summary Keratins are established tumor markers and are widely used as noninvasive markers of liver injury. In addition, the data that have become available in recent years have greatly advanced our understanding of keratins as modifiers of liver disease development.

Technical Advance: Inhibition of neutrophil chemotaxis by colchicine is modulated through viscoelastic properties of subcellular compartments

Colchicine is an efficient drug for the management of inflammatory diseases, such as gouty arthritis and familial Mediterranean fever. It affects neutrophil activity by interfering with the formation of microtubules. To test the hypothesis that therapeutic concentrations of colchicine modulate the mechanical properties of these cells, we applied a combination of biophysical techniques (optical stretching and microrheology) to analyze cellular deformability. The contribution of the subcellular compartments to the regulation of cell mechanics was determined by fitting a multicomponent model of cellular viscoelasticity to time‐dependent deformation curves. Neutrophils were found to be less deformable in response to 10 ng/ml colchicine. The model‐based analysis of cellular deformation revealed a decrease in cytoplasmatic elasticity and a substantial increase in both elasticity and viscosity of the cell membrane compartment in response to colchicine. These results correlate with a reduced number of cytoplasmatic microtubules and an increase in subcortical actin filaments. The latter finding was confirmed by microrheology and fluorescence microscopy. Neutrophil migration through small pores requiring substantial cellular deformations, but not through large pores, was significantly impaired by colchicine. These data demonstrate that colchicine determines mechanics of neutrophils and, thereby, motility in confined spaces, which is crucial during extravasation of neutrophils in response to inflammatory stimuli.

Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages

The quantity and activation state of adipose tissue macrophages (ATMs) impact the development of obesity-induced metabolic diseases. Appetite-controlling hormones play key roles in obesity; however, our understanding of their effects on ATMs is limited. Here, we have shown that human and mouse ATMs express NPFFR2, a receptor for the appetite-reducing neuropeptide FF (NPFF), and that NPFFR2 expression is upregulated by IL-4, an M2-polarizing cytokine. Plasma levels of NPFF decreased in obese patients and high-fat diet–fed mice and increased following caloric restriction. NPFF promoted M2 activation and increased the proliferation of murine and human ATMs. Both M2 activation and increased ATM proliferation were abolished in NPFFR2-deficient ATMs. Mechanistically, the effects of NPFF involved the suppression of E3 ubiquitin ligase RNF128 expression, resulting in enhanced stability of phosphorylated STAT6 and increased transcription of the M2 macrophage–associated genes IL-4 receptor &agr; (Il4ra), arginase 1 (Arg1), IL-10 (Il10), and alkylglycerol monooxygenase (Agmo). NPFF induced ATM proliferation concomitantly with the increase in N-Myc downstream-regulated gene 2 (Ndrg2) expression and suppressed the transcription of Ifi200 cell-cycle inhibitor family members and MAF bZIP transcription factor B (Mafb), a negative regulator of macrophage proliferation. NPFF thus plays an important role in supporting healthy adipose tissue via the maintenance of metabolically beneficial ATMs.

Complement C5a-induced changes in neutrophil morphology during inflammation

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose‐ and time‐dependently resulted in a rapid C5a receptor‐1 (C5aR1)‐dependent shape change, indicated by enhanced flow cytometric forward‐scatter area values. Similar changes were observed after incubation with zymosan‐activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high‐resolution digital imaging revealed a C5a‐induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a‐induced shape changes were inhibited by pharmacological blockade of either the Cl−/HCO3− ‐exchanger or the Cl−‐channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP–P2X/Y‐purinoceptor interaction was also involved in the C5a‐induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a‐dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin‐cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation

ABSTRACT Dynamic regulation of cell–cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell–cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions. Summary: Cortactin and its phosphorylation by protein kinase D1 at S298 modulate E-cadherin adhesion complex composition and F-actin linkage during ligation and dissolution of adherence junctions.

Microrheology of keratin networks in cancer cells

Microrheology is a valuable tool to determine viscoelastic properties of polymer networks. For this purpose measurements with embedded tracer beads inside the extracted network of pancreatic cancer cells were performed. Observing the beads motion with a CCD-high-speed-camera leads to the dynamic shear modulus. The complex shear modulus is divided into real and imaginary parts which give insight into the mechanical properties of the cell. The dependency on the distance of the embedded beads to the rim of the nucleus shows a tendency for a decreasing storage modulus. We draw conclusions on the network topology of the keratin network types based on the mechanical behavior.

Population Pharmacokinetics and Target Attainment of Ertapenem in Plasma and Tissue Assessed via Microdialysis in Morbidly Obese Patients after Laparoscopic Visceral Surgery

ABSTRACT Ertapenem provides broad-spectrum activity against many pathogens, and its use is relevant for the prophylaxis and treatment of infections in morbidly obese patients undergoing surgery. However, its pharmacokinetics and tissue penetration in these patients are not well defined. We assessed the population pharmacokinetics and target attainment for ertapenem in the plasma, subcutaneous tissue, and peritoneal fluid of morbidly obese patients. Six female patients (body mass index, 43.7 to 55.9 kg/m2) received 1,000 mg ertapenem as 15-min infusions at 0 and 26 h. On day 2, the unbound ertapenem concentrations in plasma, subcutaneous tissue, and peritoneal fluid were measured by microdialysis; total plasma concentrations were additionally quantified. The probability of attaining a target of an unbound ertapenem concentration above the MIC for at least 40% of the dosing interval was predicted via Monte Carlo simulations. The population pharmacokinetic model contained two disposition compartments and simultaneously described all concentrations. For unbound ertapenem, total clearance was 12.3 liters/h (coefficient of variation, 21.6% for between-patient variability) and the volume of distribution at steady state was 57.8 liters in patients with a 53-kg fat-free mass. The area under the concentration-time curve (AUC) for ertapenem was 49% lower in subcutaneous tissue and 25% lower in peritoneal fluid than the unbound AUC in plasma. Tissue penetration was rapid (equilibration half-life, <15 min) and was variable in subcutaneous tissue. Short-term ertapenem infusions (1,000 mg every 24 h) achieved robust (>90%) target attainment probabilities for MICs of up to 1 mg/liter in plasma, 0.25 to 0.5 mg/liter in subcutaneous tissue, and 0.5 mg/liter in peritoneal fluid. Ertapenem presents an attractive choice for many pathogens relevant to morbidly obese patients undergoing surgery. (This study has been registered at ClinicalTrials.gov under identifier NCT01407965.)