Effective targeting of breast cancer by the inhibition of P-glycoprotein mediated removal of toxic lipid peroxidation byproducts from drug tolerant persister cells.

Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1-/-;p53-/- mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.

Diverse Interactions: Root-Nodule Formation and Herb-Layer Composition in Black Locust (Robinia pseudoacacia) Stands.

The black locust (Robinia pseudoacacia L.) is the second-most abundant deciduous tree in forest plantations, and one of the most important invasive woody species worldwide. The species has a strong transformer capacity, especially expressed by its nitrogen enrichment effect caused by nitrogen-fixing bacteria living in its root-nodules. The aim of this study was to explore the mutually interacting factors of nitrogen-fixing root-nodules, site characteristics, and herb-layer composition of 28 North Hungarian black locust stands. In the herb-layers of the study sites, a total of 121 plant species were identified, representing a relatively low species richness. The studied black locust stands showed high variability both in their herb-layer compositions and root-nodule formation, but no clear relationship could be demonstrated between these characteristics. The PCA component with which the species richness and Shannon-Wiener diversity index were strongly correlated was negatively associated with all root-nodule parameters (number, surface area, and weight), supporting the biodiversity-reducing effect of black locust by its nitrogen-fixing bacteria. All of the root-nodule parameters were negatively correlated with the PCA factor predominantly determined by stand age, confirming that the root-nodule biomass decreases as time progresses.

Inducible overexpression of a FAM3C/ILEI transgene has pleiotropic effects with shortened life span, liver fibrosis and anemia in mice.

FAM3C/ILEI is an important factor in epithelial-to-mesenchymal transition (EMT) induction, tumor progression and metastasis. Overexpressed in many cancers, elevated ILEI levels and secretion correlate with poor patient survival. Although ILEI's causative role in invasive tumor growth and metastasis has been demonstrated in several cellular tumor models, there are no available transgenic mice to study these effects in the context of a complex organism. Here, we describe the generation and initial characterization of a Tet-ON inducible Fam3c/ILEI transgenic mouse strain. We find that ubiquitous induction of ILEI overexpression (R26-ILEIind) at weaning age leads to a shortened lifespan, reduced body weight and microcytic hypochromic anemia. The anemia was reversible at a young age within a week upon withdrawal of ILEI induction. Vav1-driven overexpression of the ILEIind transgene in all hematopoietic cells (Vav-ILEIind) did not render mice anemic or lower overall fitness, demonstrating that no intrinsic mechanisms of erythroid development were dysregulated by ILEI and that hematopoietic ILEI hyperfunction did not contribute to death. Reduced serum iron levels of R26-ILEIind mice were indicative for a malfunction in iron uptake or homeostasis. Accordingly, the liver, the main organ of iron metabolism, was severely affected in moribund ILEI overexpressing mice: increased alanine transaminase and aspartate aminotransferase levels indicated liver dysfunction, the liver was reduced in size, showed increased apoptosis, reduced cellular iron content, and had a fibrotic phenotype. These data indicate that high ILEI expression in the liver might reduce hepatoprotection and induce liver fibrosis, which leads to liver dysfunction, disturbed iron metabolism and eventually to death. Overall, we show here that the novel Tet-ON inducible Fam3c/ILEI transgenic mouse strain allows tissue specific timely controlled overexpression of ILEI and thus, will serve as a versatile tool to model the effect of elevated ILEI expression in diverse tissue entities and disease conditions, including cancer.

FAM3C/ILEI protein is elevated in psoriatic lesions and triggers psoriasiform hyperproliferation in mice.

FAM3C/ILEI is an important cytokine for tumor progression and metastasis. However, its involvement in inflammation remains elusive. Here, we show that ILEI protein is highly expressed in psoriatic lesions. Inducible keratinocyte-specific ILEI overexpression in mice (K5-ILEIind ) recapitulates many aspects of psoriasis following TPA challenge, primarily manifested by impaired epidermal differentiation and increased neutrophil recruitment. Mechanistically, ILEI triggers Erk and Akt signaling, which then activates STAT3 via Ser727 phosphorylation. Keratinocyte-specific ILEI deletion ameliorates TPA-induced skin inflammation. A transcriptomic ILEI signature obtained from the K5-ILEIind model shows enrichment in several signaling pathways also found in psoriasis and identifies urokinase as a targetable enzyme to counteract ILEI activity. Pharmacological inhibition of urokinase in TPA-induced K5-ILEIind mice results in significant improvement of psoriasiform symptoms by reducing ILEI secretion. The ILEI signature distinguishes psoriasis from healthy skin with uPA ranking among the top "separator" genes. Our study identifies ILEI as a key driver in psoriasis, indicates the relevance of ILEI-regulated genes for disease manifestation, and shows the clinical impact of ILEI and urokinase as novel potential therapeutic targets in psoriasis.

Smuggling on the Nanoscale-Fusogenic Liposomes Enable Efficient RNA-Transfer with Negligible Immune Response In Vitro and In Vivo.

The efficient and biocompatible transfer of nucleic acids into mammalian cells for research applications or medical purposes is a long-standing, challenging task. Viral transduction is the most efficient transfer system, but often entails high safety levels for research and potential health impairments for patients in medical applications. Lipo- or polyplexes are commonly used transfer systems but result in comparably low transfer efficiencies. Moreover, inflammatory responses caused by cytotoxic side effects were reported for these transfer methods. Often accountable for these effects are various recognition mechanisms for transferred nucleic acids. Using commercially available fusogenic liposomes (Fuse-It-mRNA), we established highly efficient and fully biocompatible transfer of RNA molecules for in vitro as well as in vivo applications. We demonstrated bypassing of endosomal uptake routes and, therefore, of pattern recognition receptors that recognize nucleic acids with high efficiency. This may underlie the observed almost complete abolishment of inflammatory cytokine responses. RNA transfer experiments into zebrafish embryos and adult animals fully confirmed the functional mechanism and the wide range of applications from single cells to organisms.

Membrane tension controls the phase equilibrium in fusogenic liposomes.

Fusogenic liposomes have been widely used for molecule delivery to cell membranes and cell interior. However, their physicochemical state is still little understood. We tested mechanical material behavior by micropipette aspiration of giant vesicles from fusogenic lipid mixtures and found that the membranes of these vesicles are fluid and under high mechanical tension even before aspiration. Based on this result, we developed a theoretical framework to determine the area expansion modulus and membrane tension of such pre-tensed vesicles from aspiration experiments. Surprisingly high membrane tension of 2.1 mN m-1 and very low area expansion modulus of 63 mN m-1 were found. We interpret these peculiar material properties as the result of a mechanically driven phase transition between the usual lamellar phase and an, as of now, not finally determined three dimensional phase of the lipid mixture. The free enthalpy of transition between these phases is very low, i.e. on the order of the thermal energy.

Passive Macromolecular Translocation Mechanism through Lipid Membranes.

The translocation of biologically active macromolecules through cell membranes is of vital importance for cells and is a key process for drug delivery. Proteins exploit specific conformational changes in their secondary structure to facilitate membrane translocation. For the large class of biological and synthetic macromolecules, where such conformational adaptions are not possible, guidelines to tailor the structure of monomers and macromolecules to aid membrane translocation and cross-membrane drug delivery would be highly desirable. Here, we use alternating amphiphilic macromolecules to systematically investigate the relation between polarity, polymer chain length, lipid chain length, polymer concentration, and temperature on membrane partition and translocation rate. We employed pulse field gradient NMR and confocal fluorescence microscopy to determine membrane adsorption and desorption rate constants and partitioning coefficients. We find that translocation is a two-step process involving a fast adsorption and membrane insertion process and a slower desorption process. Membrane insertion is a key step that determines the molecular weight, concentration, and temperature dependences. Passive translocation is possible on time scales from minutes to hours. Macromolecules with different adapted hydrophilic/hydrophobic comonomer sequences show the same translocation rate, indicating that common optimized translocation conditions can be realized with a variety of monomer chemical structures. The investigated copolymers are biocompatible, biodegradable, and capable of transporting a hydrophobic payload through the lipid membrane. This detailed understanding of the macromolecular translocation mechanism enables to better tailor the delivery of active agents using macromolecular carriers.

PEGylation and folic-acid functionalization of cationic lipoplexes-Improved nucleic acid transfer into cancer cells.

Efficient and reliable transfer of nucleic acids for therapy applications is a major challenge. Stabilization of lipo- and polyplexes has already been successfully achieved by PEGylation. This modification reduces the interaction with serum proteins and thus prevents the lipoplexes from being cleared by the reticuloendothelial system. Problematically, this stabilization of lipoplexes simultaneously leads to reduced transfer efficiencies compared to non-PEGylated complexes. However, this reduction in transfer efficiency can be used to advantage since additional modification of PEGylated lipoplexes with functional groups enables improved selective transfer into target cells. Cancer cells overexpress folate receptors because of a significantly increased need of folate due to high cell proliferation rates. Thus, additional folate functionalization of PEGylated lipoplexes improves uptake into cancer cells. We demonstrate herein that NHS coupling chemistries can be used to modify two commercially available transfection reagents (Fuse-It-DNA and Lipofectamine® 3000) with NHS-PEG-folate for increased uptake of nucleic acids into cancer cells. Lipoplex characterization and functional analysis in cultures of cancer- and healthy cells clearly demonstrate that functionalization of PEGylated lipoplexes offers a promising method to generate efficient, stable and selective nucleic acid transfer systems.

The FAM3C locus that encodes interleukin-like EMT inducer (ILEI) is frequently co-amplified in MET-amplified cancers and contributes to invasiveness.

BACKGROUND: Gene amplification of MET, which encodes for the receptor tyrosine kinase c-MET, occurs in a variety of human cancers. High c-MET levels often correlate with poor cancer prognosis. Interleukin-like EMT inducer (ILEI) is also overexpressed in many cancers and is associated with metastasis and poor survival. The gene for ILEI, FAM3C, is located close to MET on chromosome 7q31 in an amplification "hotspot", but it is unclear whether FAMC3 amplification contributes to elevated ILEI expression in cancer. In this study we have investigated FAMC3 copy number gain in different cancers and its potential connection to MET amplifications. METHODS: FAMC3 and MET copy numbers were investigated in various cancer samples and 200 cancer cell lines. Copy numbers of the two genes were correlated with mRNA levels, with relapse-free survival in lung cancer patient samples as well as with clinicopathological parameters in primary samples from 49 advanced stage colorectal cancer patients. ILEI knock-down and c-MET inhibition effects on proliferation and invasiveness of five cancer cell lines and growth of xenograft tumors in mice were then investigated. RESULTS: FAMC3 was amplified in strict association with MET amplification in several human cancers and cancer cell lines. Increased FAM3C and MET copy numbers were tightly linked and correlated with increased gene expression and poor survival in human lung cancer and with extramural invasion in colorectal carcinoma. Stable ILEI shRNA knock-down did not influence proliferation or sensitivity towards c-MET-inhibitor induced proliferation arrest in cancer cells, but impaired both c-MET-independent and -dependent cancer cell invasion. c-MET inhibition reduced ILEI secretion, and shRNA mediated ILEI knock-down prevented c-MET-signaling induced elevated expression and secretion of matrix metalloproteinase (MMP)-2 and MMP-9. Combination of ILEI knock-down and c-MET-inhibition significantly reduced the invasive outgrowth of NCI-H441 and NCI-H1993 lung tumor xenografts by inhibiting proliferation, MMP expression and E-cadherin membrane localization. CONCLUSIONS: These novel findings suggest MET amplifications are often in reality MET-FAM3C co-amplifications with tight functional cooperation. Therefore, the clinical relevance of this frequent cancer amplification hotspot, so far dedicated purely to c-MET function, should be re-evaluated to include ILEI as a target in the therapy of c-MET-amplified human carcinomas.

Delivery of the Radionuclide 131I Using Cationic Fusogenic Liposomes as Nanocarriers.

Liposomes are highly biocompatible and versatile drug carriers with an increasing number of applications in the field of nuclear medicine and diagnostics. So far, only negatively charged liposomes with intercalated radiometals, e.g., 64Cu, 99mTc, have been reported. However, the process of cellular uptake of liposomes by endocytosis is rather slow. Cellular uptake can be accelerated by recently developed cationic liposomes, which exhibit extraordinarily high membrane fusion ability. The aim of the present study was the development of the formulation and the characterization of such cationic fusogenic liposomes with intercalated radioactive [131I]I- for potential use in therapeutic applications. The epithelial human breast cancer cell line MDA-MB-231 was used as a model for invasive cancer cells and cellular uptake of [131I]I- was monitored in vitro. Delivery efficiencies of cationic and neutral liposomes were compared with uptake of free iodide. The best cargo delivery efficiency (~10%) was achieved using cationic fusogenic liposomes due to their special delivery pathway of membrane fusion. Additionally, human blood cells were also incubated with cationic control liposomes and free [131I]I-. In these cases, iodide delivery efficiencies remained below 3%.

Fluorescence Correlation Spectroscopy Reveals Interaction of Some Microdomain-Associated Lipids with Cellular Focal Adhesion Sites.

Cells adhere to the extracellular matrix at distinct anchoring points, mostly focal adhesions. These are rich in immobile transmembrane- and cytoskeletal-associated proteins, some of which are known to interact with lipids of the plasma membrane. To investigate their effect on lipid mobility and molecular interactions, fluorescently labeled lipids were incorporated into the plasma membranes of primary myofibroblasts using fusogenic liposomes. With fluorescence correlation spectroscopy, we tested mobilities of labeled microdomain-associated lipids such as sphingomyelin (SM), ganglioside (GM1), and cholesterol as well as of a microdomain-excluded phospholipid (PC) and a lipid-like molecule (DiIC18(7)) in focal adhesions (FAs) and in neighboring non-adherent membrane areas. We found significantly slower diffusion of SM and GM1 inside FAs but no effect on cholesterol, PC, and DiIC18(7). These data were compared to the molecular behavior in Lo/Ld-phase separated giant unilamellar vesicles, which served as a model system for microdomain containing lipid membranes. In contrast to the model system, lipid mobility changes in FAs were molecularly selective, and no particle enrichment occurred. Our findings suggest that lipid behavior in FAs cannot be described by Lo/Ld-phase separation. The observed slow-down of some molecules in FAs is potentially due to transient binding between lipids and some molecular constituent(s).

The Basement Membrane in a 3D Breast Acini Model Modulates Delivery and Anti-Proliferative Effects of Liposomal Anthracyclines.

Breast cancer progression is marked by cancer cell invasion and infiltration, which can be closely linked to sites of tumor-connected basement membrane thinning, lesion, or infiltration. Bad treatment prognosis frequently accompanies lack of markers for targeted therapy, which brings traditional chemotherapy into play, despite its adverse effects like therapy-related toxicities. In the present work, we compared different liposomal formulations for the delivery of two anthracyclines, doxorubicin and aclacinomycin A, to a 2D cell culture and a 3D breast acini model. One formulation was the classical phospholipid liposome with a polyethylene glycol (PEG) layer serving as a stealth coating. The other formulation was fusogenic liposomes, a biocompatible, cationic, three-component system of liposomes able to fuse with the plasma membrane of target cells. For the lysosome entrapment-sensitive doxorubicin, membrane fusion enabled an increased anti-proliferative effect in 2D cell culture by circumventing the endocytic route. In the 3D breast acini model, this process was found to be limited to cells beneath a thinned or compromised basement membrane. In acini with compromised basement membrane, the encapsulation of doxorubicin in fusogenic liposomes increased the anti-proliferative effect of the drug in comparison to a formulation in PEGylated liposomes, while this effect was negligible in the presence of intact basement membranes.

Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes.

Highly efficient, biocompatible, and fast nucleic acid delivery methods are essential for biomedical applications and research. At present, two main strategies are used to this end. In non-viral transfection liposome- or polymer-based formulations are used to transfer cargo into cells via endocytosis, whereas viral carriers enable direct nucleic acid delivery into the cell cytoplasm. Here, we introduce a new generation of liposomes for nucleic acid delivery, which immediately fuse with the cellular plasma membrane upon contact to transfer the functional nucleic acid directly into the cell cytoplasm. For maximum fusion efficiency combined with high cargo transfer, nucleic acids had to be complexed and partially neutralized before incorporation into fusogenic liposomes. Among the various neutralization agents tested, small, linear, and positively charged polymers yielded the best complex properties. Systematic variation of liposomal composition and nucleic acid complexation identified surface charge as well as particle size as essential parameters for cargo-liposome interaction and subsequent fusion induction. Optimized protocols were tested for the efficient transfer of different kinds of nucleic acids like plasmid DNA, messenger RNA, and short-interfering RNA into various mammalian cells in culture and into primary tissues.

Fusogenic liposomes effectively deliver resveratrol to the cerebral microcirculation and improve endothelium-dependent neurovascular coupling responses in aged mice.

Adjustment of cerebral blood flow (CBF) to the increased oxygen and nutrient demands of active brain regions via neurovascular coupling (NVC) has an essential role in maintenance of healthy cognitive function. In advanced age, cerebromicrovascular oxidative stress and endothelial dysfunction impair neurovascular coupling, contributing to age-related cognitive decline. Recently we developed a resveratrol (3,4',5-trihydroxystilbene)-containing fusogenic liposome (FL-RSV)-based molecular delivery system that can effectively target cultured cerebromicrovascular endothelial cells, attenuating age-related oxidative stress. To assess the cerebromicrovascular protective effects of FL-RSV in vivo, aged (24-month-old) C57BL/6 mice were treated with FL-RSV for four days. To demonstrate effective cellular uptake of FL-RSV, accumulation of the lipophilic tracer dyes in cells of the neurovascular unit was confirmed using two-photon imaging (through a chronic cranial window). NVC was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that NVC responses were significantly impaired in aged mice. Treatment with FL-RSV significantly improved NVC responses by increasing NO-mediated vasodilation. These findings are paralleled by the protective effects of FL-RSV on endothelium-dependent relaxation in the aorta. Thus, treatment with FL-RSV rescues endothelial function and NVC responses in aged mice. We propose that resveratrol containing fusogenic liposomes could also be used for combined delivery of various anti-geronic factors, including proteins, small molecules, DNA vectors and mRNAs targeting key pathways involved in microvascular aging and neurovascular dysfunction for the prevention/treatment of age-related cerebromicrovascular pathologies and development of vascular cognitive impairment (VCI) in aging.

Influence of Environmental Conditions on the Fusion of Cationic Liposomes with Living Mammalian Cells.

Lipid-based nanoparticles, also called vesicles or liposomes, can be used as carriers for drugs or many types of biological macromolecules, including DNA and proteins. Efficiency and speed of cargo delivery are especially high for carrier vesicles that fuse with the cellular plasma membrane. This occurs for lipid mixture containing equal amounts of the cationic lipid DOTAP and a neutral lipid with an additional few percents of an aromatic substance. The fusion ability of such particles depends on lipid composition with phosphoethanolamine (PE) lipids favoring fusion and phosphatidyl-choline (PC) lipids endocytosis. Here, we examined the effects of temperature, ionic strength, osmolality, and pH on fusion efficiency of cationic liposomes with Chinese hamster ovary (CHO) cells. The phase state of liposomes was analyzed by small angle neutron scattering (SANS). Our results showed that PC containing lipid membranes were organized in the lamellar phase. Here, fusion efficiency depended on buffer conditions and remained vanishingly small at physiological conditions. In contrast, SANS indicated the coexistence of very small (~50 nm) objects with larger, most likely lamellar structures for PE containing lipid particles. The fusion of such particles to cell membranes occurred with very high efficiency at all buffer conditions. We hypothesize that the altered phase state resulted in a highly reduced energetic barrier against fusion.

Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins.

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA- /E-/H- and racE- mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.

Changing the Way of Entrance: Highly Efficient Transfer of mRNA and siRNA via Fusogenic Nano-Carriers.

Transferring nucleic acids into mammalian cells heavily influences life science for decades. While first applications mainly dealt with DNA transfer for various purposes as e.g., plasmid encoded protein expression or generation of mutant strains, subsequent applications additionally transferred RNA molecules of mainly small lengths for specific knockdown (RNAi) or site-specific genome modification (gRNA). Significant improvements in full length mRNA generation and extension of mRNA lifetimes additionally allows their use for transient expression in latest times. For all of these types of nucleic acids the most common cell incorporation method is based on complexation and subsequent endosomal uptake. This so-called lipofection can be used theoretically for almost any mammalian cell type and a tremendous number of different product compositions exist in order to deal with drawbacks as transfer efficiency, cell type selectivity, endosomal degradation, slow uptake and cytotoxicity. In contrast, new methods transfer complexed RNA molecules directly into the cytoplasm using liposomal nano-carriers that fuse with cellular plasma membranes immediately upon contact to free functional nucleic acids directly into the cytoplasm. Here, we compare both methods in detail with special focus on robustness, short- and long-term cytotoxicity, efficiency and functionality for various types of transferred RNA. Our data clearly indicate that direct RNA incorporation via fusogenic nano-carriers circumvents most endosomal uptake-based challenges, making it to a most promising alternative for nucleic acid transfer.

Sensitivity to Strain and Shear Stress of Isolated Mechanosensitive Enteric Neurons.

Within the enteric nervous system, the neurons in charge to control motility of the gastrointestinal tract reside in a particular location nestled between two perpendicular muscle layers which contract and relax. We used primary cultured myenteric neurons of male guinea pigs to study mechanosensitivity of enteric neurons in isolation. Ultrafast Neuroimaging with a voltage-sensitive dye technique was used to record neuronal activity in response to shear stress and strain. Strain was induced by locally deforming the elastic cell culture substrate next to a neuron. Measurements showed that substrate strain was mostly elongating cells. Shear stress was exerted by hydrodynamic forces in a microchannel. Both stimuli induced excitatory responses. Strain activated 14% of the stimulated myenteric neurons that responded with a spike frequency of 1.9 (0.7/3.2) Hz, whereas shear stress excited only a few neurons (5.6%) with a very low spike frequency of 0 (0/0.6) Hz. Thus, shear stress does not seem to be an adequate stimulus for mechanosensitive enteric neurons (MEN) while strain activates enteric neurons in a relevant manner. Analyzing the adaptation behavior of MEN showed that shear stress activated rapidly/slowly/ultraslowly adapting MEN (2/62/36%) whereas strain only slowly (46%) and ultraslowly (54%) MEN. Paired experiments with strain and normal stress revealed three mechanosensitive enteric neuronal populations: one strain-sensitive (37%), one normal stress-sensitive (17%) and one strain- and stress-sensitive (46%). These results indicate that shear stress does not play a role in the neuronal control of motility but normal stress and strain.

Deciphering the Functional Composition of Fusogenic Liposomes.

Cationic liposomes are frequently used as carrier particles for nucleic acid delivery. The most popular formulation is the equimolar mixture of two components, a cationic lipid and a neutral phosphoethanolamine. Its uptake pathway has been described as endocytosis. The presence of an aromatic molecule as a third component strongly influences the cellular uptake process and results in complete membrane fusion instead of endocytosis. Here, we systematically varied all three components of this lipid mixture and determined how efficiently the resulting particles fused with the plasma membrane of living mammalian cells. Our results show that an aromatic molecule and a cationic lipid component with conical molecular shape are essential for efficient fusion induction. While a neutral lipid is not mandatory, it can be used to control fusion efficiency and, in the most extreme case, to revert the uptake mechanism back to endocytosis.

Covalent dimerization of interleukin-like epithelial-to-mesenchymal transition (EMT) inducer (ILEI) facilitates EMT, invasion, and late aspects of metastasis.

The interleukin-like epithelial-to-mesenchymal transition (EMT) inducer (ILEI)/FAM3C is a member of the highly homologous FAM3 family and is essential for EMT and metastasis formation. It is upregulated in several cancers, and its altered subcellular localization strongly correlates with poor survival. However, the mechanism of ILEI action, including the structural requirements for ILEI activity, remains elusive. Here, we show that ILEI formed both monomers and covalent dimers in cancer cell lines and in tumors. Using mutational analysis and pulse-chase experiments, we found that the four ILEI cysteines, conserved throughout the FAM3 family and involved in disulfide bond formation were essential for extracellular ILEI accumulation in cultured cells. Modification of a fifth cysteine (C185), unique for ILEI, did not alter protein secretion, but completely inhibited ILEI dimerization. Wild-type ILEI monomers, but not C185A mutants, could be converted into covalent dimers extracellularly upon overexpression by intramolecular-to-intermolecular disulfide bond isomerization. Incubation of purified ILEI with cell culture medium showed that dimerization was triggered by bovine serum in a dose- and time-dependent manner. Purified ILEI dimers induced EMT and trans-well invasion of cancer cells in vitro. In contrast, ILEI monomers and the dimerization-defective C185A mutant affected only cell motility as detected by scratch assays and cell tracking via time-lapse microscopy. Importantly, tumor cells overexpressing wild-type ILEI caused large tumors and lung metastases in nude mice, while cells overexpressing the dimerization-defective C185A mutant behaved similar to control cells. These data show that covalent ILEI self-assembly is essential for EMT induction, elevated tumor growth, and metastasis.