Segmentation and Multi-Timepoint Tracking of 3D Cancer Organoids from Optical Coherence Tomography Images Using Deep Neural Networks.

Recent years have ushered in a transformative era in in vitro modeling with the advent of organoids, three-dimensional structures derived from stem cells or patient tumor cells. Still, fully harnessing the potential of organoids requires advanced imaging technologies and analytical tools to quantitatively monitor organoid growth. Optical coherence tomography (OCT) is a promising imaging modality for organoid analysis due to its high-resolution, label-free, non-destructive, and real-time 3D imaging capabilities, but accurately identifying and quantifying organoids in OCT images remain challenging due to various factors. Here, we propose an automatic deep learning-based pipeline with convolutional neural networks that synergistically includes optimized preprocessing steps, the implementation of a state-of-the-art deep learning model, and ad-hoc postprocessing methods, showcasing good generalizability and tracking capabilities over an extended period of 13 days. The proposed tracking algorithm thoroughly documents organoid evolution, utilizing reference volumes, a dual branch analysis, key attribute evaluation, and probability scoring for match identification. The proposed comprehensive approach enables the accurate tracking of organoid growth and morphological changes over time, advancing organoid analysis and serving as a solid foundation for future studies for drug screening and tumor drug sensitivity detection based on organoids.

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.

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.

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.

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%.

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.

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.

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.

The interleukin-like epithelial-mesenchymal transition inducer ILEI exhibits a non-interleukin-like fold and is active as a domain-swapped dimer.

Production and secretion of pro-metastatic proteins is a feature of many tumor cells. The FAM3C interleukin-like epithelial-to-mesenchymal-transition (EMT) inducer (ILEI) has been shown to be strongly up-regulated in several cancers and to be essential for tumor formation and metastasis in epithelial cells, correlating with a significant decrease in overall survival in colon and breast cancer patients. ILEI has been seen to interact with the γ-secretase presenilin 1 subunit (PS1). However, not much is known about the mechanism-of-action or the detailed ILEI structure. We present here the crystal structures of FAM3C ILEI and show that it exists as monomers but also as covalent dimers. The observed ILEI ß-ß-α fold confirmed previous indications that the FAM3C proteins do not form classical four-helix-bundle structures as was initially predicted. This provides the first experimental evidence that the interleukin-like EMT inducers are not evolutionarily related to the interleukins. However, more surprisingly, the ILEI dimer structure was found to feature a trans-linked domain swap, converting an intramolecular disulfide to intermolecular. Interestingly, dimeric but not monomeric ILEI was subsequently found to cause a dose-dependent increase in EpRas cell invasiveness comparable with TGF-ß, indicating that the dimer might be the active ILEI species. This is in line with a parallel study showing that covalent oligomerization of ILEI is essential for EMT and tumor progression in vivo The structures and the activity data give some first insight into the relationship between dimerization and ILEI function as well as indicate an intriguing link between ILEI, the PS1-protease, TGF-ß, and the TGF-ß receptor 1.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins.

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.

Resveratrol encapsulated in novel fusogenic liposomes activates Nrf2 and attenuates oxidative stress in cerebromicrovascular endothelial cells from aged rats.

Resveratrol (3,4',5-trihydroxystilbene) is a plant-derived polyphenolic trans-stilbenoid, which exerts multifaceted antiaging effects. Here, we propose a novel delivery system for resveratrol, which significantly increases its cellular uptake into aged cells. Combination of resveratrol with a positively charged lipid component to "conventional" liposomes converts these lipid vesicles to a robust fusogenic system. To study their cellular uptake and cellular effects, we treated primary cerebromicrovascular endothelial cells isolated from aged F344xBN rats with resveratrol encapsulated in fusogenic liposomes (FL-RSV). To demonstrate effective cellular uptake of FL-RSV, accumulation of the lipophilic tracer dye, DiR, and resveratrol in cerebromicrovascular endothelial cells was confirmed using flow cytometry and confocal microscopy and high-performance liquid chromatography electrochemical detection. Treatment of aged cerebromicrovascular endothelial cells with FL-RSV activated Nrf2 (assessed with a reporter gene assay), significantly decreased cellular production of reactive oxygen species (assessed by a flow cytometry-based H2DCFDA fluorescence method), and inhibited apoptosis. Taken together, encapsulation of resveratrol into novel fusogenic liposomes significantly enhances the delivery of resveratrol into aged cells, which subsequently results in rapid activation of cellular Nrf2-driven antioxidant defense mechanisms. Our studies provide proof-of-concept for the development of a novel, translationally relevant interventional strategy for prevention and/or control of oxidative stress-related pathophysiological conditions in aging.

Interleukin-like epithelial-to-mesenchymal transition inducer activity is controlled by proteolytic processing and plasminogen-urokinase plasminogen activator receptor system-regulated secretion during breast cancer progression.

INTRODUCTION: Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) is an essential cytokine in tumor progression that is upregulated in several cancers, and its altered subcellular localization is a predictor of poor survival in human breast cancer. However, the regulation of ILEI activity and the molecular meaning of its altered localization remain elusive. METHODS: The influence of serum withdrawal, broad-specificity protease inhibitors, different serine proteases and plasminogen depletion on the size and amount of the secreted ILEI protein was investigated by Western blot analysis of EpRas cells. Proteases with ILEI-processing capacity were identified by carrying out an in vitro cleavage assay. Murine mammary tumor and metastasis models of EpC40 and 4T1 cells overexpressing different mutant forms of ILEI were used-extended with in vivo aprotinin treatment for the inhibition of ILEI-processing proteases-to test the in vivo relevance of proteolytic cleavage. Stable knockdown of urokinase plasminogen activator receptor (uPAR) in EpRas cells was performed to investigate the involvement of uPAR in ILEI secretion. The subcellular localization of the ILEI protein in tumor cell lines was analyzed by immunofluorescence. Immunohistochemistry for ILEI localization and uPAR expression was performed on two human breast cancer arrays, and ILEI and uPAR scores were correlated with the metastasis-free survival of patients. RESULTS: We demonstrate that secreted ILEI requires site-specific proteolytic maturation into its short form for its tumor-promoting function, which is executed by serine proteases, most efficiently by plasmin. Noncleaved ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through a propeptide-dependent interaction. In addition to ILEI processing, plasmin rapidly increases ILEI secretion by mobilizing its intracellular protein pool in a uPAR-dependent manner. Elevated ILEI secretion correlates with an altered subcellular localization of the protein, most likely representing a shift into secretory vesicles. Moreover, altered subcellular ILEI localization strongly correlates with high tumor cell-associated uPAR protein expression, as well as with poor survival, in human breast cancer. CONCLUSIONS: Our findings point out extracellular serine proteases, in particular plasmin, and uPAR as valuable therapeutic targets against ILEI-driven tumor progression and emphasize the prognostic relevance of ILEI localization and a combined ILEI-uPAR marker analysis in human breast cancer.

CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance.

The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance.

Novel fusogenic liposomes for fluorescent cell labeling and membrane modification.

Efficient delivery of biomolecules into membranes of living cells as well as cell surface modifications are major biotechnological challenges. Here, novel liposome systems based on neutral and cationic lipids in combination with lipids modified by aromatic groups are introduced for such applications. The fusion efficiency of these liposome systems was tested on single cells in culture like HEK293, myofibroblasts, cortical neurons, human macrophages, smooth muscle cells, and even on tissue. Fusogenic liposomes enabled highly efficient incorporation of molecules into mammalian cell membranes within 1 to 30 min at fully unchanged cell growth conditions and did not affect cell behavior. We hypothesize that membrane fusions were induced in all cases by the interaction of the positively charged lipids and the delocalized electron system of the aromatic group generating local dipoles and membrane instabilities. Selected applications ranging from basic research to biotechnology are envisaged here.

A novel conserved phosphotyrosine motif in the Drosophila fibroblast growth factor signaling adaptor Dof with a redundant role in signal transmission.

The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. In this study, we identify a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. We show that these motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (i) a Csw binding site, (ii) four consensus Grb2 recognition sites, and (iii) four novel tyrosine motifs. We show that Src64B binds to Dof and that Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events.

The role of apoptosis in shaping the tracheal system in the Drosophila embryo.

The tubular network of the tracheal system in the Drosophila embryo is created from a set of epithelial placodes by cell migration, rearrangements, fusions and shape changes. A designated number of cells is initially allocated to each branch of the system. We show here that the final cell number in the dorsal branches is not only determined by early patterning events and subsequent cell rearrangements but also by elimination of cells from the developing branch. Extruded cells die and are engulfed by macrophages. Our results suggest that the pattern of cell extrusion and death is not hard-wired, but is determined by environmental cues.

Variation in sequence-specific repair of UV damage in human pericentromeric heterochromatin of different cell lines.

Damaged nucleotides are removed from the condensed non-coding, or transcriptionally inactive regions of the genome by the relatively slow global genome repair system. Since few data are available for the repair of the pericentric heterochromatin region our aim was to study the repair of a specific sequence, known to be located in this region. We applied a PCR based method to monitor UV damage and repair in chAB4, a human pericentromeric heterochromatin sequence in 10 human cell lines. We here present evidence that excision repair of a sequence in the pericentromeric heterochomatin also varies between cell lines in a manner inconsistent with the canonical model. In some cell lines repair rates were efficient in heterochromatin, comparable to transcription coupled repair, but in some tumour-derived and repair-deficient cell lines we have detected deficient repair.