Jenny Eichhorst

Apical membrane maturation and cellular rosette formation during morphogenesis of the zebrafish lateral line

Tissue morphogenesis and cell sorting are major forces during organ development. Here, we characterize the process of tissue morphogenesis within the zebrafish lateral line primordium, a migratory sheet of cells that gives rise to the neuromasts of the posterior lateral line organ. We find that cells within this epithelial tissue constrict actin-rich membranes and enrich apical junction proteins at apical focal points. The coordinated apical membrane constriction in single Delta D-positive hair cell progenitors and in their neighbouring prospective support cells generates cellular rosettes. Live imaging reveals that cellular rosettes subsequently separate from each other and give rise to individual neuromasts. Genetic analysis uncovers an involvement of Lethal giant larvae proteins in the maturation of apical junction belts during cellular rosette formation. Our findings suggest that apical constriction of cell membranes spatially confines regions of strong cell-cell adhesion and restricts the number of tightly interconnected cells into cellular rosettes, which ensures the correct deposition of neuromasts during morphogenesis of the posterior lateral line organ.

Photocontrol of Contracting Muscle Fibers

The light-controlled inhibition of physiologically relevant protein–protein interactions by appropriate photoresponsive ligands in living cells or small organs (skeletal muscle fibers, vessels) could make it possible to investigate signaling pathways under high spatiotemporal control. Recently, we have reported a cyclic peptide that mimics the b-finger motif in neural NO synthase (nNOS) which is crucial for binding of nNOS to a-1-syntrophin. When a photoswitchable unit is embedded into this peptide, the binding can be controlled in vitro simply by light. In skeletal muscle the extended PDZ domain of nNOS interacts with the PDZ domain of a-1syntrophin to recruit nNOS to the dystrophin-associated protein complex in the plasma membrane, thus coupling the production of the second messenger nitric oxide (NO) to muscle contraction. Loss of sarcolemmal nNOS is known to result in functional ischemia during muscle contraction, which is commonly observed in muscle diseases as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Herein we show that the photoswitchable peptide ligand 1 (Figure 1) is able to translocate into cells, is sufficiently stable towards intracellular conditions, and can be used in vivo to photocontrol contracting muscle fibers. The recently described nNOS-derived, photoswitchable peptide ligand 1 of a-1-syntrophin contains the azobenzenew-amino acid 3-((4’-aminomethyl)phenylazo)benzoic acid (3,4’-AMPB), which in its trans form led to a ligand that showed no affinity to the PDZ domain of a-1-syntrophin while photoisomerization to the cis form resulted in a remarkable affinity of the peptide (KD = 10.6 mm). [1a] The finding has addressed the question of the applicability of the light-controlled ligand under physiological conditions to investigate the native interference in living skeletal muscles. Light-directed binding of the cis form of the photoswitchable ligand 1 to syntrophin was expected to inhibit the native syntrophin–nNOS interaction in the skeletal muscle followed by the dislocation of nNOS from the sarcolemma which may result in reduced NO release from skeletal muscle cells and thus in light-controlled muscle contraction. Azobenzene units have been used extensively for the photomodulation of biomolecules (peptides, proteins, and nucleic acids) and biological processes in vitro and in vivo (as in ion channels). The feasibility of using azobenzene systems in living organisms to photocontrol biological events has been confirmed by the in vivo imaging of the isomerization process in zebrafish. An intrinsic hindrance for applications of azobenzene in living cells derives from its susceptibility to reduction. The azo unit may be subject to reduction by enzymes or thiols such as glutathione (GSH) which is present in most cells at millimolar concentration (0.5–10 mm). The reduction rate of the cis isomer of a parasubstituted AMPB amino acid in a model tripeptide is about 100-fold higher than for the corresponding trans isomer. To determine the stability of the AMPB switch unit in the peptide ligand 1 we incubated the cis form of the photoswitchable ligand at the photostationary state (pss) in buffer solution (pH 7.5) containing reduced glutathione (10 mm). After 1 h exposure to GSH no reduced material was detectable by LC–MS analysis (Figure 2, dashed line). Even after 16 h the reduced material amounted to only 5% (Figure 2, dotted line, signal marked with an arrow; Figure S3 in the Supporting Information). In addition, when changes in the UV/Vis spectra of the photoswitchable ligand were followed during irradiation in buffer solution (pH 7.5) containing GSH (10 mm), isosbestic points were retained, indicating the stability of the 3,4’-AMPB unit in the peptide ligand 1 (Figure S1 in the Supporting Information). As expected, the thermal cis!trans isomerization of the photoswitchable ligand 1 in the presence of 10 mm glutathione was Figure 1. Structure of the cis form of the photoswitchable peptide ligand related to the b-finger peptide of nNOS.

Dimerization of Corticotropin-Releasing Factor Receptor Type 1 Is Not Coupled to Ligand Binding

As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF1R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF1R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF1R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)

GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix α7 packing against switch II. In the absence of α7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of α7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes.

Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein.

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways.

Saponins modulate the intracellular trafficking of protein toxins

Type I ribosome inactivating proteins such as saporin from the plant Saponaria officinalis L. are widely used as toxin moieties of targeted anti-tumor toxins. For exerting cytotoxicity the toxin moieties have to be released into the cytosol of tumor cells. However the cytosolic transfer of toxin molecules into the cytosol is mostly an inefficient process. In this report we demonstrate that certain saponins, which are also biosynthesized by Saponaria officinalis L., specifically mediate the release of saporin out of the intracellular compartments into the cytosol without affecting the integrity of the plasma membrane. The relevant cellular compartments were identified as late endosomes and lysosomes. Further studies revealed that endosomal acidification is a prerequisite for the saponin-mediated release of saporin. Binding analysis demonstrated an association of the saponins with saporin in a pH-dependent manner. The applicability of the saponin-mediated effect was demonstrated in vivo in a syngeneic tumor model using a saporin-based targeted anti-tumor toxin in combination with characterized saponins.

N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

Objective—The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ETB receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs). Methods and Results—VSMCs expressing either the full-length ETB receptor or an N-terminally truncated ETB receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ETB receptor, IRL1620 (an ETB-selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22α, and α-actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of Gi proteins. In contrast, in VSMCs expressing N-terminally truncated ETB receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression. Conclusions—This study is the first to show that stimulation of full-length ETB receptors promotes expression of contractile proteins and may thus participate in the differentiation of VSMCs.

Xirp proteins mark injured skeletal muscle in zebrafish.

Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does not involve cell proliferation or Pax7+ cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now become accessible to detailed molecular and cellular characterizations.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Modified trastuzumab and cetuximab mediate efficient toxin delivery while retaining antibody-dependent cell-mediated cytotoxicity in target cells.

Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.