Hedgehog (Hh) shape up development by playing important role in signaling, and thereby controlling growth and pattern formation. It is for this reason that their spatial distribution is tightly regulated. The 19kDa active form of Hh is modified with a palmitate at its N-terminal and with cholesterol at its C-terminal. This dually lipid modified form of Hh act as a morphogen, and is also referred to as HhNp (Mann and Beachy, 2004). In most cases, they are released from producing cells and spread into adjacent non-expressing cells within the tissue, where it activates target gene expression in a concentration-dependent manner. In Drosophila, Lipophorin (Lpp) particles carry these lipid-modified forms of Hh and play a role in long range signaling in the developing wing disc. Further, these particles circulate throughout the larvae in the hemolymph to distribute nutrients mostly in the form of lipids to different tissues of the animal. Thus, Lpp plays important role in metabolism and development. Hh as a morphogen plays a very important role in development and patterning of embryo and imaginal discs in Drosophila. We wanted to understand the role of Hh in overall development of Drosophila. In my thesis work, I discovered a new form of Hh that is systemically circulating in the 3rd instar larva of Drosophila. I show that imaginal tissues do not produce this form of circulating Hh. Our experiments strongly suggest that systemic Hh can travel from one tissue to another, a feature that was previously unknown. I also show that it could rescue the growth of the imaginal disc, implying its ability to influence cell proliferation. Since the concentration of systemic Hh is low it fails to up regulate the target genes. I characterized fat body as a target of systemically circulating Hh. I clearly demonstrate that fat body transcribes most of the components of Hh signaling pathway except Hh. Further, Hh accumulates in the fat body during late 3rd instar larvae. That makes the fat body an ideal target of systemic Hh. This could shed light in understanding the role of Hh in overall development of Drosophila melanogaster that includes tissue-based interaction.

Cytoskeletal motor proteins are essential to the function of a wide range of intra-cellular mechano-systems. The biophysical characterization of the movement of motor proteins along their filamentous tracks is therefore of large importance. Towards this end, in vitro stepping motility assays are commonly used to determine the motor’s velocities and runlengths. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental setups, the experimental conditions and the data analysis methods. This work describes a novel unified method to evaluate traces of fluorescently-labeled, processive dimeric motor proteins and proposes an algorithm to correct the measurements for finite filament length as well as photobleaching. Statistical errors of the proposed evaluation method are estimated by a bootstrap method. Numerical simulation and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules was used to verify the proposed approach and it was shown (i) that the velocity distribution should be fitted by a t location-scale probability density function rather than a normal distribution, (ii) that the temperature during the experiments should be controlled with a precision well below 1 K, (iii) that the impossibility to measure events shorter than the image acquisition time needs to be accounted for, (iv) that the motor’s runlength can be estimated independent of the filament length distribution, and (v) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. This allows for a better statistical comparison of motor proteins influenced by other external factors e.g. ionic strength, ATP concentration, or post-translational modifications of the filaments. In this context, the described method was then applied to experimental data to investigate the influence of the nucleotide state of the microtubule on the motility behavior of the kinesin-1 motor proteins. Here, a small but significant difference in the velocity measurements was found, but no significant difference in the runlength and interaction time measurements. Consequently, this work provides a framework for the evaluation of a wide range of experiments with single fluorescently-labeled motor proteins.

Apicobasal polarity is essential for epithelia formation and maintenance. Cell junctions, namely the zonula adherens in Drosophila melanogaster, are the morphological landmarks that define and distinguish the apical from the basal surface. This resulting compartmentalisation is key for the cell and consequently the epithelia. To maintain proper junctions, cells make use of several protein complexes and their interactions. Among these complexes, the Crumbs (Crb) network stands out. Mutations in Crumbs (crb11A22) lead to zonula adherens collapse, consequent loss of apical surface and disaggregation of the epithelia. However, the mechanisms behind this are not known and havenʼt been addressed using modern techniques such as live imaging. Several things came out of the dataset obtained from the FRAP experiments. Firstly, protein kinetics are better described when a double exponential fit curve is used, which raises the possibility that two cell processes might be involved in the recovery observed for the different markers. Another finding was the fact that the kinetics of some polarised protein markers is not the same in every region of the embryo. Distinct areas of the embryo with different morphogenetic activity levels show different kinetics for the same compartment marker. That was the case with SpiderGFP (whole plasma membrane marker) and SASVenus (apical plasma membrane marker) where τ2 was lower in the posterior region of the embryo which is characterised by intense cell movements resulting from convergence extension. DE-CadGFP (zonula adherens marker) and lacGFP (basolateral marker) behaved similarly in the whole embryo. This indicates that convergence extension shows different trafficking needs for the apical surface. In crb11A22, SpiderGFP kinetic spatial differences were not observed. τ2 in the anterior (low level of morphogenesis) is affected and similar to wild type τ2 levels in the posterior. This could pinpoint the fact that the epithelia disaggregation is a result of trafficking failure of apical components. Live imaging of DE-CadGFP in crb11A22 background revealed initial disaggregation in the anterior part of the embryo, which strengthens the idea that Crb is required for adherens junction stabilisation and maintenance.

Wnt proteins have key roles in patterning of multicellular animals, acting at a distance from their sites of production. However, it is not well understood how these molecules propagate. This question has become even more puzzling by the discovery that Wnts harbour post-translational lipid-modifications, which enhance association with membranes and may therefore limit propagation by simple diffusion in an aqueous environment. The cellular mechanisms involved in Wnt propagation are largely unknown for vertebrate organisms. Here, I discuss my findings on the cellular localization of zebrafish Wnt8, as an example of a vertebrate Wnt. Wnt8 is a key signal for positioning the midbrain-hindbrain brain boundary (MHB) organizer along the anterior-posterior axis of the developing brain in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells, in the prospective neural plate. For this purpose, I have analysed a biologically active, fluorescently tagged Wnt8 in live zebrafish embryos. Wnt8 was present in live tissue in membrane associated punctate structures. In Wnt8 expressing cells these puncta localise to filopodial cellular processes, from which the protein is released to neighbouring cells. This filopodial release requires posttranslational palmitoylation. Although palmitoylation-defective Wnt8 retains auto- and juxtacrine signaling activity, it fails to signal over a long-range. Additionally, this Wnt8 palmitoylation is necessary for regulation of its neural plate target genes. These results suggest that vertebrate Wnt proteins use cell-to-cell contact through filopodia as a shortrange propagation mechanism while released palmitoylated Wnt is required for longrange signaling activity. Furthermore, I show that a Wnt8 receptor, Frizzled9 can negatively influence Wnt8 propagation and signaling range. Finally, I was able to determine the presence of an endogenous Wnt8 gradient in the neurectoderm. I discuss these findings in the context of Wnt8 signaling function in mediating anterior-posterior patterning during early brain development.

Polyelectrolyte complexation is a rapidly growing field with applications in functional multilayer (PEM) and nanoparticle (PEC) generation, where PEM films are deposited using Layer-by-Layer technique initiated by Decher and PECs are prepared using mixing-centrifugation technique initiated by our group. Its advantages (e.g. easy preparation) result in various applications in aqueous solution, especially in pharmaceutical and biomedical fields. Therefore, the objectives in this study are to explore interesting applications of polyelectrolyte complexation in the field of low molecular chiral compound and high molecular protein binding. Due to the rapidly growing demands for preparing optically pure compounds in the pharmaceutical field, herein, enantiospecific PEM and PEC were prepared using chiral polyelectrolytes (e.g. homo-polypeptide) and their ability of chiral recognition was investigated by ATR-FTIR, UV/Vis etc.. Chiral PEM and PEC showed pronounced enantiospecificity for both small (amino acids, vitamin) and large (protein) chiral compounds. This chiral recognition is performed by a diffusion process of chiral compounds into PEM based on the structures of chiral selector (PEM, PEC) and chiral probes (chiral compounds). However, the influences, e.g. pH value, ionic strength, surface orientation etc., were found to affect significantly the enantiospecificity. Beside planar substrates, porous membranes (e.g. PTFE) were modified using chiral PEM and successfully applied in enantiospecific permeation. Additionally, protein binding properties of PEC particle dispersions or PEC particle films were also studied. Due to the properties of polyelectrolytes used for PEC (e.g. molecular weight, charge density) and proteins (e.g. isoelectric point, size, hydrophobicity), PEC showed different uptake characteristics towards different proteins. Electrostatic and hydrophobic interaction as well as counterion release force were considered as possible driving forces for protein binding.

Conventional kinesin (kinesin-1) transports membrane-bounded cargos such as mitochondria and vesicles along microtubules. In vivo it is likely that several kinesins move a single organelle and it is important that they operate in a coordinated fashion so that they do not interfere with each other. Evidence for coordination comes from in vitro assays, which show that the gliding speed of a microtubule driven by many kinesins is as high as one driven by just a single kinesin molecule. Coordination is thought to be facilitated by flexible domains so that when one motor is bound another can work irrespectively of their orientations. The tail of kinesin-1 is predicted to be composed of a coiled-coil with two main breaks, the “swivel” (380-442 Dm numbering) and the hinge (560-624). The rotational Brownian motion of microtubules attached to a glass surface by single kinesin molecules was analyzed and measured the torsion elasticity constant. The deletion of the hinge and subsequent tail domains increase the stiffness of the motor (8±1 kBT/rad) compared to the full length (0.06±0.01 kBT/rad measured previously), but does not impair motor cooperation (700±16nm/s vs. full length 756±55nm/s - speed in high motor density motility assays). Removal of the swivel domain generates a stiff construct (7±1 kBT/rad), which is fully functional at single molecule (657±63nm/s), but it cannot work in large numbers (151±46nm/s). Due to the similar value of flexibility for both short construct (8±11 kBT/rad vs 7±1 1 kBT/rad) and their different behavior at high density (700±16 nm/s vs. 151±46 nm/s) a new hypothesis is presented, the swivel might have a strain dependent conformation. Using Circular Dichroism and Fluorescence the secondary structure of this tail region was studied. The central part of the swivel is dimeric α-helical and it is surrounded by random coils, thereby named helix-coil (HC) region. Furthermore, an experimental set-up is developed to exert a torque on individual kinesin molecules using hydrodynamic flow. The results obtained suggest for the first time the possibility that a structural element within the kinesin tail (HC region) has a force-dependent conformation and that this allows motor cooperation.

Expression of the Tudor domain containing protein 6 (TDRD6), which is restricted to the male germ line, starts at day 16 of spermatogenesis, i.e. in pachytene spermatocytes. TDRD6 is a 250 kDa protein, which we recently found to be cleaved at the C-terminal end during germ cell development, resulting in a 230 kDa product. Neither is the process of cleavage itself nor are the functions of the two different forms known. The 230 kDa isoform is the most prominent form in round spermatids, where it localizes to the chromatoid body (CB), i.e. a single filamentous, perinuclear granule. One characteristic component of the CB is the RNA helicase MVH. CBs contain components of the microRNA (miRNA) pathway, including Piwi-interacting RNAs (piRNAs), as well as MIWI, MIWI2, and MILI, the mouse homologs of the Piwi proteins, which bind piRNAs and also act in transposon regulation. We showed that TDRD6 interacts with MIWI and MILI in vitro, and a direct interaction with MVH was shown before. To reveal the function of TDRD6, we generated Tdrd6-/- mice, which lack the protein. These mice are generally healthy but the males are sterile, due to the absence of mature spermatozoa. The most striking intracellular phenotype of Tdrd6-/- mice is the highly aberrant architecture of chromatoid bodies in round spermatids. Tdrd6-/- CBs appear as diffuse, disrupted, and less condensed structures. Their interior is largely missing, and only a “ghost”-like structure remains, expected to be significantly impaired in function. Other CB components like MVH, MIWI and MILI are expressed in Tdrd6-/- testes, but they cannot localize to the disrupted CBs. This suggests a role for TDRD6 in assembling the chromatoid body complex by recruiting other proteins. The CB is important for storage and translational regulation of mRNA, through interaction with miRNAs. In Tdrd6-deficient testes 10 % of all known murine miRNAs are differently expressed, whereas most of the mature miRNAs are up-regulated, indicating less turnover, and thus, accumulation of mature miRNAs. Since some precursor miRNAs are up-regulated as well, we assume, that TDRD6 affects miRNA transcription most likely by indirectly influencing transcriptional regulation of miRNA genes. In Tdrd6-/- mice an overall abnormal mRNA gene expression pattern was observed by microarray analyses. Of all mis-regulated genes 36 % are located to the centromer-proximal region of Chr 8, and 11 % are located to the distal end of Chr 1. This mis-regulation might be due to a common transcriptional regulation. The orthologous regions on the human chromosomes show altered chromosomal structures in many different carcinomas. If TDRD6 plays a role in carcinogenesis has to be investigated.

Understanding the molecular basis of protein function is a challenging task that lays the foundation for the pharmacological intervention in many diseases originating in altered structural states of the involved proteins. Dissecting a complex functional machinery into modules is a promising approach to protein function. The motivation for this work was to identify minimal requirements for “local” switching processes in the function of multidomain proteins that can adopt a variety of structural substates of different biological activity or representing intermediates of a complex reaction path. For example, modular switches are involved in signal transduction, where receptors respond to ligand-activation by specific conformational changes that are allosterically transmitted to “effector recognition sites” distant from the actual ligand-binding site. Heptahelical receptors have attracted particular attention due to their ubiquitous role in a large variety of pharmacologically relevant processes. Although constituting switches in their own right, it has become clear through mutagenesis and functional studies that receptors exhibit substates of partial active/inactive structure that can explain biological phenotypes of different levels of activity. Here, the notion that microdomains undergo individual switching processes that are integrated in the overall response of structurally regulated proteins is addressed by studies on the molecular basis of proton-dependent (chemical) and force-dependent (mechanical) conformational transitions. A combination of peptide synthesis, biochemical analysis, and secondary structure sensitive spectroscopy (Infrared, Circular dichroism, Fluorescence) was used to prove the switching capability of putative functional modules derived from three selected proteins, in which conformational transitions determine their function in transmembrane signaling (rhodopsin), transmembrane transport (bacteriorhodopsin) and chemical force generation (kinesin-1). The data are then related to the phenotypes of the corresponding full length-systems. In the first two systems the chemical potential of protons is crucial in linking proton exchange reactions to transmembrane protein conformation. This work addresses the hypothesized involvement of lipid protein interactions in this linkage (1). It is shown here that the lipidic phase is a key player in coupling proton uptake at a highly conserved carboxylic acid (DRY motif located at the C-terminus of helix 3) to conformation during activation of class-1 G protein coupled receptors (GPCRs) independently from ligand protein interactions and interhelical contacts. The data rationalize how evolutionary diversity underlying ligand-specifity can be reconciled with the conservation of a cytosolic ‘proton switch’, that is adapted to the general physical constraints of a lipidic bilayer described here for the prototypical class-1 GPCR rhodopsin (2). Whereas the exact sequence of modular switching events is of minor importance for rhodopsin as long as the final overall active conformation is reached, the related heptahelical light-transducing proton pump bacteriorhodopsin (bR), requires the precise relative timing in coupling protonation events to conformationtional switching at the cytosolic, transmembrane, and extracellular domains to guarantee vectorial proton transport. This study has focused on the cytosolic proton uptake site of this retinal protein whose proton exchange reactions at the cytosolic halfchannel resemble that of rhodopsin. It was a prime task in this work to monitor in real time the allosteric coupling between different protein regions. A novel powerful method based on the correlation of simultaneously recorded infrared absorption and fluorescence emission changes during bR function was established here (3), to study the switching kinetics in the cytosolic proton uptake domain relative to internal proton transfer reactions at the retinal and its counter ion. Using an uptake-impaired bR mutant the data proves the modular nature of domain couplings and shows that the energy barrier of the conformational transition in the cytosolic half but not its detailed structure is under the control of proton transfer reactions at the retinal Schiff base and its counter ion Asp85 (4). Despite the different functions of the two studied retinal proteins, the protonation is coupled to local switching mechanisms studied here at two levels of complexity, [a] a single carboxylic acid side chain acting as a lipid-dependent proton switch [b] a full-length system, where concerted modular regions orchestrate the functional coupling of proton translocation reactions. Switching on the level of an individual amino acid is shown to rely on localizable chemical properties (charge state, hydrophobicity, rotamer state). In contrast, switching processes involving longer stretches of amino acids are less understood, less generalizable, and can constitute switches of mechanical, rather than chemical nature. This applies particularly to molecular motors, where local structural switching processes are directly involved in force generation. A controversy exists with respect to the structural requirements for the cooperation of many molecular motors attached to a single cargo. The mechanical properties of the Hinge 1 domain of kinesin-1 linking the “neck” and motor domain to the “tail” were addressed here to complement single molecule data on torsional flexibility with secondary structure analysis and thermal stability of peptides derived from Hinge 1 (5). It is shown that the Hinge 1 exhibits an unexpected helix-forming propensity that resists thermal forces but unfolds under load. The data resolve the paradox that the hinge is required for motor cooperation, whereas it is dispensable for single motor processivity, clearly emphasizing the modular function of the holoprotein. However, the secondary-structural data reveal the functional importance of providing high compliance by force-dependent unfolding, i.e. in a fundamentally different way than disordered domains that are flexible but yet do not support cooperativity.

For the development of smart surfaces high attention is focused on stimuli-responsive polymers. Since type and rate of response to environmental stimuli can be regulated by chain length, composition, architecture and topology, polymer films offer a variety of opportunities to develop such stimuli-responsive surfaces. Here polymer brush surfaces designed for a controlled adsorption of proteins and a switchable activity of immobilized enzymes are presented. The work is focused on temperature as well as pH-sensitive binary brushes, consisting of poly(N-isopropylacrylamide) (PNIPAAm) and poly(acrylic acid) (PAA), and their swelling behavior as well as their protein adsorption affinity is compared to the corresponding homopolymer brushes. All polymer brushes are covalently grafted by ester bonds to an anchoring layer of poly(glycidyl methacrylate), that itself is grafted via ether bonds to a silicon surface. Methodical investigations of layer thickness and refractive index of the brushes in the swollen state and after protein adsorption are carried out with in-situ spectroscopic ellipsometry, varying the brush composition and the solution parameters pH, salt concentration and temperature. The ellipsometric findings are correlated to results of contact angle, atomic force microscopy and zeta-potential measurements as well as colorimetric assays of enzyme activities at the brush surface. Furthermore the swelling of PNIPAAm brushes and protein adsorption at PAA Guiselin brushes are investigated in more detail with attenuated total reflexion Fourier-transform infrared spectroscopy and quartz crystal microbalance with dissipation, respectively.

Cox11 (cytochrome c oxidase 11) is an ancient and conserved protein family present in most respiring organisms. Studies of several family members, mainly in yeast and bacteria, have revealed that these proteins are in charge of Cu+ delivery to the respiratory complex IV (COX). Absence of Cox11 leads to a non-functional COX complex and a complete respiratory deficiency. Although it is assumed that homologues in other species perform the same function, experimental data supporting this notion are lacking. The aim of this work was to characterize the putative Arabidopsis homologue AtCox11 (encoded by locus At1g02410) and to determine its functions. Comparison of AtCox11 with the well-studied ScCox11 in yeast revealed that the two proteins share high similarity in their sequences (32% amino acid identity) and in the predicted secondary structures. Surprisingly, despite this high similarity AtCox11 proved not to be able to functionally replace the yeast protein in ΔSccox11 yeast deletion strains. As presumed, AtCox11 is localized to mitochondria, probably tethered to the inner mitochondrial membrane with its C-terminus facing the intermembrane space. The subsequent experimental work addressed the functions of AtCox11. To this end AtCOX11 knock-down (KD) and overexpression lines (OE) were generated and their impact on plant phenotype was investigated. KD lines that were obtained by artificial micro RNA technology, possess approximately 30% of the WT AtCOX11 mRNA levels. Overexpression resulting in 4-6 fold higher AtCOX11 mRNA levels, was achieved by placing AtCOX11 under the control of the 35S promoter. Remarkably, both KD and OE plants had reduced levels of COX complex activity (~45% and ~80%, respectively) indicating that AtCox11 is, as expected, involved in COX complex assembly. The KD and OE plants exhibited reduced root lengths and pollen germination rates (compared to WT). As both processes are dependent on respiratory energy, these phenotypic changes seemingly result from the reduced COX activity. Interestingly, the short-root phenotype in OE plants was rescued by a surplus of copper in the media, whereas copper deficiency intensified the phenotype. By contrast, KD plants did not respond to changes of the copper concentration. This difference in the copper response between KD and OE plants hints at a different cause for the reduced COX activity. It is proposed that the concentration of AtCox11 in KD plants limits the efficient insertion of Cu+ into COX, independent of the available copper concentration. In OE plants, binding of the limited copper by the high AtCox11 level may lead to a copper deficiency for the copper chaperone AtHcc1 that is required to load copper to subunit AtCoxII. Indeed, addition of copper to the media was able to rescue the phenotype. In line with these data, the analysis of the expression pattern of AtCOX11 revealed that it is expressed in tissues which require substantial mitochondrial and COX biogenesis to sustain their high metabolic and/or cell division rates. Furthermore AtCOX11 was shown to be up-regulated as part of the plant’s response to increased oxidative stress induced by the addition to the plant media of peroxides or inhibitors of respiratory complexes. The up-regulation of AtCOX11 in response to oxidative stress was corroborated with publicly available RNA microarray data and analysis of the AtCOX11 promoter, which revealed the presence of a number of potential oxidative stress responsive elements. Taken together, the experimental results presented in this thesis support the conclusion that AtCox11 is a member of the conserved Cox11 protein family. Most probably, this mitochondrial protein participates in the assembly of the COX complex by inserting Cu+ into the CuB center of the AtCoxI subunit. In addition to this expected role, the data indicate that AtCox11 might participate in cellular oxidative stress response and defense via a yet unknown mechanism.