@phdthesis{Liu2022, author = {Liu, Ruiqi}, title = {Dynamic regulation of the melanocortin 4 receptor system in body weight homeostasis and reproductive maturation in fish}, doi = {10.25972/OPUS-20653}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206536}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Puberty is an important period of life with physiological changes to enable animals to reproduce. Xiphophorus fish exhibit polymorphism in body size, puberty timing, and reproductive tactics. These phenotypical polymorphisms are controlled by the Puberty (P) locus. In X. nigrensis and X. multilineatus, the P locus encodes the melanocortin 4 receptor (Mc4r) with high genetic polymorphisms. Mc4r is a member of the melanocortin receptors, belonging to class A G-protein coupled receptors. The Mc4r signaling system consists of Mc4r, the agonist Pomc (precursor of various MSH and of ACTH), the antagonist Agrp and accessory protein Mrap2. In humans, MC4R has a role in energy homeostasis. MC4R and MRAP2 mutations are linked to human obesity but not to puberty. Mc4rs in X. nigrensis and X. multilineatus are present in three allele classes, A, B1 and B2, of which the X-linked A alleles express functional receptors and the male-specific Y-linked B alleles encode defective receptors. Male body sizes are correlated with B allele type and B allele copy numbers. Late-maturing large males carry B alleles in high copy number while early-maturing small males carry B alleles in low copy number or only A alleles. Cell culture co-expression experiments indicated that B alleles may act as dominant negative receptor mutants on A alleles. In this study, the main aim was to biochemically characterize the mechanism of puberty regulation by Mc4r in X. nigrensis and X. multilineatus, whether it is by Mc4r dimerization and/or Mrap2 interaction with Mc4r or other mechanisms. Furthermore, Mc4r in X. hellerii (another swordtail species) and medaka (a model organism phylogenetically close to Xiphophorus) were investigated to understand if the investigated mechanisms are conserved in other species. In medaka, the Mc4r signaling system genes (mc4r, mrap2, pomc, agrp1) are expressed before hatching, with agrp1 being highly upregulated during hatching and first feeding. These genes are mainly expressed in adult brain, and the transcripts of mrap2 co-localize with mc4r indicating a function in modulating Mc4r signaling. Functional comparison between wild-type and mc4r knockout medaka showed that Mc4r knockout does not affect puberty timing but significantly delays hatching due to the retarded embryonic development of knockout medaka. Hence, the Mc4r system in medaka is involved in regulation of growth rather than puberty. In Xiphophorus, expression co-localization of mc4r and mrap2 in X. nigrensis and X. hellerii fish adult brains was characterized by in situ hybridization. In both species, large males exhibit strikingly high expression of mc4r while mrap2 shows similar expression level in the large and small male and female. Differently, X. hellerii has only A-type alleles indicating that the puberty regulation mechanisms evolved independently in Xiphophorus genus. Functional analysis of Mrap2 and Mc4r A/B1/B2 alleles of X. multilineatus showed that increased Mrap2 amounts induce higher cAMP response but EC50 values do not change much upon Mrap2 co-expression with Mc4r (expressing only A allele or A and B1 alleles). A and B1 alleles were expressed higher in large male brains, while B2 alleles were only barely expressed. Mc4r A-B1 cells have lower cAMP production than Mc4r A cells. Together, this indicates a role of Mc4r alleles, but not Mrap2, in puberty onset regulation signaling. Interaction studies by FRET approach evidenced that Mc4r A and B alleles can form heterodimers and homodimers in vitro, but only for a certain fraction of the expressed receptors. Single-molecule colocalization study using super-resolution microscope dSTORM confirmed that only few Mc4r A and B1 receptors co-localized on the membrane. Altogether, the species-specific puberty onset regulation in X. nigrensis and X. multilineatus is linked to the presence of Mc4r B alleles and to some extent to its interaction with A allele gene products. This is reasoned to result in certain levels of cAMP signaling which reaches the dynamic or static threshold to permit late puberty in large males. In summary, puberty onset regulation by dominant negative effect of Mc4r mutant alleles is a special mechanism that is found so far only in X. nigrensis and X. multilineatus. Other Xiphophorus species obviously evolved the same function of the pathway by diverse mechanisms. Mc4r in other fish (medaka) has a role in regulation of growth, reminiscent of its role in energy homeostasis in humans. The results of this study will contribute to better understand the biochemical and physiological functions of the Mc4r system in vertebrates including human.}, subject = {Japank{\"a}rpfling}, language = {en} } @phdthesis{Pasch2016, author = {Pasch, Elisabeth}, title = {The role of SUN4 and related proteins in sperm head formation and fertility}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139092}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Spermiogenesis describes the differentiation of haploid germ cells into motile, fertilization-competent spermatozoa. During this fundamental transition the species-specific sperm head is formed, which necessitates profound nuclear restructuring coincident with the assembly of sperm-specific structures and chromatin compaction. In the case of the mouse, it is characterized by reshaping of the early round spermatid nucleus into an elongated sickle-shaped sperm head. This tremendous shape change requires the transduction of cytoskeletal forces onto the nuclear envelope (NE) or even further into the nuclear interior. LINC (linkers of nucleoskeleton and cytoskeleton) complexes might be involved in this process, due to their general function in bridging the NE and thereby physically connecting the nucleus to the peripheral cytoskeleton. LINC complexes consist of inner nuclear membrane integral SUN-domain proteins and outer nuclear membrane KASH-domain counterparts. SUN- and KASH-domain proteins are directly connected to each other within the perinuclear space, and are thus capable of transferring forces across the NE. To date, these protein complexes are known for their essential functions in nuclear migration, anchoring and positioning of the nucleus, and even for chromosome movements and the maintenance of cell polarity and nuclear shape. In this study LINC complexes were investigated with regard to their potential role in sperm head formation, in order to gain further insight into the processes occurring during spermiogenesis. To this end, the behavior and function of the testis-specific SUN4 protein was studied. The SUN-domain protein SUN4, which had received limited characterization prior to this work, was found to be exclusively expressed in haploid stages during germ cell development. In these cell stages, it specifically localized to the posterior NE at regions decorated by the manchette, a spermatid-specific structure which was previously shown to be involved in nuclear shaping. Mice deficient for SUN4 exhibited severely disorganized manchette residues and gravely misshapen sperm heads. These defects resulted in a globozoospermia-like phenotype and male mice infertility. Therefore, SUN4 was not only found to be mandatory for the correct assembly and anchorage of the manchette, but also for the correct localization of SUN3 and Nesprin1, as well as of other NE components. Interaction studies revealed that SUN4 had the potential to interact with SUN3, Nesprin1, and itself, and as such is likely to build functional LINC complexes that anchor the manchette and transfer cytoskeletal forces onto the nucleus. Taken together, the severe impact of SUN4 deficiency on the nucleocytoplasmic junction during sperm development provided direct evidence for a crucial role of SUN4 and other LINC complex components in mammalian sperm head formation and fertility.}, subject = {Maus}, language = {en} } @phdthesis{Scholl2015, author = {Scholl, Christina}, title = {Cellular and molecular mechanisms contributing to behavioral transitions and learning in the honeybee}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115527}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {The honeybee Apis mellifera is a social insect well known for its complex behavior and the ability to learn tasks associated with central place foraging, such as visual navigation or to learn and remember odor-reward associations. Although its brain is smaller than 1mm² with only 8.2 x 105 neurons compared to ~ 20 x 109 in humans, bees still show amazing social, cognitive and learning skills. They express an age - related division of labor with nurse bees staying inside the hive and performing tasks like caring for the brood or cleaning, and foragers who collect food and water outside the hive. This challenges foragers with new responsibilities like sophisticated navigation skills to find and remember food sources, drastic changes in the sensory environment and to communicate new information to other bees. Associated with this plasticity of the behavior, the brain and especially the mushroom bodies (MBs) - sensory integration and association centers involved in learning and memory formation - undergo massive structural and functional neuronal alterations. Related to this background my thesis on one hand focuses on neuronal plasticity and underlying molecular mechanisms in the MBs that accompany the nurse - forager transition. In the first part I investigated an endogenous and an internal factor that may contribute to the nurse - forager phenotype plasticity and the correlating changes in neuronal network in the MBs: sensory exposure (light) and juvenile hormone (JH). Young bees were precociously exposed to light and subsequently synaptic complexes (microglomeruli, MG) in the MBs or respectively hemolymph juvenile hormone (JH) levels were quantified. The results show that light input indeed triggered a significant decrease in MG density, and mass spectrometry JH detection revealed an increase in JH titer. Interestingly light stimulation in young bees (presumably nurse bees) triggered changes in MG density and JH levels comparable to natural foragers. This indicates that both sensory stimuli as well as the endocrine system may play a part in preparing bees for the behavioral transition to foraging. Considering a connection between the JH levels and synaptic remodeling I used gene knockdown to disturb JH pathways and artificially increase the JH level. Even though the knockdown was successful, the results show that MG densities remained unchanged, showing no direct effect of JH on synaptic restructuring. To find a potential mediator of structural synaptic plasticity I focused on the calcium-calmodulin-dependent protein kinase II (CaMKII) in the second part of my thesis. CaMKII is a protein known to be involved in neuronal and behavioral plasticity and also plays an important part in structural plasticity reorganizing synapses. Therefore it is an interesting candidate for molecular mechanisms underlying MG reorganization in the MBs in the honeybee. Corresponding to the high abundance of CaMKII in the learning center in vertebrates (hippocampus), CaMKII was shown to be enriched in the MBs of the honeybee. Here I first investigated the function of CaMKII in learning and memory formation as from vertebrate work CaMKII is known to be associated with the strengthening of synaptic connections inducing long term potentiation and memory formation. The experimental approach included manipulating CaMKII function using 2 different inhibitors and a specific siRNA to create a CaMKII knockdown phenotype. Afterwards bees were subjected to classical olfactory conditioning which is known to induce stable long-term memory. All bees showed normal learning curves and an intact memory acquisition, short-term and mid-term memory (1 hour retention). However, in all cases long-term memory formation was significantly disrupted (24 and 72 hour retention). These results suggests the necessity of functional CaMKII in the MBs for the induction of both early and late phases of long-term memory in honeybees. The neuronal and molecular bases underlying long-term memory and the resulting plasticity in behavior is key to understanding higher brain function and phenotype plasticity. In this context CaMKII may be an important mediator inducing structural synaptic and neuronal changes in the MB synaptic network.}, subject = {Biene}, language = {en} } @phdthesis{Vona2014, author = {Vona, Barbara C.}, title = {Molecular Characterization of Genes Involved in Hearing Loss}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112170}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The auditory system is an exquisitely complex sensory organ dependent upon the synchronization of numerous processes for proper function. The molecular characterization of hereditary hearing loss is complicated by extreme genetic heterogeneity, wherein hundreds of genes dispersed genome-wide play a central and irreplaceable role in normal hearing function. The present study explores this area on a genome-wide and single gene basis for the detection of genetic mutations playing critical roles in human hearing. This work initiated with a high resolution SNP array study involving 109 individuals. A 6.9 Mb heterozygous deletion on chromosome 4q35.1q35.2 was identified in a syndromic patient that was in agreement with a chromosome 4q deletion syndrome diagnosis. A 99.9 kb heterozygous deletion of exons 58-64 in USH2A was identified in one patient. Two homozygous deletions and five heterozygous deletions in STRC (DFNB16) were also detected. The homozygous deletions alone were enough to resolve the hearing impairment in the two patients. A Sanger sequencing assay was developed to exclude a pseudogene with a high percentage sequence identity to STRC from the analysis, which further solved three of the six heterozygous deletion patients with the hemizygous, in silico predicted pathogenic mutations c.2726A>T (p.H909L), c.4918C>T (p.L1640F), and c.4402C>T (p.R1468X). A single patient who was copy neutral for STRC and without pathogenic copy number variations had compound heterozygous mutations [c. 2303_2313+1del12 (p.G768Vfs*77) and c.5125A>G (p.T1709A)] in STRC. It has been shown that STRC has been previously underestimated as a hearing loss gene. One additional patient is described who does not have pathogenic copy number variation but is the only affected member of his family having hearing loss with a paternally segregating translocation t(10;15)(q26.13;q21.1). Twenty-four patients without chromosomal aberrations and the above described patient with an USH2A heterozygous deletion were subjected to a targeted hearing loss gene next generation sequencing panel consisting of either 80 or 129 hearing-relevant genes. The patient having the USH2A heterozygous deletion also disclosed a second mutation in this gene [c.2276G>T (p.C759F)]. This compound heterozygous mutation is the most likely cause of hearing loss in this patient. Nine mutations in genes conferring autosomal dominant hearing loss [ACTG1 (DFNA20/26); CCDC50 (DFNA44); EYA4 (DFNA10); GRHL2 (DFNA28); MYH14 (DFNA4A); MYO6 (DFNA22); TCF21 and twice in MYO1A (DFNA48)] and four genes causing autosomal recessive hearing loss were detected [GJB2 (DFNB1A); MYO7A (DFNB2); MYO15A (DFNB3), and USH2A]. Nine normal hearing controls were also included. Statistical significance was achieved comparing controls and patients that revealed an excess of mutations in the hearing loss patients compared to the control group. The family with the GRHL2 c.1258-1G>A mutation is only the second family published worldwide with a mutation described in this gene to date, supporting the initial claim of this gene causing DFNA28 hearing loss. Audiogram analysis of five affected family members uncovered the progressive nature of DFNA28 hearing impairment. Regression analysis predicted the annual threshold deterioration in each of the five family members with multiple audiograms available over a number of years.}, subject = {Molekularbiologie}, language = {en} } @phdthesis{Vona2014, author = {Vona, Barbara C.}, title = {Molecular Characterization of Genes Involved in Hearing Loss}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-98031}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {The auditory system is an exquisitely complex sensory organ dependent upon the synchronization of numerous processes for proper function. The molecular characterization of hereditary hearing loss is complicated by extreme genetic heterogeneity, wherein hundreds of genes dispersed genome-wide play a central and irreplaceable role in normal hearing function. The present study explores this area on a genome-wide and single gene basis for the detection of genetic mutations playing critical roles in human hearing. This work initiated with a high resolution SNP array study involving 109 individuals. A 6.9 Mb heterozygous deletion on chromosome 4q35.1q35.2 was identified in a syndromic patient that was in agreement with a chromosome 4q deletion syndrome diagnosis. A 99.9 kb heterozygous deletion of exons 58-64 in USH2A was identified in one patient. Two homozygous deletions and five heterozygous deletions in STRC (DFNB16) were also detected. The homozygous deletions alone were enough to resolve the hearing impairment in the two patients. A Sanger sequencing assay was developed to exclude a pseudogene with a high percentage sequence identity to STRC from the analysis, which further solved three of the six heterozygous deletion patients with the hemizygous, in silico predicted pathogenic mutations c.2726A>T (p.H909L), c.4918C>T (p.L1640F), and c.4402C>T (p.R1468X). A single patient who was copy neutral for STRC and without pathogenic copy number variations had compound heterozygous mutations [c. 2303_2313+1del12 (p.G768Vfs*77) and c.5125A>G (p.T1709A)] in STRC. It has been shown that STRC has been previously underestimated as a hearing loss gene. One additional patient is described who does not have pathogenic copy number variation but is the only affected member of his family having hearing loss with a paternally segregating translocation t(10;15)(q26.13;q21.1). Twenty-four patients without chromosomal aberrations and the above described patient with an USH2A heterozygous deletion were subjected to a targeted hearing loss gene next generation sequencing panel consisting of either 80 or 129 hearing-relevant genes. The patient having the USH2A heterozygous deletion also disclosed a second mutation in this gene [c.2276G>T (p.C759F)]. This compound heterozygous mutation is the most likely cause of hearing loss in this patient. Nine mutations in genes conferring autosomal dominant hearing loss [ACTG1 (DFNA20/26); CCDC50 (DFNA44); EYA4 (DFNA10); GRHL2 (DFNA28); MYH14 (DFNA4A); MYO6 (DFNA22); TCF21 and twice in MYO1A (DFNA48)] and four genes causing autosomal recessive hearing loss were detected [GJB2 (DFNB1A); MYO7A (DFNB2); MYO15A (DFNB3), and USH2A]. Nine normal hearing controls were also included. Statistical significance was achieved comparing controls and patients that revealed an excess of mutations in the hearing loss patients compared to the control group. The family with the GRHL2 c.1258-1G>A mutation is only the second family published worldwide with a mutation described in this gene to date, supporting the initial claim of this gene causing DFNA28 hearing loss. Audiogram analysis of five affected family members uncovered the progressive nature of DFNA28 hearing impairment. Regression analysis predicted the annual threshold deterioration in each of the five family members with multiple audiograms available over a number of years.}, subject = {Molekularbiologie}, language = {en} } @phdthesis{Ott2013, author = {Ott, Christine Kornelia}, title = {Diverse Aspects of the Sorting and Assembly Machinery in Human Mitochondria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85462}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Mitochondria are organelles of endosymbiotic origin, which play many important roles in eukaryotic cells. Mitochondria are surrounded by two membranes and, considering that most of the mitochondrial proteins are produced in the cytosol, possess import machineries, which transport mitochondria-targeted proteins to their designated location. A special class of outer mitochondrial membrane (OMM) proteins, the β-barrel proteins, require the sorting and assembly machinery (SAM) for their OMM integration. Both mitochondrial β-barrel proteins and the central component of the SAM complex, Sam50, have homologs in gram-negative bacteria. In yeast mitochondria, bacterial β-barrel proteins can be imported and assembled into the OMM. Our group demonstrated that this, however, is not the case for human mitochondria, which import only neisserial β barrel proteins, but not those of Escherichia coli and Salmonella enterica. As a part of this study, I could demonstrate that β-barrel proteins such as Omp85 and PorB of different Neisseria species are targeted to human mitochondria. Interestingly, only proteins belonging to the neisserial Omp85 family were integrated into the OMM, whereas PorB was imported into mitochondria but not assembled. By exchanging parts of homologous neisserial Omp85 and E. coli BamA and, similarly, of neisserial PorB and E. coli OmpC, it could be demonstrated in this work that the mitochondrial import signal of bacterial β barrel proteins cannot be limited to one short linear sequence, but rather secondary structure and protein charge seem to play an important role, as well as specific residues in the last β-strand of Omp85. Omp85 possesses five conserved POTRA domains in its amino-terminal part. This work additionally demonstrated that in human mitochondria, at least two POTRA domains of Omp85 are necessary for membrane integration and functionality of Omp85. In the second part of this work, the influence of Sam50 on the mitochondrial cristae structure was investigated. This work contributed to a study performed by our group in which it was confirmed that Sam50 is present in a high molecular weight complex together with mitofilin, CHCHD3, CHCHD6, DnaJC11, metaxin 1 and metaxin 2. This connection between the inner and outer mitochondrial membrane was shown to be crucial for the maintenance of the mitochondrial cristae structure. In addition, a role of Sam50 in respiratory complex assembly, suggested by a SILAC experiment conducted in our group, could be confirmed by in vitro import studies. An influence of Sam50 not only on respiratory complexes but also on the recently described respiratory complex assembly factor TTC19 was demonstrated. It was shown that TTC19 not only plays a role in complex III assembly as published, but also influences the assembly of respiratory complex IV. Thus, in this part of the work a connection between the OMM protein Sam50 and maintenance of cristae structure, respiratory complex assembly and an assembly factor could be established.}, subject = {Mitochondrien}, language = {en} } @phdthesis{Knapek2010, author = {Knapek, Stephan}, title = {Synapsin and Bruchpilot, two synaptic proteins underlying specific phases of olfactory aversive memory in Drosophila melanogaster}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-49726}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Memory is dynamic: shortly after acquisition it is susceptible to amnesic treatments, gets gradually consolidated, and becomes resistant to retrograde amnesia (McGaugh, 2000). Associative olfactory memory of the fruit fly Drosophila melanogaster also shows these features. After a single associative training where an odor is paired with electric shock (Quinn et al., 1974; Tully and Quinn, 1985), flies form an aversive odor memory that lasts for several hours, consisting of qualitatively different components. These components can be dissociated by mutations, their underlying neuronal circuitry and susceptibility to amnesic treatments (Dubnau and Tully, 1998; Isabel et al., 2004; Keene and Waddell, 2007; Masek and Heisenberg, 2008; Xia and Tully, 2007). A component that is susceptible to an amnesic treatment, i.e. anesthesia-sensitive memory (ASM), dominates early memory, but decays rapidly (Margulies et al., 2005; Quinn and Dudai, 1976). A consolidated anesthesia-resistant memory component (ARM) is built gradually within the following hours and lasts significantly longer (Margulies et al., 2005; Quinn and Dudai, 1976). I showed here that the establishment of ARM requires less intensity of shock reinforcement than ASM. ARM and ASM rely on different molecular and/or neuronal processes: ARM is selectively impaired in the radish mutant, whereas for example the amnesiac and rutabaga genes are specifically required for ASM (Dudai et al., 1988; Folkers et al., 1993; Isabel et al., 2004; Quinn and Dudai, 1976; Schwaerzel et al., 2007; Tully et al., 1994). The latter comprise the cAMP signaling pathway in the fly, with the PKA being its supposed major target (Levin et al., 1992). Here I showed that a synapsin null-mutant encoding the evolutionary conserved phosphoprotein Synapsin is selectively impaired in the labile ASM. Further experiments suggested Synapsin as a potential downstream effector of the cAMP/PKA cascade. Similar to my results, Synapsin plays a role for different learning tasks in vertebrates (Gitler et al., 2004; Silva et al., 1996). Also in Aplysia, PKA-dependent phosphorylation of Synapsin has been proposed to be involved in regulation of neurotransmitter release and short-term plasticity (Angers et al., 2002; Fiumara et al., 2004). Synapsin is associated with a reserve pool of vesicles at the presynapse and is required to maintain vesicle release specifically under sustained high frequency nerve stimulation (Akbergenova and Bykhovskaia, 2007; Li et al., 1995; Pieribone et al., 1995; Sun et al., 2006). In contrast, the requirement of Bruchpilot, which is homologous to the mammalian active zone proteins ELKS/CAST (Wagh et al., 2006), is most pronounced in immediate vesicle release (Kittel et al., 2006). Under repeated stimulation of a bruchpilot mutant motor neuron, immediate vesicle release is severely impaired whereas the following steady-state release is still possible (Kittel et al., 2006). In line with that, knockdown of the Bruchpilot protein causes impairment in clustering of Ca2+ channels to the active zones and a lack of electron-dense projections at presynaptic terminals (T-bars). Thus, less synaptic vesicles of the readily-releasable pool are accumulated to the release sites and their release probability is severely impaired (Kittel et al., 2006; Wagh et al., 2006). First, I showed that Bruchpilot is required for aversive olfactory memory and localized the requirement of Bruchpilot to the Kenyon cells of the mushroom body, the second-order olfactory interneurons in Drosophila. Furthermore, I demonstrated that Bruchpilot selectively functions for the consolidated anesthesia-resistant memory. Since Synapsin is specifically required for the labile anesthesia sensitive memory, different synaptic proteins can dissociate consolidated and labile components of olfactory memory and two different modes of neurotransmission (high- vs. low frequency dependent) might differentiate ASM and ARM.}, subject = {Taufliege}, language = {en} } @phdthesis{Nuwal2010, author = {Nuwal, Tulip}, title = {Characterization of Synapsin, Tubulin-Binding Chaperone E-like, And Their Putative Interactions With Synapse Associated Protein Of 47 kDa In Drosophila melanogaster}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-51683}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {In this thesis we have used Drosophila melanogaster as a model organism to investigate proteins and their putative interacting partners that are directly or indirectly involved in the release of neurotransmitters at the synapse. We have used molecular techniques to investigate conserved synaptic proteins, synapsin and synapse associated protein of 47 kD (SAP47), and a putative interaction partner of SAP47, tubulin binding chaperone E-like (TBCEL). SAP47 and synapsins are highly conserved synaptic vesicle associated proteins in Drosophila melanogaster. To further investigate the role and function of Sap47 and Syn genes, we had earlier generated the null mutants by P-element mutagenesis (Funk et al., 2004; Godenschwege et al., 2004). Western blots and ELISA of brain homogenates from Sap47156 null mutants showed the presence of up-regulated phospho-synapsin in comparison to wild-type (CS) and the presence of up-regulated phospho-synapsin was partially abolished when a pan-neuronal rescue of SAP47 was performed by the Gal4- UAS technique. Thus, the results suggest a qualitative and quantitative modulation of synapsin by SAP47. At the transcript level, we did not observe any difference in content of Syn transcript in Sap47156 and wild-type CS flies. The question of a direct molecular interaction between SAP47 and synapsin was investigated by co-immunoprecipitation (Co-IP) experiments and we did not find any stable interactions under the several IP conditions we tested. The possibility of Sap47 as a modifier of Syn at the genetic level was investigated by generating and testing homozygous double null mutants of Sap47 and Syn. The Syn97, Sap47156 double mutants are viable but have a reduced life span and decreased locomotion when compared to CS. In 2D-PAGE analysis of synapsins we identified trains of spots corresponding to synapsins, suggesting that synapsin has several isoforms and each one of them is posttranslationally modified. In an analysis by Blue native-SDS-PAGE (BN-SDS-2D- PAGE) and Western blot we observed synapsin and SAP47 signals to be present at 700-900 kDa and 200-250 kDa, respectively, suggesting that they are part of large but different complexes. We also report the possibility of Drosophila synapsin forming homo- and heteromultimers, which has also been reported for synapsins of vertebrates. In parallel to the above experiments, phosphorylation of synapsins in Drosophila was studied by IP techniques followed by 1D-SDS gel electrophoresis and mass spectrometry (in collaboration with S. Heo and G. Lubec). We identified and verified 5 unique phosphorylation sites in Drosophila synapsin from our MS analysis. Apart from phosphorylation modifications we identified several other PTMs which have not been verified. The significance of these phosphorylations and other identified PTMs needs to be investigated further and their implications for synapsin function and Drosophila behavior has to be elucidated by further experiments. In a collaborative project with S. Kneitz and N. Nuwal, we investigated the effects of Sap156 and Syn97 mutations by performing a whole Drosophila transcriptome microarray analysis of the individual null mutants and the double mutants (V2 and V3). We obtained several candidates which were significantly altered in the mutants. These genes need to be investigated further to elucidate their interactions with Sap47 and Syn. In another project, we investigated the role and function of Drosophila tubulin- binding chaperone E-like (Tbcel, CG12214). The TBCEL protein has high homology to vertebrate TBCE-like (or E-like) which has high sequence similarity to tubulin-binding chaperone E (TBCE) (hence the name TBCE-Like). We generated an anti-TBCEL polyclonal antiserum (in collaboration with G. Krohne). According to flybase, the Tbcel gene has only one exon and codes for two different transcripts by alternative transcription start sites. The longer transcript RB is present only in males whereas the shorter transcript RA is present only in females. In order to study the gene function we performed P- element jump-out mutagenesis to generate deletion mutants. We used the NP4786 (NP) stock which has a P(GawB) insertion in the 5' UTR of the Tbcel gene. NP4786 flies are homozygous lethal due to a second-site lethality as the flies are viable over a deficiency (Df) chromosome (a deletion of genomic region spanning the Tbcel gene and other upstream and downstream genes). We performed the P-element mutagenesis twice. In the first trial we obtained only revertants and the second experiment is still in progress. In the second attempt, jump-out was performed over the deficiency chromosome to prevent homologous chromosome mediated double stranded DNA repair. During the second mutagenesis an insertion stock G18151 became available. These flies had a P-element insertion in the open reading frame (ORF) of the Tbcel gene but was homozygous viable. Western blots of fresh tissue homogenates of NP/Df and G18151 flies probed with anti-TBCEL antiserum showed no TBCEL signal, suggesting that these flies are Tbcel null mutants. We used these flies for further immunohistochemical analyses and found that TBCEL is specifically expressed in the cytoplasm of cyst cells of the testes and is associated with the tubulin of spermatid tails in wild-type CS, whereas in NP/Df and G18151 flies the TBCEL staining in the cyst cells was absent and there was a disruption of actin investment cones. We also found enrichment of TBCEL staining around the actin investment cone. These results are also supported by the observation that the enhancer trap expression of the NP4786 line is localised to the cyst cells, similar to TBCEL expression. Also, male fertility of NP/Df and G18151 flies was tested and they were found to be sterile with few escapers. Thus, these results suggest that TBCEL is involved in Drosophila spermatogenesis with a possible role in the spermatid elongation and individualisation process.}, subject = {Taufliege}, language = {en} } @phdthesis{Milenkovic2006, author = {Milenkovic, Vladimir M.}, title = {Structural and functional analysis of bestrophin}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-19372}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Morbus Best (OMIM 153700), auch als vitelliforme Makuladystrophie Typ 2 (VMD2) bezeichnet, ist eine autosomal dominant vererbte Makuladystrophie mit juvenilem Beginn. Die Erkrankung geht einher mit einer Ansammlung von Lipofuscin-{\"a}hnlichem Material im sowie unterhalb des retinalen Pigmentepithels (RPE). Das bei Morbus Best mutierte VMD2- Gen kodiert f{\"u}r ein 585 Aminos{\"a}uren langes Transmembranprotein, genannt Bestrophin, und wird vorwiegend im RPE exprimiert. Das Protein hat eine komplexe Membrantopologie mit 4-6 putativen Transmembrandom{\"a}nen (TMD) und ist vermutlich in den Ca2+-abh{\"a}ngigen Transport von Chloridionen durch die Plasmamembran involviert. Die {\"u}berwiegende Mehrheit der krankheitsassoziierten Ver{\"a}nderungen bei M. Best Patienten sind Missense-Mutationen, die innerhalb der hochkonservierten N-terminalen H{\"a}lfte des Proteins nahe der mutmaßlichen Transmembrandom{\"a}nen akkumulieren. Der Zusammenhang zwischen Pathologie und identifizierter Mutationen bzw. der Chloridkanal- Funktion von Bestrophin-1 ist noch unklar. Um die biologische Funktion von Bestrohin-1 weiter aufzukl{\"a}ren und die zugrunde liegenden molekularen Mechanismen der BMD besser zu verstehen, wurde mit Hilfe des GAL4-basierenden Hefe-Zwei-Hybridsystems (Y2H) nach interagierenden Partnern von Bestrophin-1 gesucht. Ein Screen in einer bovinen RPE cDNABank mit verschiedenen verk{\"u}rzten Fragmenten von Bestrophin-1 ergab 53 m{\"o}gliche interagierende Partner. Allerdings schlossen anschließende Verifikationsexperimente die Kandidatengene aus. Somit deuten die Resultate dieser umfangreichen YH2-Studie daraufhin, dass Bestrophin f{\"u}r das herk{\"o}mmliche Zwei-Hybrid-System nicht geeignet ist. Zum einen k{\"o}nnte dies daran liegen, dass das Protein ein integraler Bestandteil der Membran ist und zum anderen, dass m{\"o}glicherweise der Transport der gew{\"a}hlten Bestrophin-Fragmente zum Nukleus nicht stattfindet. Dies gilt jedoch als Grundvoraussetzung f{\"u}r eine Proteininteraktion im Hefe-2-Hybridsystem. Bestrophin geh{\"o}rt zu einer großen Familie von integralen Membranproteinen, von der bis heute bereits {\"u}ber 100 Mitglieder bei verschiedenen Organismengruppen wie denS{\"a}ugern, Insekten und W{\"u}rmern identifiziert werden konnten. Als auff{\"a}lligste Besonderheit in der Familie der Bestrophine zeigt sich neben einer nicht-variablen RFP-Dom{\"a}ne (Arginin- Phenylalanin-Prolin) eine evolution{\"a}r hochkonservierte N-terminale Region. Um die phylogenetische Beziehung der Bestrophine zu untersuchen sowie den Aufbau und die Funktion von konservierten Motiven innerhalb der Familienmitglieder zu identifizieren, wurde diese konservierte N-terminale Region sowohl bioinformatisch wie auch Chapter Two: Zusammenfassung 4 phylogenetisch weiter untersucht. Die phylogenetische Analyse der Bestrophin Homologen brachte vier evolution{\"a}r konservierte Familienmitglieder in S{\"a}ugern hervor, die jeweils eine starke Homologie zu den Proteinen VMD2, VMD2-L1 bis VMD2-L3 des Menschen zeigen. Die signifikante {\"A}hnlichkeit der Proteinsequenz innerhalb der vier Familienmitglieder l{\"a}sst die Schlussfolgerungen zu, dass zum einen jedes einzelne Familienmitglied ihre eigene evolution{\"a}r konservierte Funktion hat und zum anderen dass die Divergenz des Bestrophins in verschiedene Familienmitglieder zeitlich vor der Divergenz der verschiedenen S{\"a}ugerspezien erfolgt sein muss.}, subject = {Makuladegeneration}, language = {en} } @phdthesis{Berkane2005, author = {Berkane, Emir}, title = {Etude de l'interaction entre GpJ, une prot{\´e}ine du bact{\´e}riophage Lambda, et LamB, une prot{\´e}ine de la membrane externe des bact{\´e}ries gram-n{\´e}gatives}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-12889}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {La fixation du bact{\´e}riophage Lambda sur son r{\´e}cepteur cellulaire, LamB, est d{\^u}e {\`a} une prot{\´e}ine de sa queue appel{\´e}e GpJ. Le but des travaux est d'{\´e}tudier l'int{\´e}raction entre le bact{\´e}riophage Lambda et LamB {\`a} travers l'{\´e}tude du complexe entre LamB et GpJ exprim{\´e}e en prot{\´e}ine de fusion. Pour ce faire, deux prot{\´e}ines de fusion sont utilis{\´e}es : MBP-gpJ et HisgpJ. MBP-gpJ est une prot{\´e}ine de fusion entre la Maltose Binding Prot{\´e}ine et l'extr{\^e}mit{\´e} Cterminale de la prot{\´e}ine GpJ (r{\´e}sidu 684 {\`a} 1132), gr{\^a}cieusement fournie par le Pr. Charbit (Paris, France). Gr{\^a}ce {\`a} la Technique du Film Noir (BLM), il a {\´e}t{\´e} permis d'observer que MBP-gpJ, apr{\`e}s expression dans E.coli et purification, int{\´e}ragit gr{\^a}ce au fragment de GpJ avec l'extr{\^e}mit{\´e} extracellulaire de LamB. Cette int{\´e}raction se traduit par un blocage complet et r{\´e}versible des canaux de LamB sauvage, mais {\´e}galement de mutants: LamB de Shigella sonnei, LamB Y118G et LamB D4+D6+D9v. Afin d'obtenir des informations sur la liaison de LamB avec uniquement le fragment de GpJ sans la partie MBP, une autre prot{\´e}ine de fusion a {\´e}t{\´e} r{\´e}alis{\´e}e: His-GpJ. His-gpJ repr{\´e}sente l'extr{\^e}mit{\´e} C-terminale de GpJ (684-1132) en fusion avec un 6×Histidine-tag. Cette prot{\´e}ine est exprim{\´e}e sous forme de corps d'inclusion dans E.coli. Apr{\`e}s purification et renaturation, une prot{\´e}ine de nouveau soluble peut {\^e}tre obtenue. Lors d'exp{\´e}riences de Film Noir, His-gpJ int{\´e}ragit certes avec LamB, mais n'induit pas le blocage des canaux comme pr{\´e}cedemment observ{\´e} apr{\`e}s ajout de MBP-gpJ. En parall{\`e}le, la formation d'un complexe entre His-gpJ et LamB sauvage, ainsi que de mutants a pu {\^e}tre confirm{\´e}e au travers de travaux de SDS-PAGE et d'immunod{\´e}tection par la pr{\´e}sence de bandes de masse mol{\´e}culaire {\´e}lev{\´e}e. L'utilisation de mutants de LamB a par ailleurs permis d'essayer d'identifier la partie de LamB impliqu{\´e}e dans l'interaction avec le fragment C-terminal de GpJ, qui se r{\´e}v{\`e}le {\^e}tre diff{\´e}rente de celle de GpJ dans la queue du bact{\´e}riophage Lambda. Mots cl{\´e}s: bact{\´e}riophage Lambda, gpJ, LamB, technique du film noir (BLM), immunod{\´e}tection.}, subject = {Bakteriophage Lambda}, language = {en} }