Role of cell cycle regulators in development of the inner ear

The inner ear originates from an ectodermal thickening called the otic placode. The otic placode invaginates and closes to an otic vesicle, the otocyst. The otocyst epithelium undergoes morphogenetic changes and cell differentiation, leading to the formation of the labyrinth-like mature inner ear. Epithelial-mesenchymal interactions control inner ear morphogenesis, but the modes and molecules are largely unresolved. The expressions of negative cell cycle regulators in the epithelium of the early-developing inner ear have also not been elucidated. The mature inner ear comprises the hearing (cochlea) and balance (vestibular) organs that contain the nonsensory and sensory cells. In mammals, the inner ear sensory cells, called hair cells, exit the cell cycle during embryogenesis and are mitotically quiescent during late-embryonic differentiation stages and postnatally. The mechanisms that maintain this hair cell quiescense are largely unresolved. In this work I examined 1) the epithelial-mesenchymal interactions involved in inner ear morphogenesis, 2) expression of negative cell cycle regulators in the epithelium of the early developing inner ear and 3) the molecular mechanisms that maintain the postmitotic state of inner ear sensory cells. We observed that during otocyst stages, epithelial fibroblast growth factor 9 (Fgf9) communicates with the surrounding mesenchyme, where its receptors are expressed. Fgf9 inactivation leads to reduced proliferation of the surrounding vestibular mesenchyme and to the absence of semicircular canals. Semicircular canal development is blocked, since fusion plates do not form. These results show that the mesenchyme directs fusion plate formation and give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of proliferation. We show that the members of the Cip/Kip family of CKIs (p21Cip1, p27Kip1 and p57Kip2) are expressed in the early-developing inner ear. Our expression data suggest that CKIs divide the otic epithelium into proliferative and nonproliferative compartments that may underlie shaping of the otocyst. At later stages, CKIs regulate proliferation of the vestibular appendages, and this may regulate their continual growth. In addition to restricting proliferation, CKIs may play a role in regional differentiation of various epithelial cells. Differentiating and adult inner ear hair cells are postmitotic and do not proliferate in response to serum or mitogenic growth factors. In our study, we show that this is the result of the activity of negative cell cycle regulators. Based on expression profiles, we first focused on the retinoblastoma (Rb) gene, which functions downstream of the CKIs. Analysis of the inner ear phenotype of Rb mutant mice show, that the retinoblastoma protein regulates the postmitotic state of hair cells. Rb inactivation leads to hyperplasia of vestibular and cochlear sensory epithelia that is a result of abnormal cell cycle entry of differentiated hair cells and of delayed cell cycle exit of the hair cell precursor cells. In addition, we show that p21Cip1 and p19Ink4d cooperate in maintaining the postmitotic state of postnatal auditory hair cells. Whereas inactivation of p19Ink4d alone leads to low-level S-phase entry (Chen et al., 2003) and p21Cip1 null mutant mice have a normal inner ear phenotype, codeletion of p19Ink4d and p21Cip1 triggers high-level S-phase entry of auditory hair cells during early postnatal life, which leads to supernumerary hair cells. The ectopic hair cells undergo apoptosis in all of the mutant mice studied, DNA damage being the immediate cause of this death. These findings demonstrate that the maintenance of the postmitotic state of hair cells is regulated by Rb and several CKIs, and that these cell cycle regulators are critical for the lifelong survival of hair cells. These data have implications for the future design of therapies to induce hair cell regrowth.

Structural and Functional Studies on Trimeric Autotransporters

Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.

Oxygen reduction and proton translocation by cytochrome c oxidase

Energy conversion by living organisms is central dogma of bioenergetics. The effectiveness of the energy extraction by aerobic organisms is much greater than by anaerobic ones. In aerobic organisms the final stage of energy conversion occurs in respiratory chain that is located in the inner membrane of mitochondria or cell membrane of some aerobic bacteria. The terminal complex of the respiratory chain is cytochrome c oxidase (CcO) - the subject of this study. The primary function of CcO is to reduce oxygen to water. For this, CcO accepts electrons from a small soluble enzyme cytochrome c from one side of the membrane and protons from another side. Moreover, CcO translocates protons across the membrane. Both oxygen reduction and proton translocation contributes to generation of transmembrane electrochemical gradient that is used for ATP synthesis and different types of work in the cell. Although the structure of CcO is defined with a relatively high atomic resolution (1.8 Å), its function can hardly be elucidated from the structure. The electron transfer route within CcO and its steps are very well defined. Meanwhile, the proton transfer roots were predicted from the site-specific mutagenesis and later proved by X-ray crystallography, however, the more strong proof of the players of the proton translocation machine is still required. In this work we developed new methods to study CcO function based on FTIR (Fourier Transform Infrared) spectroscopy. Mainly with use of these methods we answered several questions that were controversial for many years: [i] the donor of H+ for dioxygen bond splitting was identified and [ii] the protolytic transitions of Glu-278 one of the key amino acid in proton translocation mechanism was shown for the first time.

U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

The genome packaging machinery of dsDNA bacteriophage PRD1

Viral genomes are encapsidated within protective protein shells. This encapsidation can be achieved either by a co-condensation reaction of the nucleic acid and coat proteins, or by first forming empty viral particles which are subsequently packaged with nucleic acid, the latter mechanism being typical for many dsDNA bacteriophages. Bacteriophage PRD1 is an icosahedral, non-tailed dsDNA virus that has an internal lipid membrane, the hallmark of the Tectiviridae family. Although PRD1 has been known to assemble empty particles into which the genome is subsequently packaged, the mechanism for this has been unknown, and there has been no evidence for a separate packaging vertex, similar to the portal structures used for packaging in the tailed bacteriophages and herpesviruses. In this study, a unique DNA packaging vertex was identified for PRD1, containing the packaging ATPase P9, packaging factor P6 and two small membrane proteins, P20 and P22, extending the packaging vertex to the internal membrane. Lack of small membrane protein P20 was shown to totally abolish packaging, making it an essential part of the PRD1 packaging mechanism. The minor capsid proteins P6 was shown to be an important packaging factor, its absence leading to greatly reduced packaging efficiency. An in vitro DNA packaging mechanism consisting of recombinant packaging ATPase P9, empty procapsids and mutant PRD1 DNA with a LacZ-insert was developed for the analysis of PRD1 packaging, the first such system ever for a virus containing an internal membrane. A new tectiviral sequence, a linear plasmid called pBClin15, was identified in Bacillus cereus, providing material for sequence analysis of the tectiviruses. Analysis of PRD1 P9 and other putative tectiviral ATPase sequences revealed several conserved sequence motifs, among them a new tectiviral packaging ATPase motif. Mutagenesis studies on PRD1 P9 were used to confirm the significance of the motifs. P9-type putative ATPase sequences carrying a similar sequence motif were identified in several other membrane containing dsDNA viruses of bacterial, archaeal and eukaryotic hosts, suggesting that these viruses may have similar packaging mechanisms. Interestingly, almost the same set of viruses that were found to have similar putative packaging ATPases had earlier been found to share similar coat protein folds and capsid structures, and a common origin for these viruses had been suggested. The finding in this study of similar packaging proteins further supports the idea that these viruses are descendants of a common ancestor.

Mycorrhizal fungi and nitrogen dynamics in drained peatland

Fungi have a fundamental role in carbon and nutrient transformations in the acids soils of boreal regions, such as peatlands, where high amounts of carbon (C) and nutrients are stored in peat, the pH is relatively low and the nutrient uptake of trees is highly dependent on mycorrhizae. In this thesis, the aim was to examine nitrogen (N) transformations and the availability of dissolved N compounds in forestry-drained peatlands, to compare the fungal community biomass and structure at various peat N levels, to investigate the growth of ectomycorrhizal fungi with variable P and K availability and to assess how the ectomycorrhizal fungi (ECM) affect N transformations. Both field and laboratory experiments were carried out. The peat N concentration did not affect the soil fungal community structure within a site. Phosphorus (P) and potassium (K) deficiency of the trees as well as the degree of decomposition and dissolved organic nitrogen (DON) concentration of the peat were shown to affect the fungal community structure and biomass of ECMs, highlighting the complexity of the below ground system on drained peatlands. The biomass of extrametrical mycorrhizal mycelia (EMM) was enhanced by P and/or K deficiency of the trees, and ECM biomass in the roots was increased by P deficiency. Thus, PK deficiency in drained peatlands may increase the allocation of C by the tree to ECMs. It was also observed that fungi can alter N mineralization processes in the rhizosphere but variously depending on fungal species and fertility level of peat. Gross N mineralization did not vary but the net N mineralization rate significantly increased along the N gradient in both field and laboratory experiments. Gross N immobilization also significantly increased when the peat N concentration increased. Nitrification was hardly detectable in either field or laboratory experiments. During the growing season, dissolved inorganic N (DIN) fluctuated much more than the relatively stable DON. Special methodological challenges associated with sampling and analysis in microbial studies on peatlands are discussed.

The effects of eutrophication on alternative reproductive tactics in threespine sticklebacks

When organisms compete for mates and fertilisations, the process of sexual selection drives the evolution of traits that increase reproductive success. The traits targeted by selection, and the extent to which they change, are constrained by the local environment. Sexual selection due to female mate choice can be undermined by alternative reproductive tactics (ARTs), which refers to discontinuous variation in traits or behaviours used in reproduction. As human activities are rapidly changing our planet, this raises the question how ARTs will be affected. Fish show a bewildering diversity of ARTs, which make them good model organisms to answer these questions. One example of human-induced environmental change, which is affecting aquatic ecosystems around the world, is eutrophication, the over-enrichment of water bodies with nutrients. One of its effects is decreased underwater visibility due to increases in both turbidity and vegetation density. The aims of this thesis were to investigate the effects increased turbidity and vegetation density have on an ART in sticklebacks, a fish common to marine and fresh water bodies of the Northern hemisphere. I furthermore investigated how this affected sexual selection for male size, a trait commonly under selection. I used a combination of behavioural observations in microcosms, where I manipulated underwater visibility, with collection of genetic material to reconstruct parentage of broods, and thus identify sneak fertilisations. The results show that turbidity might have weak negative effects on the frequency of sneaking behaviour, although this behaviour was rather infrequent in these experiments, which complicates firm conclusions. In dense vegetation the number of sneak fertilisations decreased slightly, as fewer nesting males sneaked, while the number of non-nesting males sneaking remained constant. The paternity analyses revealed that a significantly smaller fraction of eggs was sneak fertilised under dense vegetation. Furthermore, amongst the nesting males that sneaked, the amount of eggs sneak fertilised correlated positively with courtship success. A reduction in sneaking by these males under dense vegetation equalised the distribution of fertilisation success, in turn contributing to a decrease in the opportunity for selection. Under dense vegetation significantly more males built nests, which has also been observed in previous field studies. In a separate experiment we addressed if such changes in the proportion of nesters and non-nesters, without changes in visibility, affected the incidence of sneak fertilisation. My results show this was not the case, likely because sneaking is an opportunistic tactic shown by both nesters and non-nesters. Non-nesters did sneak proportionately more when there were many of them, which could be due to changes in the cost-benefit ratio of sneaking. As nesters can only attack one intruder at a time, the costs and risks per sneaker will decrease as the number of sneakers increases. The defensive behaviours shown by the nesters before spawning shifted to a more aggressive form of nest defence. This could be because less aggressive behaviours lose their effectiveness when the number of intruders increases. It could also indicate that the risks associated with aggressive behaviours decrease when there are fewer fellow nesters, as other studies indicate nesters are competitive and aggressive individuals. Under turbid conditions I did not detect changes in the opportunity for selection, based on fertilisation success, nor was male size under significant selection under clear or turbid conditions. More thorough analyses under densely vegetated conditions across the nesting, courtship and fertilisation stages revealed a decrease in the opportunity for selection across all stages. A reduction in sneaking by nesters contributed to this. During the nesting stage, but not during later stages, body size was under significant directional selection under sparse, but not dense vegetation. This illustrates the importance of considering all selection stages to get a complete picture of how environmental changes affect sexual selection. Leaving out certain stages or subgroups can result in incomplete or misleading results.

Flight metabolic rate in the Glanville fritillary butterfly

Dispersal is a highly important life history trait. In fragmented landscapes the long-term persistence of populations depends on dispersal. Evolution of dispersal is affected by costs and benefits and these may differ between different landscapes. This results in differences in the strength and direction of natural selection on dispersal in fragmented landscapes. Dispersal has been shown to be a nonrandom process that is associated with traits such as flight ability in insects. This thesis examines genetic and physiological traits affecting dispersal in the Glanville fritillary butterfly (Melitaea cinxia). Flight metabolic rate is a repeatable trait representing flight ability. Unlike in many vertebrates, resting metabolic rate cannot be used as a surrogate of maximum metabolic rate as no strong correlation between the two was found in the Glanville fritillary. Resting and flight metabolic rate are affected by environmental variables, most notably temperature. However, only flight metabolic rate has a strong genetic component. Molecular variation in the much-studied candidate locus phosphoglucose isomerase (Pgi), which encodes the glycolytic enzyme PGI, has an effect on carbohydrate metabolism in flight. This effect is temperature dependent: in low to moderate temperatures individuals with the heterozygous genotype at the single nucleotide polymorphism (SNP) AA111 have higher flight metabolic rate than the common homozygous genotype. At high temperatures the situation is reversed. This finding suggests that variation in enzyme properties is indeed translated to organismal performance. High-resolution data on individual female Glanville fritillaries moving freely in the field were recorded using harmonic radar. There was a strong positive correlation between flight metabolic rate and dispersal rate. Flight metabolic rate explained one third of the observed variation in the one-hour movement distance. A fine-scaled analysis of mobility showed that mobility peaked at intermediate ambient temperatures but the two common Pgi genotypes differed in their reaction norms to temperature. As with flight metabolic rate, heterozygotes at SNP AA111 were the most active genotype in low to moderate temperatures. The results show that molecular variation is associated with variation in dispersal rate through the link of flight physiology under the influence of environmental conditions. The evolutionary pressures for dispersal differ between males and females. The effect of flight metabolic rate on dispersal was examined in both sexes in field and laboratory conditions. The relationship between flight metabolic rate and dispersal rate in the field and flight duration in the laboratory were found to differ between the two sexes. In females the relationship was positive, but in males the longest distances and flight durations were recorded for individuals with low flight metabolic rate. These findings may reflect male investment in mate locating. Instead of dispersing, males with high flight metabolic rate may establish territories and follow a perching strategy when locating females and hence move less on the landscape level. Males with low metabolic rate may be forced to disperse due to low competitive success or may show adaptations to an alternative strategy: patrolling. In the light of life history trade-offs and the rate of living theory having high metabolic rate may carry a cost in the form of shortened lifespan. Experiments relating flight metabolic rate to longevity showed a clear correlation in the opposite direction: high flight metabolic rate was associated with long lifespan. This suggests that individuals with high metabolic rate do not pay an extra physiological cost for their high flight capacity, rather there are positive correlations between different measures of fitness. These results highlight the importance of condition.

Spatial ecology of a specialist insect herbivore : the leaf-mining moth Tischeria ekebladella on the pedunculate oak Quercus robur

Herbivorous insects comprise a major part of terrestrial biodiversity, and their interactions with their host plants and natural enemies are of vast ecological importance. A large body of research demonstrates that the ecology and evolution of these insects may be affected by trophic interactions, by abiotic influences, and by intraspecific processes, but so far research on these individual aspects has rarely been combined. This thesis uses the leaf-mining moth Tischeria ekebladella and the pedunculate oak (Quercus robur) as a case study to assess how spatial variation in trophic interactions and the physical distribution of host trees jointly affect the distribution, dynamics and evolution of a host-specific herbivore. With respect to habitat quality, Tischeria ekebladella experiences abundant variation at several spatial scales. Most of this variation occurs at small scales notably among leaves and shoots within individual trees. While hypothetically this could cause moths to evolve an ability to select leaves and shoots of high quality, I did not find any coupling between female preference and offspring performance. Based on my studies on temporal variation in resource quality I therefore propose that unpredictable temporal changes in the relative rankings of individual resource units may render it difficult for females to predict the fate of their developing offspring. With respect to intraspecific processes, my results suggest that limited moth dispersal in relation to the spatial distribution of oak trees plays a key role in determining the regional distribution of Tischeria ekebladella. The distribution of the moth is aggregated at the landscape level, where local leaf miner populations are less likely to be present where oaks are scarce. A modelling exercise based on empirical dispersal estimates revealed that the moth population on Wattkast an island in south-western Finland is spatially structured overall, but that the relative importance of local and regional processes on tree-specific moth dynamics varies drastically across the landscape. To conclude, my work in the oak-Tischeria ekebladella system demonstrates that the local abundance and regional distribution of a herbivore may be more strongly influenced by the spatial location of host trees than by their relative quality. Hence, it reveals the importance of considering spatial context in the study of herbivorous insects, and forms a bridge between the classical fields of plant-insect interactions and spatial ecology.

Electron cryo-microscopy studies of bacteriophage phi8 and archaeal virus SH1

Symmetry is a key principle in viral structures, especially the protein capsid shells. However, symmetry mismatches are very common, and often correlate with dynamic functionality of biological significance. The three-dimensional structures of two isometric viruses, bacteriophage phi8 and the archaeal virus SH1 were reconstructed using electron cryo-microscopy. Two image reconstruction methods were used: the classical icosahedral method yielded high resolution models for the symmetrical parts of the structures, and a novel asymmetric in-situ reconstruction method allowed us to resolve the symmetry mismatches at the vertices of the viruses. Evidence was found that the hexameric packaging enzyme at the vertices of phi8 does not rotate relative to the capsid. The large two-fold symmetric spikes of SH1 were found not to be responsible for infectivity. Both virus structures provided insight into the evolution of viruses. Comparison of the phi8 polymerase complex capsid with those of phi6 and other dsRNA viruses suggests that the quaternary structure in dsRNA bacteriophages differs from other dsRNA viruses. SH1 is unusual because there are two major types of capsomers building up the capsid, both of which seem to be composed mainly of single beta-barrels perpendicular to the capsid surface. This indicates that the beta-barrel may be ancestral to the double beta-barrel fold.