Browsing by Organization "Institute of Biotechnology"

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  • Würtz, Peter (Helsingin yliopisto, 2008)
    NMR spectroscopy enables the study of biomolecules from peptides and carbohydrates to proteins at atomic resolution. The technique uniquely allows for structure determination of molecules in solution-state. It also gives insights into dynamics and intermolecular interactions important for determining biological function. Detailed molecular information is entangled in the nuclear spin states. The information can be extracted by pulse sequences designed to measure the desired molecular parameters. Advancement of pulse sequence methodology therefore plays a key role in the development of biomolecular NMR spectroscopy. A range of novel pulse sequences for solution-state NMR spectroscopy are presented in this thesis. The pulse sequences are described in relation to the molecular information they provide. The pulse sequence experiments represent several advances in NMR spectroscopy with particular emphasis on applications for proteins. Some of the novel methods are focusing on methyl-containing amino acids which are pivotal for structure determination. Methyl-specific assignment schemes are introduced for increasing the size range of 13C,15N labeled proteins amenable to structure determination without resolving to more elaborate labeling schemes. Furthermore, cost-effective means are presented for monitoring amide and methyl correlations simultaneously. Residual dipolar couplings can be applied for structure refinement as well as for studying dynamics. Accurate methods for measuring residual dipolar couplings in small proteins are devised along with special techniques applicable when proteins require high pH or high temperature solvent conditions. Finally, a new technique is demonstrated to diminish strong-coupling induced artifacts in HMBC, a routine experiment for establishing long-range correlations in unlabeled molecules. The presented experiments facilitate structural studies of biomolecules by NMR spectroscopy.
  • Saarikangas, Juha (Helsingin yliopisto, 2010)
    Plasma membrane adopts myriad of different shapes to carry out essential cellular processes such as nutrient uptake, immunological defence mechanisms and cell migration. Therefore, the details how different plasma membrane structures are made and remodelled are of the upmost importance. Bending of plasma membrane into different shapes requires substantial amount of force, which can be provided by the actin cytoskeleton, however, the molecules that regulate the interplay between the actin cytoskeleton and plasma membrane have remained elusive. Recent findings have placed new types of effectors at sites of plasma membrane remodelling, including BAR proteins, which can directly bind and deform plasma membrane into different shapes. In addition to their membrane-bending abilities, BAR proteins also harbor protein domains that intimately link them to the actin cytoskeleton. The ancient BAR domain fold has evolved into at least three structurally and functionally different sub-groups: the BAR, F-BAR and I-BAR domains. This thesis work describes the discovery and functional characterization of the Inverse-BAR domains (I-BARs). Using synthetic model membranes, we have shown that I-BAR domains bind and deform membranes into tubular structures through a binding-surface composed of positively charged amino acids. Importantly, the membrane-binding surface of I-BAR domains displays an inverse geometry to that of the BAR and F-BAR domains, and these structural differences explain why I-BAR domains induce cell protrusions whereas BAR and most F-BAR domains induce cell invaginations. In addition, our results indicate that the binding of I-BAR domains to membranes can alter the spatial organization of phosphoinositides within membranes. Intriguingly, we also found that some I-BAR domains can insert helical motifs into the membrane bilayer, which has important consequences for their membrane binding/bending functions. In mammals there are five I-BAR domain containing proteins. Cell biological studies on ABBA revealed that it is highly expressed in radial glial cells during the development of the central nervous system and plays an important role in the extension process of radial glia-like C6R cells by regulating lamellipodial dynamics through its I-BAR domain. To reveal the role of these proteins in the context of animals, we analyzed MIM knockout mice and found that MIM is required for proper renal functions in adult mice. MIM deficient mice displayed a severe urine concentration defect due to defective intercellular junctions of the kidney epithelia. Consistently, MIM localized to adherens junctions in cultured kidney epithelial cells, where it promoted actin assembly through its I-BAR andWH2 domains. In summary, this thesis describes the mechanism how I-BAR proteins deform membranes and provides information about the biological role of these proteins, which to our knowledge are the first proteins that have been shown to directly deform plasma membrane to make cell protrusions.
  • Voutilainen, Merja (Helsingin yliopisto, 2010)
    Parkinson s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra (SN). Current therapies of PD do not stop the progression of the disease and the efficacy of these treatments wanes over time. Neurotrophic factors are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. Neurotrophic factors are attractive candidates for neuroprotective or even neurorestorative treatment of PD. Thus, searching for and characterizing trophic factors are highly important approaches to degenerative diseases. CDNF (cerebral dopamine neurotrophic factor) and MANF (mesencephalic astrocyte-derived neurotrophic factor) are secreted proteins that constitute a novel, evolutionarily conserved neurotrophic factor family expressed in vertebrates and invertebrates. The present study investigated the neuroprotective and restorative effects of human CDNF and MANF in rats with unilateral partial lesion of dopamine neurons by 6-hydroxydopamine (6-OHDA) using both behavioral (amphetamine-induced rotation) and immunohistochemical analyses. We also investigated the distribution and transportation profiles of intrastriatally injected CDNF and MANF in rats. Intrastriatal CDNF and MANF protected nigrostriatal dopaminergic neurons when administered six hours before or four weeks after the neurotoxin 6-OHDA. More importantly, the function of the lesioned nigrostriatal dopaminergic system was partially restored even when the neurotrophic factors were administered four weeks after 6-OHDA. A 14-day continuous infusion of CDNF but not of MANF restored the function of the midbrain neural circuits controlling movement when initiated two weeks after unilateral injection of 6-OHDA. Continuous infusion of CDNF also protected dopaminergic TH-positive cell bodies from toxin-induced degeneration in the substantia nigra pars compacta (SNpc) and fibers in the striatum. When injected into the striatum, CDNF and GDNF had similar transportation profiles from the striatum to the SNpc; thus CDNF may act via the same nerve tracts as GDNF. Intrastriatal MANF was transported to cortical areas which may reflect a mechanism of neurorestorative action that is different from that of CDNF and GDNF. CDNF and MANF were also shown to distribute more readily than GDNF. In conclusion, CDNF and MANF are potential therapeutic proteins for the treatment of PD.
  • Kirjavainen, Anna (Helsingin yliopisto, 2014)
    Development of the sensory epithelia of the inner ear and their primary cell types, hair cells and supporting cells, is a complex process under tight molecular regulation. These cells arise from common progenitors that are guided to follow cell-type-specific differentiation program, and undergo prominent structural changes to reach mature morphologies. The mechanisms regulating this cellular differentiation in the developing inner ear are not fully understood. The focus of this thesis has been in understanding the molecular control of the stepwise development of hair cells and supporting cells. Sequential expression of transcription factors has a central role in the control of development of the cells and tissues. Here we show that transcription factor Prox1 participates in the molecular cascade directing cellular differentiation in the inner ear. During early development, Prox1 is expressed in the progenitors of hair cells and supporting cells, and later maintained only in the supporting cells. We found novel interactions between Prox1 and hair cell-specific transcription factors Atoh1, the master regulator of hair cell development, and Gfi1, an essential survival factor of the cochlear hair cells. When overexpressed in hair cells, Prox1 suppressed the expression of Atoh1 and Gfi1, illustrating the possibility of transcriptional reprogramming of hair cells. This downregulation had functional consequences, resulting in auditory hair cell death during a restricted period at late-embryogenesis. Furthermore, when we studied Gfi1-knock-in mice, the model in which auditory hair cells die shortly after differentiation, we found positive interaction between Gfi1 and p57Kip2. Thus, p57Kip2 is introduced as a new candidate to mediate the survival-promoting function of Gfi1 in the auditory hair cells. Rho GTPases integrate signals from different molecular pathways to regulate cell cytoskeleton, intercellular junctions and polarity, all properties that are heavily modulated in the epithelial cells of the developing inner ear. A member of Rho GTPase family, Cdc42, was found to be expressed in the developing auditory sensory epithelium. Analysis of Cdc42 mutant mice revealed a versatile role of this protein, demonstrating its importance in 1) the formation of proper cellular patterning in the auditory sensory epithelium, 2) the regulation of apical-basal and planar polarities of the sensory epithelial cells, and 3) the regulation of apical cytoskeleton in these cells. In the absence of Cdc42, mechanosensory hair bundles at the apices of hair cells failed to develop normally, indicating Cdc42 s significance in hearing function. In addition, Cdc42 regulates the maturation of adherens junctions and apical actin cytoskeleton in postnatal supporting cells. Cdc42-deficient supporting cells lacked the ability for normal wound healing, showing that properly developed apical module is needed for epithelium repair following injury to the hearing organ. This thesis presents new pieces to the molecular network controlling cellular differentiation of the inner ear sensory epithelia. Understanding the regulation of this stepwise development may have therapeutic value. It may help to explain the fundamental reasons why mammalian hair cells do not regenerate and, to identify the mechanisms and factors that could be applied to promote hair cell regeneration in the future.
  • Kukkaro, Petra (Helsingin yliopisto, 2009)
    Viruses of Archaea are the least studied group of viruses. Fewer than 50 archaeal viruses have been reported which constitutes less than one percent of all the isolated prokaryotic viruses. Only about one third of the isolated archaeal viruses infect halophiles. The diversity of haloviruses, virus ecology in highly saline environments and the interactions of haloviruses with their hosts have been little studied. The exiguous knowledge available on halophilic systems is not only due to inadequate sampling but also reflects the extra challenge highly saline systems set on biochemical studies. In this study six new haloviruses were isolated and characterized. Viruses included four archaeal viruses and two bacteriophages. All of the other isolates exhibited head-tail morphology, except SH1 which was the first tailless icosahedral virus isolated from a high salt environment. Production and purification procedures were set up for all of these viruses and they were subjected to stability determinations. Archaeal virus SH1 was studied in more detail. Biochemical studies revealed an internal membrane underneath the protein capsid and a linear dsDNA genome. The overall structure of SH1 resembles phages PRD1, PM2 and Bam35 as well as an archaeal virus STIV. SH1 possesses about 15 structural proteins that form complexes under non-reducing conditions. Quantitative dissociation provided information about the positions of these proteins in the virion. The life cycle of SH1 was also studied. This lytic virus infects Haloarcula hispanica. Adsorption to the host cells is fairly inefficient and the life cycle rather long. Finally, virus responses in a variety of ionic conditions were studied. It was discovered that all of the studied viruses from low salt, marine and high salt environments tolerated larger range of salinities than their bacterial or archaeal hosts. The adsorption efficiency was not determined by the natural environment of a virus. Even though viruses with the slowest binding kinetics were among the haloviruses, fast binders were observed in viruses from all environments. When the salinity was altered, the virus adsorption responses were diverse. Four different behavioral patterns were observed: virus binding increased or decreased in increasing salinity, adsorption maximum was at a particular salt concentration or the salinity did not affect the binding. The way the virus binding was affected did not correlate with the environment, virus morphology or the organism the virus infects.
  • Saariaho, Anna-Helena (Helsingin yliopisto, 2006)
    Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.
  • Ta, Hung (Helsingin yliopisto, 2012)
    Living systems, which are composed of biological components such as molecules, cells, organisms or entire species, are dynamic and complex. Their behaviors are difficult to study with respect to the properties of individual elements. To study their behaviors, we use quantitative techniques in the "omic" fields such as genomics, bioinformatics and proteomics to measure the behavior of groups of interacting components, and we use mathematical and computational modeling to describe and predict their dynamical behavior. The first step in the understanding of a biological system is to investigate how its individual elements interact with each other. This step consist of drawing a static wiring diagram that connects the individual parts. Experimental techniques that are used - are designed to observe interactions among the biological components in the laboratory while computational approaches are designed to predict interactions among the individual elements based on their properties. In the first part of this thesis, we present techniques for network inference that are particularly targeted at protein-protein interaction networks. These techniques include comparative genomics, structure-based, biological context methods and integrated frameworks. We evaluate and compare the prediction methods that have been most often used for domain-domain interactions and we discuss the limitations of the methods and data resources. We introduce the concept of the Enhanced Phylogenetic Tree, which is a new graphical presentation of the evolutionary history of protein families; then, we propose a novel method for assigning functional linkages to proteins. This method was applied to predicting both human and yeast protein functional linkages. The next step is to obtain insights into the dynamical aspects of the biological systems. One of the outreaching goals of systems biology is to understand the emergent properties of living systems, i.e., to understand how the individual components of a system come together to form distinct, collective and interactive properties and functions. The emergent properties of a system are neither to be found in nor are directly deducible from the lower-level properties of that system. An example of the emergent properties is synchronization, a dynamical state of complex network systems in which the individual components of the systems behave coherently, almost in unison. In the second part of the thesis, we apply computational modeling to mimic and simplify real-life complex systems. We focus on clarifying how the network topology determines the initiation and propagation of synchronization. A simple but efficient method is proposed to reconstruct network structures from functional behaviors for oscillatory systems such as brain. We study the feasibility of network reconstruction systematically for different regimes of coupling and for different network topologies. We utilize the Kuramoto model, an interacting system of oscillators, which is simple but relevant enough to address our questions.
  • Sharma, Vivek (Helsingin yliopisto, 2012)
    Heme-copper oxidases terminate the respiratory chain in many eukaryotes and prokaryotes as the final electron acceptors. They catalyze the reduction of molecular oxygen to water, and conserve the free-energy by proton pumping across the inner mitochondrial membrane or plasma membrane of bacteria. This leads to the generation of an electrochemical gradient across the membrane, which is utilized in the synthesis of ATP. The catalytic mechanism of oxidase is a complex coupling of electrons and protons, which has been studied with the help of numerous biophysical and biochemical methods. The superfamily of oxidases is classified into three different subfamilies; A-, B- and C-type. The A- and B-type oxidases have been studied in great depth, whereas relatively less is known about the molecular mechanism of distinct C-type (or cbb3-type) oxidases. The latter enzymes, which are known to possess unusually high oxygen affinity relative to the former class of enzymes, also share little sequence or structural similarity with the A- and B-type oxidases. In the work presented in this thesis, C-type oxidases have been studied using a variety of computational procedures, such as homology modeling, molecular dynamics simulations, density functional theory calculations and continuum electrostatics. Homology models of the C-type oxidase correctly predicts the side-chain orientation of the cross-linked tyrosine and a proton-channel. The active-site region is also modelled with high accuracy in the models, which are subsequently used in the DFT calculations. With the help of these calculations it is proposed that the different orientation of the cross-linked tyrosine, and a strong hydrogen bond in the proximal side of the high-spin heme are responsible for the higher apparent oxygen affinity and a more rhombic EPR signal in the C-type oxidases. Furthermore, the pKa profiles of two amino acid residues, which are located close to the active-site, suggest a strong electron-proton coupling and a unique proton pumping route. Molecular dynamics simulations on the two-subunit C-type oxidase allowed for the first time to observe redox state dependent water-chain formation in the protein interior, which can be utilized for the redox coupled proton transfer.
  • Yu, Liying (Helsingin yliopisto, 2009)
    Programed cell death (PCD) is a fundamental biological process that is as essential for the development and tissue homeostasis as cell proliferation, differentiation and adaptation. The main mode of PCD - apoptosis - occurs via specifi c pathways, such as mitochondrial or death receptor pathway. In the developing nervous system, programed death broadly occurs, mainly triggered by the defi ciency of different survival-promoting neurotrophic factors, but the respective death pathways are poorly studied. In one of the best-characterized models, sympathetic neurons deprived of nerve growth factor (NGF) die via the classical mitochondrial apoptotic pathway. The main aim of this study was to describe the death programs activated in these and other neuronal populations by using neuronal cultures deprived of other neurotrophic factors. First, this study showed that the cultured sympathetic neurons deprived of glial cell line-derived neurotrophic factor (GDNF) die via a novel non-classical death pathway, in which mitochondria and death receptors are not involved. Indeed, cytochrome c was not released into the cytosol, Bax, caspase-9, and caspase-3 were not involved, and Bcl-xL overexpression did not prevent the death. This pathway involved activation of mixed lineage kinases and c-jun, and crucially requires caspase-2 and -7. Second, it was shown that deprivation of neurotrophin-3 (NT-3) from cultured sensory neurons of the dorsal root ganglia kills them via a dependence receptor pathway, including cleavage of the NT- 3 receptor TrkC and liberation of a pro-apoptotic dependence domain. Indeed, death of NT-3-deprived neurons was blocked by a dominant-negative construct interfering with TrkC cleavage. Also, the uncleavable mutant of TrkC, replacing the siRNA-silenced endogeneous TrkC, was not able to trigger death upon NT-3 removal. Such a pathway was not activated in another subpopulation of sensory neurons deprived of NGF. Third, it was shown that cultured midbrain dopaminergic neurons deprived of GDNF or brainderived neurotrophic factor (BDNF) kills them by still a different pathway, in which death receptors and caspases, but not mitochondria, are activated. Indeed, cytochrome c was not released into the cytosol, Bax was not activated, and Bcl-xL did not block the death, but caspases were necessary for the death of these neurons. Blocking the components of the death receptor pathway - caspase-8, FADD, or Fas - blocked the death, whereas activation of Fas accelerated it. The activity of Fas in the dopaminergic neurons could be controlled by the apoptosis inhibitory molecule FAIML. For these studies we developed a novel assay to study apoptosis in the transfected dopaminergic neurons. Thus, a novel death pathway, characteristic for the dopaminergic neurons was described. The study suggests death receptors as possible targets for the treatment of Parkinson s disease, which is caused by the degeneration of dopaminergic neurons.
  • Pietilä, Maija (Helsingin yliopisto, 2013)
    Extremophiles are found in all three domains of cellular life but especially archaea are able to withstand harsh conditions. Halophilic archaea thrive in hypersaline environments like salt lakes and salterns which have been shown to contain high abundance of virus-like particles. So far, head-tailed viruses are the most common isolates infecting haloarchaea, which is in contrast to a variety of morphologies described for the viruses of hyper-thermophilic archaea. Altogether, approximately 100 archaeal viruses have been isolated but only a fraction of them has been subjected to detailed structural analyses. In this thesis, a novel haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1), was isolated from a solar saltern. This virus was shown to have a flexible, pleomorphic vesicle-like virion devoid of a rigid protein capsid. The genome analyses revealed that HRPV-1 is the first archaeal virus to be isolated which does not have a double-stranded but a single-stranded DNA genome. A genomic region of HRPV-1 showed similarity to the genome of another haloarchaeal virus, Haloarcula hispanica virus 2 (His2), as well as to the genome of Haloarcula marismortui and Natronomonas pharaonis indicating that HRPV-1-like elements are widespread. Consistent with this, pleomorphic viruses resembling HRPV-1 and infecting haloarchaea of the genera Haloarcula, Halorubrum and Halogeometricum have subsequently been isolated from geographically distant locations, and this study was extended to altogether seven viruses. All these viruses were sensitive to lowered ionic strength confirming their halophilic nature. Based on the virion properties, these haloviruses were defined as pleolipoviruses. Life-cycle studies showed that the pleolipoviruses are nonlytic and progeny virions are produced continuously resulting in host growth retardation. The most likely exit mechanism is budding which is consistent with the observation that the pleolipoviruses acquire their lipids unselectively from the host lipid pool. All pleolipoviruses have two major structural protein species, and biochemical dissociation studies showed that the larger-sized proteins form spike-like protrusions on the virion surface and the smaller-sized proteins are embedded in the inner surface of the membrane vesicle. The three-dimensional virion structure of HRPV-1 revealed that the spike structures are randomly distributed on the virion surface. The genome of the pleolipoviruses is enclosed in a lipid vesicle without associated nucleoproteins. Although the pleolipoviruses have different genome types, single- or double-stranded, circular or linear DNA, the membrane vesicle-based virion architecture is conserved. This work introduced a novel group of pleomorphic viruses infecting extremely halophilic archaea and showed that vesicle-like virion architecture is common in hypersaline environments. Interestingly, the archaeal pleolipoviruses were observed to share several similarities with a bacterial mycoplasmavirus indicating that these viruses may form a viral lineage with an ancient origin.
  • Ziedaite, Gabija (Helsingin yliopisto, 2008)
    The object of this study is a tailless internal membrane-containing bacteriophage PRD1. It has a dsDNA genome with covalently bound terminal proteins required for replication. The uniqueness of the structure makes this phage a desirable object of research. PRD1 has been studied for some 30 years during which time a lot of information has accumulated on its structure and life-cycle. The two least characterised steps of the PRD1 life-cycle, the genome packaging and virus release are investigated here. PRD1 shares the main principles of virion assembly (DNA packaging in particular) and host cell lysis with other dsDNA bacteriophages. However, this phage has some fascinating individual peculiarities, such as DNA packaging into a membrane vesicle inside the capsid, absence of apparent portal protein, holin inhibitor and procapsid expansion. In the course of this study we have identified the components of the DNA packaging vertex of the capsid, and determined the function of protein P6 in packaging. We managed to purify the procapsids for an in vitro packaging system, optimise the reaction and significantly increase its efficiency. We developed a new method to determine DNA translocation and were able to quantify the efficiency and the rate of packaging. A model for PRD1 DNA packaging was also proposed. Another part of this study covers the lysis of the host cell. As other dsDNA bacteriophages PRD1 has been proposed to utilise a two-component lysis system. The existence of this lysis system in PRD1 has been proven by experiments using recombinant proteins and the multi-step nature of the lysis process has been established.
  • Sun, Xiaoyu (Helsingin yliopisto, 2014)
    Many viruses protect their genome in a protein capsid. Viral capsid formation involves the association of multiple copies of viral capsid protein subunits, representing single or multiple protein species, and different assembly strategies are utilized. Pseudomonas phage phi6 uses an assembly pathway in which an empty capsid (procapsid, PC) is first assembled, serving as a compartment for the subsequent encapsidation of the RNA genome. During encapsidation, the compact, empty PC undergoes conformational rearrangement to reach its final expanded form. The phi6 PC is composed of the main structural protein, P1, and three minor protein species: the RNA-dependent RNA polymerase P2, the packaging nucleoside triphosphatase (NTPase) P4, and the assembly cofactor P7. In vitro systems of phi6 assembly, genome encapsidation, and transcription have been established, allowing infectious particles to be constructed from purified protein and RNA components. In this thesis, stoichiometric measurements were established to estimate the relative copy numbers of PC proteins in phi6 virions and PCs. Different concentrations of the phi6 minor proteins were employed in in vitro assembly reactions to probe potential PC binding sites. The results indicate that potential binding sites for proteins P2 and P7 are only partially occupied in phi6 virions and recombinant PCs. High P7 occupancy in self-assembled PCs resulted in reduced P2 incorporation, suggesting some correlation between P2 and P7 during PC assembly. Although high P4 hexamer occupancy was critical for initial particle formation, a large excess of P4 in the self-assembly reaction slowed the rate of PC self-assembly, which may be ascribed to excessive production of P1-P4 nucleation complexes. In addition, electrostatic interactions were demonstrated to be the main driving force in phi6 PC assembly. Furthermore, it was shown that P4 hexamers spontaneously dissociate from the empty capsid shell. P4-deficient particles have slower sedimentation velocity and an expanded appearance compared to the PC that has full-occupancy of P4. These particles are also defective in RNA packaging and transcription. However, purified P4 hexamers can efficiently assemble on P4-deficient particles, guiding the particles to their naive compact conformation and rescuing packaging and transcription activities. The results obtained from this study provide new insight into the principles of viral capsid assembly and demonstrate the reversibility of the PC maturation pathway.
  • Skarp, Kari-Pekka (Helsingin yliopisto, 2014)
    The notion of actin in the nucleus has slowly garnered popularity over the decades transforming the protein from an obscure artefact into a target of extreme curiosity in that compartment. Actin has been associated with a whole spectrum of nuclear functions, which directly or undirectly connect the protein with the most important nuclear function, transcription. However, it is currently not known how actin enters the nucleus in the first place and whether the protein is subjected to constitutive transport between the two eukaryotic compartments. The question regarding nucleocytoplasmic shuttling of actin is further complicated by the fact that actin is near the nuclear pore complex size exclusion limit, which sets a physical barrier for passive diffusion but not active transport. To investigate the nature of actin transit between the nucleus and the cytoplasm we developed microscope assays to monitor the shuttling of fluorescently labeled particles in real time in living cells. Using our assays it was possible to quantitate nuclear actin import and export rates. This immediately revealed that actin constantly shuttles in steady state cells. We used fluorescent probes of various sizes to explore the limits of passive vs. active import and found that even if the size of the fluorescent actin construct is increased, it retains the import rate. This suggests an active mechanism is behind the nuclear import of actin, because passively traveling constructs slow down when size increases as shown by controls. Longer observations of nuclear import revealed that nuclear actin exists in at least three pools of different motility and the largest one exhibits retarded exchange rates with the environment. This is consistent with the reports of actin bound to various nuclear complexes, which may render the actins in question less mobile. We were also able to clarify the status of actin export, where two export receptors have been reported. By using a small molecule inhibitor, we showed that CRM1 does not participate in the export of actin, which at least during steady state seems to fall exclusively in the domain of exportin 6. Next we identified the components of active import machinery by using RNAi vs. exportin 6 and suspected import factors. We found that actin is imported in complex with importin 9 and unphosphorylated cofilin and the process is dependent on Ran. We then showed that RNA polymerase II dependent transcription requires a suitable amount of actin in the nucleus or it is disrupted. Finally, we wanted to investigate which cellular features might correlate with the transport rates of actin and how actin shuttling is conducted in cells of varying amount of actin in the 2 nucleus in relation to cytoplasm (N/C ratio). We found that factors such as the size or shape of the nucleus or cytoplasm or the ratio of their sizes does not affect the import or export rates of actin. Instead, we made the surprising discovery the the N/C ratio of actin inversely correlates with both export and import. This means that the fastest shuttling rates can be found in cells with least actin in the nucleus while in cells of high amount of actin in the nucleus, the transport is slow. The latter situation suggests the nucleus is filled actin, which is somehow made export incompetent, perhaps by associating with nuclear complexes. To explore the matter further, we performed studies on the motility of nuclear and cytoplasmic actin pools. However, we found no relation with high N/C ratio of actin and the size of the G-actin pool. This suggests that the primary mode of regulation of nucleocytoplasmic shuttling does not take place through manipulating the size of the global G-actin pool.
  • Alakuijala, Anniina (Helsingin yliopisto, 2007)
    γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.
  • Lahti, Laura (Helsingin yliopisto, 2012)
    Embryonic midbrain and hindbrain are structures which will give rise to brain stem and cerebellum in the adult vertebrates. Brain stem contains several nuclei which are essential for the regulation of movements and behavior. They include serotonin-producing neurons, which develop in the hindbrain, and dopamine-producing neurons in the ventral midbrain. Degeneration and malfunction of these neurons leads to various neurological disorders, including schizophrenia, depression, Alzheimer s, and Parkinson s disease. Thus, understanding their development is of high interest. During embryogenesis, a local signaling center called isthmic organizer regulates the development of midbrain and anterior hindbrain. It secretes peptides belonging to fibroblast growth factor (FGF) and Wingless/Int (Wnt) families. These factors bind to their receptors in the surrounding tissues, and activate various downstream signaling pathways which lead to alterations in gene expression. This in turn affects the various developmental processes in this region, such as proliferation, survival, patterning, and neuronal differentiation. In this study we have analyzed the role of FGFs in the development of midbrain and anterior hindbrain, by using mouse as a model organism. We show that FGF receptors cooperate to receive isthmic signals, and cell-autonomously promote cell survival, proliferation, and maintenance of neuronal progenitors. FGF signaling is required for the maintenance of Sox3 and Hes1 expression in progenitors, and Hes1 in turn suppresses the activity of proneural genes. Loss of Hes1 is correlated with increased cell cycle exit and premature neuronal differentiation. We further demonstrate that FGF8 protein forms an antero-posterior gradient in the basal lamina, and might enter the neuronal progenitors via their basal processes. We also analyze the impact of FGF signaling on the various neuronal nuclei in midbrain and hindbrain. Rostral serotonergic neurons appear to require high levels of FGF signaling in order to develop. In the absence of FGF signaling, these neurons are absent. We also show that embryonic meso-diencephalic dopaminergic domain consists of two populations in the anterior-posterior direction, and that these populations display different molecular profiles. The anterior diencephalic domain appears less dependent on isthmic FGFs, and lack several genes typical of midbrain dopaminergic neurons, such as Pitx3 and DAT. In Fgfr compound mutants, midbrain dopaminergic neurons begin to develop but soon adopt characteristics which highly resemble those of diencephalic dopaminergic precursors. Our results indicate that FGF signaling regulates patterning of these two domains cell-autonomously.
  • Saarimäki-Vire, Jonna (Helsingin yliopisto, 2012)
    The embryonic midbrain and hindbrain give rise to brain stem structures and the cerebellum. The ventral midbrain and anterior hindbrain include highly important brain nuclei such as the dopaminergic substantia nigra and the ventral tegmental area, as well as serotonergic dorsal raphe neurons. These specific brain structures are affected in several disorders such as Parkinson s disease, depression, schizophrenia and drug addiction. Between the developing midbrain and hindbrain is a signalling centre called the Isthmic Organizer. This Isthmic Organizer secretes signalling molecules, such as Wnts and Fibroblast growth factors (Fgfs). Fgf8 is able to induce midbrain and anterior hindbrain characteristics in ectopic locations, and thus Fgf8 can act as an organizer molecule. Fgf signals are mediated by Fgf receptors (Fgfr). Of the four Fgfrs, Fgfr1-Fgfr3 are expressed in the nervous system. Fgfr1 is required to maintain coherence of a slowly dividing midbrain-hindbrain boundary cell population. However, the role of Fgfr2 and Fgfr3 in the development of midbrain and anterior hindbrain is poorly understood as well as cell adhesion molecules related to the maintenance of the coherent isthmic constriction. In this study, we elucidated the role of Fgfr2 and Fgfr3 during the development of the mid-brain and hindbrain. We showed that loss of either Fgfr2 or Fgfr3 alone or even both together did not result in any structural abnormalities. Thus, Fgfr1 is the major Fgf receptor in the midbrain and anterior hindbrain region. However, when Fgfr1 and Fgfr2, or all three Fgfr1, Fgfr2 and Fgfr3 were simultaneously inactivated, the defects in the midbrain-hindbrain development were much more severe than in the Fgfr1 mutants alone. Dorsal midbrain structures and the cerebellum were lost. Although some dopaminergic precursors appeared in the ventral midbrain, all dopaminergic neurons and several other ventral neuronal populations were lost by birth. We showed that Fgfr cooperatively regulate cell survival, antero-posterior patterning, and the maintenance of neural progenitor properties. Loss of Fgf signalling in the ventral midbrain resulted in a thinner ventricular zone and premature neurogenesis. This was not caused by shortened cell cycle length or abnormalities in cellular polarity, cellular architecture or the orientation of mitotic spindles. Instead, loss of Fgf signalling lead to a downregulation of neural stem cell transcription factors, which allowed upregulation of proneural genes. Thus, these gene expression changes drove neural progenitors to exit the cell cycle. In addition, we showed that Fgf8 is localized in the basal membrane. Thus, Fgf signalling may maintain proliferative identity of the midbrain neural progenitors, and the cells likely receive these guiding Fgf signals through their basal processes. Finally, we showed that an Fgf-regulated adhesion molecule Cadherin22 (Cdh22) is not essential for the maintenance of the coherent compartment boundary between the midbrain and the hindbrain. Possibly, Cdh22 acts redundantly with other type II cadherins. In addition, specific expression patterns in distinct brain nuclei suggest roles for Cdh22 in the segregation of neuronal populations cooperatively with other cadherins. In summary, these results demonstrate that Fgf signalling, and especially cooperation of the Fgf receptors, is required for proliferation, cell survival, and patterning of the neural progenitors in the midbrain and anterior hindbrain. A good understanding of developmental processes such as detailed mechanisms of signalling pathways and their regulation elucidates possibilities for therapeutic use.
  • Shmelev, Anton (Helsingin yliopisto, 2007)
    The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditions the beta-lactamase portion unfolds for translocation. Cytosolic machinery must be responsible for the unfolding. The unfolding is a prerequisite for translocation through the Sec61 channel into the lumen of the ER, where the polypeptide is again folded into a bioactive and secretion-competent conformation. Lhs1p is a member of the Hsp70 family, which functions in the conformational repair of misfolded proteins in the yeast ER. It contains Hsp70 motifs, thus it has been thought to be an ATPase, like other Hsp70 members. In order to understand its activity, authentic Lhs1p and its recombinant forms expressed in E. coli, were purified. However, no ATPase activity of Lhs1p could be detected. Nor could physical interaction between Lhs1p and activators of the ER Hsp70 chaperone Kar2p, such as the J-domain proteins Sec63p, Scj1p, and Jem1p and the nucleotide exchange factor Sil1p, be demonstrated. The domain structure of Lhs1p was modelled, and found to consist of an ATPase-like domain, a domain resembling the peptide-binding domain (PBD) of Hsp70 proteins, and a C-terminal extension. Crosslinking experiments showed that Lhs1p and Kar2p interact. The interacting domains were the C-terminal extension of Lhs1p and the ATPase domain of Kar2p, and this interaction was independent of ATPase activity of Kar2p. A model is presented where the C-terminal part of Lhs1p forms a Bag-like 3 helices bundle that might serve in the nucleotide exchange function for Kar2p in translocation and folding of secretory proteins in the ER. Exit of secretory proteins in COPII-coated vesicles is believed to be dependent of retrograde transport from the Golgi to the ER in COPI-coated vesicles. It is thought that receptors escaping to the Golgi must be recycled back to the ER exit sites to recruit cargo proteins. We found that Hsp150 leaves the ER even in the absence of functional COPI-traffic from the Golgi to the ER. Thus, an alternative, COPI-independent ER exit pathway must exists, and Hsp150 is recruited to this route. The region containing the signature guiding Hsp150 to this alternative pathway was mapped.
  • Wahlström, Gudrun (Helsingin yliopisto, 2006)
    The actin cytoskeleton is essential for a large variety of cell biological processes. Actin exists in either a monomeric or a filamentous form, and it is very important for many cellular functions that the local balance between these two actin populations is properly regulated. A large number of proteins participate in the regulation of actin dynamics in the cell, and twinfilin, one of the proteins examined in this thesis, belongs to this category. The second level of regulation involves proteins that crosslink or bundle actin filaments, thereby providing the cell with a certain shape. α-Actinin, the second protein studied, mainly acts as an actin crosslinking protein. Both proteins are conserved in organisms ranging from yeast to mammals. In this thesis, the roles of twinfilin and α-actinin in development were examined using Drosophila melanogaster as a model organism. Twinfilin is an actin monomer binding protein that is structurally related to cofilin. In vitro, twinfilin reduces actin polymerisation by sequestering actin monomers. The Drosophila twinfilin (twf) gene was identified and found to encode a protein functionally similar to yeast and mammalian twinfilins. A strong hypomorphic twf mutation was identified, and flies homozygous for this allele were viable and fertile. The adult twf mutant flies displayed reduced viability, a rough eye phenotype and severely malformed bristles. The shape of the adult bristle is determined by the actin bundles that are regularly spaced around the perimeter of the developing pupal bristles. Examination of the twf pupal bristles revealed an increased level of filamentous actin, which in turn resulted in splitting and displacement of the actin bundles. The bristle defect was rescued by twf overexpression in developing bristles. The Twinfilin protein was localised at sites of actin filament assembly, where it was required to limit actin polymerisation. A genetic interaction between twinfilin and twinstar (the gene encoding Cofilin) was detected, consistent with the model predicting that both proteins act to limit the amount of filamentous actin. α-Actinin has been implicated in several diverse cell biological processes. In Drosophila, the only function for α-actinin yet known is in the organisation of the muscle sarcomere. Muscle and non-muscle cells utilise different α-actinin isoforms, which in Drosophila are produced by alternative splicing of a single gene. In this work, novel α-actinin deletion alleles, including ActnΔ233, were generated, which specifically disrupted the transcript encoding the non-muscle α-actinin isoform. Nevertheless, ActnΔ233 homozygous mutant flies were viable and fertile with no obvious defects. By comparing α-actinin protein distribution in wild type and ActnΔ233 mutant animals, it could be concluded that non-muscle α-actinin is the only isoform expressed in young embryos, in the embryonic central nervous system and in various actin-rich structures of the ovarian germline cells. In the ActnΔ233 mutant, α-actinin was detected not only in muscle tissue, but also in embryonic epidermal cells and in certain follicle cell populations in the ovaries. The population of α-actinin protein present in non-muscle cells of the ActnΔ233 mutant is referred to as FC-α-actinin (Follicle Cell). The follicular epithelium in the Drosophila ovary is a well characterised model system for studies on patterning and morphogenesis. Therefore, α-actinin expression, regulation and function in this tissue were further analysed. Examination of the α-actinin localisation pattern revealed that the basal actin fibres of the main body follicle cells underwent an organised remodelling during the final stages of oogenesis. This involved the assembly of a transient adhesion site in the posterior of the cell, in which α-actinin and Enabled (Ena) accumulated. Follicle cells genetically manipulated to lack all α-actinin isoforms failed to remodel their cytoskeleton and translocate Ena to the posterior of the cell, while the actin fibres as such were not affected. Neither was epithelial morphogenesis disrupted. The reorganisation of the basal actin cytoskeleton was also disturbed following ectopic expression of Decapentaplegic (Dpp) or as a result of a heat shock. At late oogenesis, the main body follicle cells express both non-muscle α-actinin and FC-α-actinin, while the dorsal anterior follicle cells express only non-muscle α-actinin. The dorsal anterior cells are patterned by the Dpp and Epidermal growth factor receptor (EGFR) signalling pathways, and they will ultimately secrete the dorsal appendages of the egg. Experiments involving ectopic activation of EGFR and Dpp signalling showed that FC-α-actinin is negatively regulated by combined EGFR and Dpp signalling. Ubiquitous overexpression of the adult muscle-specific α-actinin isoform induced the formation of aberrant actin bundles in migrating follicle cells that did not normally express FC-α-actinin, provided that the EGFR signalling pathway was activated in the cells. Taken together, this work contributes new data to our knowledge of α-actinin function and regulation in Drosophila. The cytoskeletal remodelling shown to depend on α-actinin function provides the first evidence that α-actinin has a role in the organisation of the cytoskeleton in a non-muscle tissue. Furthermore, the cytoskeletal remodelling constitutes a previously undescribed morphogenetic event, which may provide us with a model system for in vivo studies on adhesion dynamics in Drosophila.
  • Nunes Bastos, Ricardo (Helsingin yliopisto, 2008)
    Eukaryotic cells are characterized by having a subset of internal membrane compartments, each one with a specifi c identity, structure and function. Proteins destined to be targeted to the exterior of the cell need to enter and progress through the secretory pathway. Transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi takes place by the selective packaging of proteins into COPII-coated vesicles at the ER membrane. Taking advantage of the extensive genetic tools available for S. cerevisiae we found that Hsp150, a yeast secretory glycoprotein, selectively exited the ER in the absence of any of the three Sec24p family members. Sec24p has been thought to be an essential component of the COPII coat and thus indispensable for exocytic membrane traffic. Next we analyzed the ability of Hsp150 to be secreted in mutants, where post-Golgi transport is temperature sensitive. We found that Hsp150 could be selectively secreted under conditions where the exocyst component Sec15p is defective. Analysis of the secretory vesicles revealed that Hsp150 was packaged into a subset of known secretory vesicles as well as in a novel pool of secretory vesicles at the level of the Golgi. Secretion of Hsp150 in the absence of Sec15p function was dependent of Mso1p, a protein capable of interacting with vesicles intended to fuse with the plasma membrane, with the SNARE machinery and with Sec1p. This work demonstrated that Hsp150 is capable of using alternative secretory pathways in ER-to-Golgi and Golgi-to-plasma membrane traffi c. The sorting signals, used at both stages of the secretory pathway, for secretion of Hsp150 were different, revealing the highly dynamic nature and spatial organization of the secretory pathway. Foreign proteins usually misfold in the yeast ER. We used Hsp150 as a carrier to assist folding and transport of heterologous proteins though the secretory pathway to the culture medium in both S. cerevisiae and P. pastoris. Using this technique we expressed Hsp150Δ-HRP and developed a staining procedure, which allowed the visualization of the organelles of the secretory pathway of S. cerevisiae.
  • Kvist, Jouni (Helsingin yliopisto, 2014)
    The glanville fritillary butterfly is an important ecological model species for habitat fragmentation, whose genetics was poorly understood. In order to expand the research of this butterfly species into the realm of functional genomics a lot genetic tools were developed. These tools were used to investigate the genetic basis of phenotypic traits that are important in the wild. Gene expression microarrays based on de novo assembled transcriptome were used to study expression differences between adult butterflies from newly established colonies and older colonies as well as gene expression variation among larval families reared in three thermal regimens during final larval instar. Colonization and larval development are crucially important in maintaining the metapopulation structure of glanville fritillary butterfly in the Åland. We identified gene expression differences than can explain the observed variation in the phenotypes in the natural population. We sequenced the full genome of the glanville fritillary butterfly and used this to do additional gene expression and allelic variation analysis variation from multiple populations around the baltic sea using rna sequencing (rna-seq). Flight induced gene expression changes were analyzed using butterflies from Åland islands and the small isolated Pieni Tytärsaari ("daughter island") populations in a forced flight experiment. Fragmented populations (Åland islands and Uppland) were compared to continuous populations (saaremaa and öland) in order to find common signatures of selection caused by habitat fragmentation. Together these four full-genome studies have revealed that habitat fragmentation causes selection pressure on an intricately connected set of genes and pathways that leads to a so called life history syndrome , where the butterflies that colonize new habitat patches have a distinct set of traits and associated expression differences in these traits that make them more successful in establishing new colonies.