Magmatic evolution of topaz-bearing granite stocks within the Wiborg rapakivi granite batholith

This study brings new insights into the magmatic evolution of natural F-enriched peraluminous granitic systems. The Artjärvi, Sääskjärvi and Kymi granite stocks within the 1.64 Ga Wiborg rapakivi granite batholith have been investigated by petrographic, geochemical, experimental and melt inclusion methods. These stocks represent late-stage leucocratic and weakly peraluminous intrusive phases typical of rapakivi granites worldwide. The Artjärvi and Sääskjärvi stocks are multiphase intrusions in which the most evolved phase is topaz granite. The Kymi stock contains topaz throughout and has a well-developed zoned structure, from the rim to the center: stockscheider pegmatite equigranular topaz granite porphyritic topaz granite. Geochemically the topaz granites are enriched in F, Li, Be, Ga, Rb, Sn and Nb and depleted in Mg, Fe, Ti, Ba, Sr, Zr and Eu. The anomalous geochemistry and mineralogy of the topaz granites are essentially magmatic in origin; postmagmatic reactions have only slightly modified the compositions. The Kymi equigranular topaz granite shows the most evolved character, and the topaz granites at Artjärvi and Sääskjärvi resemble the less evolved porphyritic topaz granite of the Kymi stock. Stockscheiders are found at the roof contacts of the Artjärvi and Kymi stocks. The stockscheider at Artjärvi is composed of biotite-rich schlieren and pegmatite layers parallel to the contact. The schlieren layering is considered to have formed by velocity-gradient sorting mechanism parallel to the flow, which led to the accumulation of mafic minerals along the upper contact of the topaz granite. Cooling and contraction of the topaz granite formed fractures parallel to the roof contact and residual pegmatite magmas were injected along the fractures and formed the pegmatite layers. The zoned structure of the Kymi stock is the result of intrusion of highly evolved residual melt from deeper parts of the magma chamber along the fractured contact between the porphyritic granite crystal mush and country rock. The equigranular topaz granite and marginal pegmatite (stockscheider) crystallized from this evolved melt. Phase relations of the Kymi equigranular topaz granite have been investigated utilizing crystallization experiments at 100 to 500 MPa as a function of water activity and F content. Fluorite and topaz can crystallize as liquidus phases in F-rich peraluminous systems, but the F content of the melt should exceed 2.5 - 3.0 wt % to facilitate crystallization of topaz. In peraluminous F-bearing melts containing more than 1 wt % F, topaz and muscovite are expected to be the first F-bearing phases to crystallize at high pressure, whereas fluorite and topaz should crystallize first at low pressure. Overall, the saturation of fluorite and topaz follows the reaction: CaAl2Si2O8 (plagioclase) + 2[AlF3]melt = CaF2 (fluorite) + 2Al2SiO4F2 (topaz). The obtained partition coefficient for F between biotite and glass D(F)Bt/glass is 1.89 to 0.80 (average 1.29) and can be used as an empirical fluormeter to determine the F content of coexisting melts. In order to study the magmatic evolution of the Kymi stock, crystallized melt inclusions in quartz and topaz grains in the porphyritic and the equigranular topaz granites and the marginal pegmatite were rehomogenized and analyzed. The homogenization conditions for the melt inclusions from the granites were 700 °C, 300 MPa, and 24 h, and for melt inclusions from the pegmatite, 700 °C, 100 MPa, and 24/96 h. The majority of the melt inclusions is chemically similar to the bulk rocks (excluding H2O content), but a few melt inclusions in the equigranular granite show clearly higher F and low K2O contents (on average 11.6 wt % F, 0.65 wt % K2O). The melt inclusion compositions indicate coexistence of two melt fractions, a prevailing peraluminous and a very volatile-rich, possibly peralkaline. Combined petrological, experimental and melt inclusion studies of the Kymi equigranular topaz granite indicate that plagioclase was the liquidus phase at nearly water-saturated (fluid-saturated) conditions and that the F content of the melt was at least 2 wt %. The early crystallization of biotite and the presence of muscovite in crystallization experiments at 200 MPa contrasts with the late-stage crystallization of biotite and the absence of muscovite in the equigranular granite, indicating that crystallization pressure may have been lower than 200 MPa for the granite.

Boundedness and convergence of singular integrals on fractal type sets

The topic of this dissertation lies in the intersection of harmonic analysis and fractal geometry. We particulary consider singular integrals in Euclidean spaces with respect to general measures, and we study how the geometric structure of the measures affects certain analytic properties of the operators. The thesis consists of three research articles and an overview. In the first article we construct singular integral operators on lower dimensional Sierpinski gaskets associated with homogeneous Calderón-Zygmund kernels. While these operators are bounded their principal values fail to exist almost everywhere. Conformal iterated function systems generate a broad range of fractal sets. In the second article we prove that many of these limit sets are porous in a very strong sense, by showing that they contain holes spread in every direction. In the following we connect these results with singular integrals. We exploit the fractal structure of these limit sets, in order to establish that singular integrals associated with very general kernels converge weakly. Boundedness questions consist a central topic of investigation in the theory of singular integrals. In the third article we study singular integrals of different measures. We prove a very general boundedness result in the case where the two underlying measures are separated by a Lipshitz graph. As a consequence we show that a certain weak convergence holds for a large class of singular integrals.

Composition operators on vector-valued BMOA and related function spaces

A composition operator is a linear operator between spaces of analytic or harmonic functions on the unit disk, which precomposes a function with a fixed self-map of the disk. A fundamental problem is to relate properties of a composition operator to the function-theoretic properties of the self-map. During the recent decades these operators have been very actively studied in connection with various function spaces. The study of composition operators lies in the intersection of two central fields of mathematical analysis; function theory and operator theory. This thesis consists of four research articles and an overview. In the first three articles the weak compactness of composition operators is studied on certain vector-valued function spaces. A vector-valued function takes its values in some complex Banach space. In the first and third article sufficient conditions are given for a composition operator to be weakly compact on different versions of vector-valued BMOA spaces. In the second article characterizations are given for the weak compactness of a composition operator on harmonic Hardy spaces and spaces of Cauchy transforms, provided the functions take values in a reflexive Banach space. Composition operators are also considered on certain weak versions of the above function spaces. In addition, the relationship of different vector-valued function spaces is analyzed. In the fourth article weighted composition operators are studied on the scalar-valued BMOA space and its subspace VMOA. A weighted composition operator is obtained by first applying a composition operator and then a pointwise multiplier. A complete characterization is given for the boundedness and compactness of a weighted composition operator on BMOA and VMOA. Moreover, the essential norm of a weighted composition operator on VMOA is estimated. These results generalize many previously known results about composition operators and pointwise multipliers on these spaces.

On the Rademacher maximal function

Tools known as maximal functions are frequently used in harmonic analysis when studying local behaviour of functions. Typically they measure the suprema of local averages of non-negative functions. It is essential that the size (more precisely, the L^p-norm) of the maximal function is comparable to the size of the original function. When dealing with families of operators between Banach spaces we are often forced to replace the uniform bound with the larger R-bound. Hence such a replacement is also needed in the maximal function for functions taking values in spaces of operators. More specifically, the suprema of norms of local averages (i.e. their uniform bound in the operator norm) has to be replaced by their R-bound. This procedure gives us the Rademacher maximal function, which was introduced by Hytönen, McIntosh and Portal in order to prove a certain vector-valued Carleson's embedding theorem. They noticed that the sizes of an operator-valued function and its Rademacher maximal function are comparable for many common range spaces, but not for all. Certain requirements on the type and cotype of the spaces involved are necessary for this comparability, henceforth referred to as the “RMF-property”. It was shown, that other objects and parameters appearing in the definition, such as the domain of functions and the exponent p of the norm, make no difference to this. After a short introduction to randomized norms and geometry in Banach spaces we study the Rademacher maximal function on Euclidean spaces. The requirements on the type and cotype are considered, providing examples of spaces without RMF. L^p-spaces are shown to have RMF not only for p greater or equal to 2 (when it is trivial) but also for 1 < p < 2. A dyadic version of Carleson's embedding theorem is proven for scalar- and operator-valued functions. As the analysis with dyadic cubes can be generalized to filtrations on sigma-finite measure spaces, we consider the Rademacher maximal function in this case as well. It turns out that the RMF-property is independent of the filtration and the underlying measure space and that it is enough to consider very simple ones known as Haar filtrations. Scalar- and operator-valued analogues of Carleson's embedding theorem are also provided. With the RMF-property proven independent of the underlying measure space, we can use probabilistic notions and formulate it for martingales. Following a similar result for UMD-spaces, a weak type inequality is shown to be (necessary and) sufficient for the RMF-property. The RMF-property is also studied using concave functions giving yet another proof of its independence from various parameters.

Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

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.

GDNF family receptors in peripheral target innervation and hormone production

Glial cell line-derived neurotrophic factor (GDNF) family ligands: GDNF, neurturin, persephin and artemin, signal through a receptor tyrosine kinase Ret by binding first to a co-receptor (GFRα1-4) that is attached to the plasma membrane. The GDNF family factors can support the survival of various peripheral and central neuronal populations and have important functions also outside the nervous system, especially in kidney development. Activating mutations in the RET gene cause tumours in neuroendocrine cells, whereas inactivating mutations in RET are found in patients with Hirschsprung s disease (HSCR) characterized by loss of ganglionic cells along the intestine. The aim of this study was to examine the in vivo functions of neurturin receptor GFRα2 and persephin receptor GFRα4 using knockout (KO) mice. Mice lacking GFRα2 grow poorly after weaning and have deficits in parasympathetic and enteric innervation. This study shows that impaired secretion of the salivary glands and exocrine pancreas contribute to growth retardation in GFRα2-KO mice. These mice have a reduced number of intrapancreatic neurons and decreased cholinergic innervation of the exocrine pancreas as well as reduced excitatory fibres in the myenteric plexus of the small intestine. This study also demonstrates that GFRα2-mediated Ret signalling is required for target innervation and maintenance of soma size of sympathetic cholinergic neurons and sensory nociceptive IB4-binding neurons. Furthermore, lack of GFRα2 in mice results in deficient perception of temperatures above and below thermoneutrality and in attenuated inflammatory pain response. GFRα4 is co-expressed with Ret predominantly in calcitonin-producing thyroid C-cells in the mouse. In this study GFRα4-deficient mice were generated. The mice show no gross developmental deficits and have a normal number of C-cells. However, young but not adult mice lacking GFRα4 have a lower production of calcitonin in thyroid tissue and consequently, an increased bone formation rate. Thus, GFRα4/Ret signalling may regulate calcitonin production. In conclusion, this study reveals that GFRα2/Ret signalling is crucial for the development and function of specific components of the peripheral nervous system and that GFRα4-mediated Ret signalling is required for controlling transmitter synthesis in thyroid C-cells.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.

Causes and consequences of variation in nestling immune function

Defence against pathogens is a vital need of all living organisms that has led to the evolution of complex immune mechanisms. However, although immunocompetence the ability to resist pathogens and control infection has in recent decades become a focus for research in evolutionary ecology, the variation in immune function observed in natural populations is relatively little understood. This thesis examines sources of this variation (environmental, genetic and maternal effects) during the nestling stage and its fitness consequences in wild populations of passerines: the blue tit (Cyanistes caeruleus) and the collared flycatcher (Ficedula albicollis). A developing organism may face a dilemma as to whether to allocate limited resources to growth or to immune defences. The optimal level of investment in immunity is shaped inherently by specific requirements of the environment. If the probability of contracting infection is low, maintaining high growth rates even at the expense of immune function may be advantageous for nestlings, as body mass is usually a good predictor of post-fledging survival. In experiments with blue tits and haematophagous hen fleas (Ceratophyllus gallinae) using two methods, methionine supplementation (to manipulate nestlings resource allocation to cellular immune function) and food supplementation (to increase resource availability), I confirmed that there is a trade-off between growth and immunity and that the abundance of ectoparasites is an environmental factor affecting allocation of resources to immune function. A cross-fostering experiment also revealed that environmental heterogeneity in terms of abundance of ectoparasites may contribute to maintaining additive genetic variation in immunity and other traits. Animal model analysis of extensive data collected from the population of collared flycatchers on Gotland (Sweden) allowed examination of the narrow-sense heritability of PHA-response the most commonly used index of cellular immunocompetence in avian studies. PHA-response is not heritable in this population, but is subject to a non-heritable origin (presumably maternal) effect. However, experimental manipulation of yolk androgen levels indicates that the mechanism of the maternal effect in PHA-response is not in ovo deposition of androgens. The relationship between PHA-response and recruitment was studied for over 1300 collared flycatcher nestlings. Multivariate selection analysis shows that it is body mass, not PHA-response, that is under direct selection. PHA-response appears to be related to recruitment because of its positive relationship with body mass. These results imply that either PHA-response fails to capture the immune mechanisms that are relevant for defence against pathogens encountered by fledglings or that the selection pressure from parasites is not as strong as commonly assumed.

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.

Effects of recreational use and fragmentation on the understorey vegetation and soil microbial communities of urban forests in southern Finland

The impacts of fragmentation and recreational use on the hemiboreal urban forest understorey vegetation and the microbial community of the humus layer (the phospholipid fatty acid (PLFA) pattern, microbial biomass and microbial activity, measured as basal respiration) were examined in the greater Helsinki area, southern Finland. Trampling tolerance of 1) herb-rich OMT, 2) mesic MT, and 3) sub-xeric VT forests (in decreasing order of fertility) was studied by comparing relative understorey vegetation cover (urban/untrampled reference ratio) of the three forest types. The trampling tolerance of forest vegetation increased with the productivity of the site (sub-xeric < mesic < herb-rich). Wear of understorey vegetation correlated positively with the number of residents (i.e., recreational pressure) around the forest patch. An increase of 15000 residents within a radius of 1 km around a forest patch was associated with ca. 30% decrease in the relative understorey vegetation cover. The cover of dwarf shrub Vaccinium myrtillus in particular decreased with increasing levels of wear. The cover of mosses in urban forests was less than half of that in untrampled reference areas. Cover of tree saplings, mainly Sorbus aucuparia, and some resilient herbs was higher than in the reference areas. In small urban forest fragments, broad-leaved trees, grasses and herbs were more abundant and mosses were scarcer than in larger urban forest areas. Thus, due to trampling and edge effects, resilient herb and grass species are replacing sensitive dwarf shrubs, mosses and lichens in urban forests. Differences in the soil microbial community structure were found between paths and untrampled areas and the effects of paths extended more than one meter from the paths. Paths supported approximately 25-30% higher microbial biomass with a transition zone of at least 1 m from the path edge. However, microbial activity per unit of biomass was lower on paths than in untrampled areas. Furthermore, microbial biomass and activity were 30-45% lower at the first 20 m into the forest fragments, due to low moisture content of humus near the edge. The decreased microbial activity detected at forest edges and paths implies decreased litter decomposition rates, and thus, a change in nutrient cycling. Changes in the decomposition and nutrient supply may in turn affect the diversity and function of plant communities in urban forests. Keywords: boreal forest vegetation, edge effects, phospholipid fatty acids, trampling, urban woodlands, wear

The biological functions of mouse twinfilin isoforms

The actin cytoskeleton is essential for many cellular processes, including motility, morphogenesis, endocytosis and signal transduction. Actin can exist in monomeric (G-actin) or filamentous (F-actin) form. Actin filaments are considered to be the functional form of actin, generating the protrusive forces characteristic for the actin cytoskeleton. The structure and dynamics of the actin filament and monomer pools are regulated by a large number of actin-binding proteins in eukaryotic cells. Twinfilin is an evolutionarily conserved small actin monomer binding protein. Twinfilin is composed of two ADF/cofilin-like domains, separated by a short linker and followed by a C-terminal tail. Twinfilin forms a stable, high affinity complex with ADP-G-actin, inhibits the nucleotide exchange on actin monomers, and prevents their assembly into filament ends. Twinfilin was originally identified from yeast and has since then been found from all organisms studied except plants. Not much was known about the role of twinfilin in the actin dynamics in mammalian cells before this study. We set out to unravel the mysteries still covering twinfilins functions using biochemistry, cell biology, and genetics. We identified and characterized two mouse isoforms for the previously identified mouse twinfilin-1. The new isoforms, twinfilin-2a and -2b, are generated from the same gene through alternative promoter usage. The three isoforms have distinctive expression patterns, but are similar biochemically. Twinfilin-1 is the major isoform during development and is expressed in high levels in almost all tissues examined. Twinfilin-2a is also expressed almost ubiquitously, but at lower levels. Twinfilin-2b turned out to be a muscle-specific isoform, with very high expression in heart and skeletal muscle. It seems all mouse tissues express at least two twinfilin isoforms, indicating that twinfilins are important regulators of actin dynamics in all cell and tissue types. A knockout mouse line was generated for twinfilin-2a. The mice homozygous for this knockout were viable and developed normally, indicating that twinfilin-2a is dispensable for mouse development. However, it is important to note that twinfilin-2a shows similar expression pattern to twinfilin-1, suggesting that these proteins play redundant roles in mice. All mouse isoforms were shown to be able to sequester actin filaments and have higher affinity for ADP-G-actin than ATP-G-actin. They are also able to directly interact with heterodimeric capping protein and PI(4,5)P2 similar to yeast twinfilin. In this study we also uncovered a novel function for mouse twinfilins; capping actin filament barbed ends. All mouse twinfilin isoforms were shown to possess this function, while yeast and Drosophila twinfilin were not able to cap filament barbed ends. Twinfilins localize to the cytoplasm but also to actin-rich regions in mammalian cells. The subcellular localizations of the isoforms are regulated differently, indicating that even though twinfilins biochemical functions in vitro are very similar, in vivo they can play different roles through different regulatory pathways. Together, this study show that twinfilins regulate actin filament assembly both by sequestering actin monomers and by capping filament barbed ends, and that mammals have three biochemically similar twinfilin isoforms with partially overlapping expression patterns.

Type III Secretion System of Phytopathogenic Bacterium Pseudomonas syringae:From Gene to Function

The type III secretion system (T3SS) is an essential requirement for the virulence of many Gram-negative bacteria which infect plants, animals and men. Pathogens use the T3SS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cells, where the effectors subvert host defenses. The best candidates for directing effector protein traffic are the bacterial type III-associated appendages, called needles or pili. In plant pathogenic bacteria, the best characterized example of a T3SS-associated appendage is the HrpA pilus of the plant pathogen Pseudomonas syringae pv. tomato DC3000. The components of the T3SS in plant pathogens are encoded by a cluster of hrp (hypersensitive reaction and pathogenicity) genes. Two major classes of T3SS-secreted proteins are: harpin proteins such as HrpZ which are exported into extracellular space, and avirulence (Avr) proteins such as AvrPto which are translocated directly to the plant cytoplasm. This study deals with the structural and functional characterization of the T3SS-associated HrpA pilus and the T3SS-secreted harpins. By insertional mutagenesis analysis of HrpA, we located the optimal epitope insertion site in the amino-terminus of HrpA, and revealed the potential application of the HrpA pilus as a carrier of antigenic determinants for vaccination. By pulse-expression of proteins combined with immuno-electron microscopy, we discovered the Hrp pilus assembly strategy as addition of HrpA subunits to the distal end of the growing pilus, and we showed for the first time that secretion of HrpZ occurs at the tip of the pilus. The pilus thus functions as a conduit delivering proteins to the extracellular milieu. By using phage-display and scanning-insertion mutagenesis methods we identified a conserved HrpZ-binding peptide and localized the peptide-binding site to the central domain of HrpZ. We also found that the HrpZ specifically interacts with a host bean protein. Taken together, the current results provide deeper insight into the molecular mechanism of T3SS-associated pilus assembly and effector protein translocation, which will be helpful for further studies on the pathogenic mechanisms of Gram-negative bacteria and for developing new strategies to prevent bacterial infection.