Functional dissection of alternative secretory pathways in the yeast S. cerevisiae

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.

Regulation of Growth by Drosophila FoxO Transcription Factor

Forkhead box class O (FoxO) transcription factors are members of the forkhead box transcription factor superfamily, with orthologues in various species such as human, worm and fly. FoxO proteins are key regulators of growth, metabolism, stress resistance and, consequently, life span. FoxOs integrate signals from different pathways, e.g. the growth controlling Insulin-TOR signaling pathway and the stress induced JNK and Hippo signaling pathways. FoxO proteins have evolved to guide the cellular response to varying energy and stress conditions by inducing the expression of genes involved in the regulation of growth and metabolism. This work has aimed to deepen the understanding of how FoxO executes its biological functions. A particular emphasis has been laid to its role in growth control. Specifically, evidence is presented indicating that FoxO restricts tissue growth in a situation when TOR signaling is high. This finding can have implications in a human condition called Tuberous sclerosis, manifested by multiple benign tumors. Further, it is shown that FoxO directly binds to the promoter and regulates the expression of a Drosophila Adenylate cyclase gene, ac76e, which in turn modulates the fly s development and growth systemically. These results strengthen FoxOs position among central size regulators as it is able to operate at the level of individual cells as well as in the whole organism. Finally, an attempt to reveal the regulatory network upstream of FoxO has been carried out. Several putative FoxO activity regulators were identified in an RNAi screen of Drosophila kinases and phosphatases. The results underscore that FoxO is regulated through an elaborate network, ensuring the correct execution of key cellular processes in metabolism and response to stress. Overall, the evidence provided in this study strengthens our view of FoxO as a key integrator of growth and stress signals.

Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

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.

The role of cyclase-associated protein (CAP) in actin dynamics during cell motility and morphogenesis

The highly dynamic remodeling of the actin cytoskeleton is responsible for most motile and morphogenetic processes in all eukaryotic cells. In order to generate appropriate spatial and temporal movements, the actin dynamics must be under tight control of an array of actin binding proteins (ABPs). Many proteins have been shown to play a specific role in actin filament growth or disassembly of older filaments. Very little is known about the proteins affecting recycling i.e. the step where newly depolymerized actin monomers are funneled into new rounds of filament assembly. A central protein family involved in the regulation of actin turnover is cyclase-associated proteins (CAP, called Srv2 in budding yeast). This 50-60 kDa protein was first identified from yeast as a suppressor of an activated RAS-allele and a factor associated with adenylyl cyclase. The CAP proteins harbor N-terminal coiled-coil (cc) domain, originally identified as a site for adenylyl cyclase binding. In the N-terminal half is also a 14-3-3 like domain, which is followed by central proline-rich domains and the WH2 domain. In the C-terminal end locates the highly conserved ADP-G-actin binding domain. In this study, we identified two previously suggested but poorly characterized interaction partners for Srv2/CAP: profilin and ADF/cofilin. Profilins are small proteins (12-16 kDa) that bind ATP-actin monomers and promote the nucleotide exchange of actin. The profilin-ATP-actin complex can be directly targeted to the growth of the filament barbed ends capped by Ena/VASP or formins. ADF/cofilins are also small (13-19 kDa) and highly conserved actin binding proteins. They depolymerize ADP-actin monomers from filament pointed ends and remain bound to ADP-actin strongly inhibiting nucleotide exchange. We revealed that the ADP-actin-cofilin complex is able to directly interact with the 14-3-3 like domain at the N-terminal region of Srv2/CAP. The C-terminal high affinity ADP-actin binding site of Srv2/CAP competes with cofilin for an actin monomer. Cofilin can thus be released from Srv2/CAP for the subsequent round of depolymerization. We also revealed that profilin interacts with the first proline-rich region of Srv2/CAP and that the binding occurs simultaneously with ADP-actin binding to C-terminal domain of Srv2/CAP. Both profilin and Srv2/CAP can promote nucleotide exchange of actin monomer. Because profilin has much higher affinity to ATP-actin than Srv2/CAP, the ATP-actin-profilin complex is released for filament polymerization. While a disruption of cofilin binding in yeast Srv2/CAP produces a severe phenotype comparable to Srv2/CAP deletion, an impairment of profilin binding from Srv2/CAP results in much milder phenotype. This suggests that the interaction with cofilin is essential for the function of Srv2/CAP, whereas profilin can also promote its function without direct interaction with Srv2/CAP. We also show that two CAP isoforms with specific expression patterns are present in mice. CAP1 is the major isoform in most tissues, while CAP2 is predominantly expressed in muscles. Deletion of CAP1 from non-muscle cells results in severe actin phenotype accompanied with mislocalization of cofilin to cytoplasmic aggregates. Together these studies suggest that Srv2/CAP recycles actin monomers from cofilin to profilin and thus it plays a central role in actin dynamics in both yeast and mammalian cells.

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

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.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

GDNF Receptors: Veterans and Novices

Neurotrophic factors play essential role in the development and functioning of the nervous system and other organs. Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands (GFLs) are of particular interest because they promote the survival of dopaminergic neurons in vitro, in Parkinson s disease animal models and in patients. GDNF is also a potent survival factor for the central motoneurons and thus is considered as a potential lead for the treatment of amyotrophic lateral sclerosis. The survival promoting receptor complex for GFLs consists of a ligand-specific co-receptor, GFRα and a signal transducing module, receptor tyrosine kinase RET. At least GDNF and persephin, a GFL, have established functions outside central nervous system. GDNF is crucial for enteric nervous system and kidney development as well as for spermatogenesis. Persephin controls calcitonin secretion. Communication between cells often occurs in the extracellular matrix (ECM), a meshwork, which is secreted and deposited by the cells and is mainly composed of fibrillar proteins and polymerized sugars. We evaluated the relationship between GFLs and extracellular matrix components and demonstrated that three GFLs - GDNF, neurturin and artemin bind heparan sulfates with nanomolar affinities. The fourth member of the family - persephin binds these polysaccharides thousand times less tightly. GDNF, neurturin and artemin also bind with high affinity to heparan sulfate proteoglycan (HSPG) isolated from the nervous system, syndecan-3. GDNF signals through HSPGs, evoking Src family kinase activation. This signaling induces cell spreading, hippocampal neurite outgrowth in vitro and cellular migration. Specifically, GDNF signaling through syndecan-3 is important for embryonic cortical neuron migration. Syndecan-3-deficient mice, similarly to mice lacking GDNF, have less GABAergic neurons in their cortex, as compared to the wild-type mice. This fact provides indirect evidence that GDNF interaction with syndecan-3 is important for cortical brain development. Noteworthy, in non-neuronal tissues GFLs may signal via other syndecans. We also present the structural model for a GDNF co-receptor, GFRα1. The X-ray structure of the GFRα1 domain 3 was solved with 1.8 Å resolution, revealing a new protein fold. Later we also solved the structure of the truncated GFRα1 in the complex with GDNF and this model was confirmed by site-directed mutagenesis. In summary, our work contributed to the structural characterization of GFRα-based receptor complex and revealed a new receptor for GDNF, neurturin and artemin the HSPG syndecan-3. This information is critically important for the development of GFRα/RET agonists for the treatment of neurodegenerative diseases.

Structural basis of cytoskeletal regulation by twinfilin

The actin cytoskeleton is required, in all eukaryotic organisms, for several key cellular functions such as cell motility, cytokinesis, and endocytosis. In cells, actin exists either in a monomeric state (G-actin) or in a filamentous form (F-actin). F-actin is the functional form, which can assemble into various structures and produce direct pushing forces that are required for different motile processes. The assembly of actin monomers into complicated three-dimensional structures is tightly regulated by a large number of actin regulating proteins. One central actin regulating protein is twinfilin. Twinfilin consists of two actin depolymerizing-factor homology (ADF-H) domains, which are capable of binding actin, and is conserved from yeast to mammals. Previously it has been shown that twinfilin binds to and sequesters G-actin, and interacts with the heterodimeric capping protein. More recently it has been found that twinfilin also binds to the fast growing actin filament ends and prevents their growth. However, the cellular role of twinfilin and the molecular mechanisms of these interactions have remained unclear. In this study we characterized the molecular mechanisms behind the functions of twinfilin. We demonstrated that twinfilin forms a high-affinity complex with ADP-bound actin monomers (ADP-G-actin). Both ADF-H domains are capable of binding G-actin, but the C-terminal domain contains the high-affinity binding site. Our biochemical analyses identified twinfilin s C-terminal tail region as the interaction site for capping protein. Contrary to G-actin binding, both ADF-H domains of twinfilin are required for the actin filament barbed end capping activity. The C-terminal domain is structurally homologous to ADF/cofilin and binds to filament sides in a similar manner, providing the main affinity for F-actin during barbed end capping. The structure of the N-terminal domain is more distant from ADF/cofilin, and thus it can only associate with G-actin or the terminal actin monomer at the filament barbed end, where it regulates twinfilin s affinity for barbed ends. These data suggest that the mechanism of barbed end capping is similar for twinfilin and gelsolin family proteins. Taken together, these studies revealed how twinfilin interacts with G-actin, filament barbed ends, and capping protein, and also provide a model for how these activities evolved through a duplication of an ancient ADF/cofilin-like domain.

From actin monomers to bundles: The roles of twinfilin and α-actinin in Drosophila melanogaster development

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.

Gene Expression : From Microarrays to Functional Genomics

The time of the large sequencing projects has enabled unprecedented possibilities of investigating more complex aspects of living organisms. Among the high-throughput technologies based on the genomic sequences, the DNA microarrays are widely used for many purposes, including the measurement of the relative quantity of the messenger RNAs. However, the reliability of microarrays has been strongly doubted as robust analysis of the complex microarray output data has been developed only after the technology had already been spread in the community. An objective of this study consisted of increasing the performance of microarrays, and was measured by the successful validation of the results by independent techniques. To this end, emphasis has been given to the possibility of selecting candidate genes with remarkable biological significance within specific experimental design. Along with literature evidence, the re-annotation of the probes and model-based normalization algorithms were found to be beneficial when analyzing Affymetrix GeneChip data. Typically, the analysis of microarrays aims at selecting genes whose expression is significantly different in different conditions followed by grouping them in functional categories, enabling a biological interpretation of the results. Another approach investigates the global differences in the expression of functionally related groups of genes. Here, this technique has been effective in discovering patterns related to temporal changes during infection of human cells. Another aspect explored in this thesis is related to the possibility of combining independent gene expression data for creating a catalog of genes that are selectively expressed in healthy human tissues. Not all the genes present in human cells are active; some involved in basic activities (named housekeeping genes) are expressed ubiquitously. Other genes (named tissue-selective genes) provide more specific functions and they are expressed preferably in certain cell types or tissues. Defining the tissue-selective genes is also important as these genes can cause disease with phenotype in the tissues where they are expressed. The hypothesis that gene expression could be used as a measure of the relatedness of the tissues has been also proved. Microarray experiments provide long lists of candidate genes that are often difficult to interpret and prioritize. Extending the power of microarray results is possible by inferring the relationships of genes under certain conditions. Gene transcription is constantly regulated by the coordinated binding of proteins, named transcription factors, to specific portions of the its promoter sequence. In this study, the analysis of promoters from groups of candidate genes has been utilized for predicting gene networks and highlighting modules of transcription factors playing a central role in the regulation of their transcription. Specific modules have been found regulating the expression of genes selectively expressed in the hippocampus, an area of the brain having a central role in the Major Depression Disorder. Similarly, gene networks derived from microarray results have elucidated aspects of the development of the mesencephalon, another region of the brain involved in Parkinson Disease.

The genome packaging machinery of dsDNA bacteriophage PRD1

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

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.