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.

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.

Functional Analysis of MrpL55, Vig and Mat1 Acting at the Crossroad of Cell Cycle, Transcription and Metabolism Regulation

Cell proliferation, transcription and metabolism are regulated by complex partly overlapping signaling networks involving proteins in various subcellular compartments. The objective of this study was to increase our knowledge on such regulatory networks and their interrelationships through analysis of MrpL55, Vig, and Mat1 representing three gene products implicated in regulation of cell cycle, transcription, and metabolism. Genome-wide and biochemical in vitro studies have previously revealed MrpL55 as a component of the large subunit of the mitochondrial ribosome and demonstrated a possible role for the protein in cell cycle regulation. Vig has been implicated in heterochromatin formation and identified as a constituent of the RNAi-induced silencing complex (RISC) involved in cell cycle regulation and RNAi-directed transcriptional gene silencing (TGS) coupled to RNA polymerase II (RNAPII) transcription. Mat1 has been characterized as a regulatory subunit of cyclin-dependent kinase 7 (Cdk7) complex phosphorylating and regulating critical targets involved in cell cycle progression, energy metabolism and transcription by RNAPII. The first part of the study explored whether mRpL55 is required for cell viability or involved in a regulation of energy metabolism and cell proliferation. The results revealed a dynamic requirement of the essential Drosophila mRpL55 gene during development and suggested a function of MrpL55 in cell cycle control either at the G1/S or G2/M transition prior to cell differentiation. This first in vivo characterization of a metazoan-specific constituent of the large subunit of mitochondrial ribosome also demonstrated forth compelling evidence of the interconnection of nuclear and mitochondrial genomes as well as complex functions of the evolutionarily young metazoan-specific mitochondrial ribosomal proteins. In studies on the Drosophila RISC complex regulation, it was noted that Vig, a protein involved in heterochromatin formation, unlike other analyzed RISC associated proteins Argonaute2 and R2D2, is dynamically phosphorylated in a dsRNA-independent manner. Vig displays similarity with a known in vivo substrate for protein kinase C (PKC), human chromatin remodeling factor Ki-1/57, and is efficiently phosphorylated by PKC on multiple sites in vitro. These results suggest that function of the RISC complex protein Vig in RNAi-directed TGS and chromatin modification may be regulated through dsRNA-independent phosphorylation by PKC. In the third part of this study the role of Mat1 in regulating RNAPII transcription was investigated using cultured murine immortal fibroblasts with a conditional allele of Mat1. The results demonstrated that phosphorylation of the carboxy-terminal domain (CTD) of the large subunit of RNAPII in the heptapeptide YSPTSPS repeat in Mat-/- cells was over 10-fold reduced on Serine-5 and subsequently on Serine-2. Occupancy of the hypophosphorylated RNAPII in gene bodies was detectably decreased, whereas capping, splicing, histone methylation and mRNA levels were generally not affected. However, a subset of transcripts in absence of Mat1 was repressed and associated with decreased occupancy of RNAPII at promoters as well as defective capping. The results identify the Cdk7-CycH-Mat1 kinase submodule of TFIIH as a stimulatory non-essential regulator of transcriptional elongation and a genespecific essential factor for stable binding of RNAPII at the promoter region and capping. The results of these studies suggest important roles for both MrpL55 and Mat1 in cell cycle progression and their possible interplay at the G2/M stage in undifferentiated cells. The identified function of Mat1 and of TFIIH kinase complex in gene-specific transcriptional repression is challenging for further studies in regard to a possible link to Vig and RISC-mediated transcriptional gene silencing.

Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.