JNK, a versatile regulator of kinesin-1 transport and cytoskeleton dynamics

C-Jun N-terminal kinase (JNK) is traditionally recognized as a crucial factor in stress response and inducer of apoptosis upon various stimulations. Three isoforms build the JNK subfamily of MAPK; generally expressed JNK1 and JNK2 and brain specific JNK3. Degenerative potency placed JNK in the spotlight as potential pharmacological option for intervention. Unfortunately, adverse effects of potential drugs and observation that expression of only JNK2 and JNK3 are induced upon stress, restrained initial enthusiasm. Notably, JNK1 demonstrated atypical high constitutive activity in neurons that is not responsive to cellular stresses and indicated existence of physiological activity. This thesis aimed at revealing the physiological functions of JNK1 in actin homeostasis through novel effector MARCKS-Like 1 (MARCKSL1) protein, neuronal trafficking mediated by major kinesin-1 motor protein and microtubule (MT) dynamics via STMN2/SCG10. The screen for novel physiological JNK substrates revealed specific phosphorylation of C-terminal end of MARCKSL1 at S120, T148 and T183 both ex vivo and in vitro. By utilizing site-specific mutagenesis, various actin dynamics and migrations assays we were able to demonstrate that JNK1 phosphorylation specifically facilitates F-actin bundling and thus filament stabilisation. Consecutively, this molecular mechanism was proved to enhance formation of filopodia; cell surface projections that allow cell sensing surrounding environment and migrate efficiently. Our results visualize JNK dependent and MARCKSL1 executed induction of filopodia in neurons and fibroblast indicating general mechanism. Subsequently, inactivation of JNK action on MARCKSL1 shifts cellular actin machinery into lamellipodial dynamic arrangement. Tuning of actin cytoskeleton inevitably melds with cell migration. We observed that both active JNK and JNK pseudo-phosphorylated form of MARCKSL1 reduce actin turnover in intact cells leading to overall diminished cell motility. We demonstrate that tumour transformed cells from breast, prostate, lung and muscle-derived cancers upregulate MARCKSL1. We showed on the example of prostate cancer PC-3 cell line that JNK phosphorylation negatively controls MARCKSL1 ability to induce migration, which precedes cancer cell metastasis. The second round of identification of JNK physiological substrates resulted in detection of predominant motor protein kinesin-1 (Kif5). Mass spectrometry detailed analysis showed evident endogenous phosphorylation of kinesin-1 on S176 within motor domain that interacts with MT. In vitro phosphorylation of bacterially expressed kinesin heavy chain by JNK isoforms displayed higher specificity of JNK1 when compared to JNK3. Since, JNK1 is constitutively active in neurons it signified physiological aspect of kinesin-1 regulation. Subsequent biochemical examination revealed that kinesin-1, when not phosphorylated on JNK site, exhibits much higher affinity toward MTs. Expression of the JNK non-phosphorable kinesin-1 mutant in intact cells as well as in vitro single molecule imaging using total internal reflection fluorescence microscopy indicated that the mutant loses normal speed and is not able to move processively into proper cellular compartments. We identify novel kinesin-1 cargo protein STMN2/SCG10, which along with known kinesin-1 cargo BDNF is showing impaired trafficking when JNK activity is inhibited. Our data postulates that constitutive JNK activity in neurons is crucial for unperturbed physiologically relevant transport of kinesin-1 dependant cargo. Additionally, my work helps to validate another novel physiological JNK1 effector STMN2/SCG10 as determinant of axodendritic neurites dynamics in the developing brain through regulation of MT turnover. We show successively that this increased MT dynamics is crucial during developmental radial migration when brain layering occurs. Successively, we are able to show that introduction of JNK phosphorylation mimicking STMN2/SCG10 S62/73D mutant rescues completely JNK1 genetic deletion migration phenotype. We prove that STMN2/SCG10 is predominant JNK effector responsible for MT depolymerising activity and neurite length during brain development. Summarizing, this work describes identification of three novel JNK substrates MARCKSL1, kinesin-1 and STMN2/SCG10 and investigation of their roles in cytoskeleton dynamics and cargo transport. This data is of high importance to understand physiological meaning of JNK activity, which might have an adverse effect during pharmaceutical intervention aiming at blocking pathological JNK action.

Integrins on the move

Cancer is a leading cause of death worldwide accounting for 13% of all deaths in 2005. The spread of cancer and formation of metastases is the major cause of mortality among cancer patients. The spread of cancer is based on the cancer cell’s ability to break away from the surrounding tissue and to migrate into new areas in the body. The ability of cells to bind its surroundings and to move is controlled by the mechanical cell surface adhesion receptors called the integrins. Integrins have a critical role in cell adhesion, cell motility and tissue homeostasis. By communicating with ECM, integrins transmit signals from the surrounding environment inside the cell and modulate the function of many important signalling pathways involved in cell survival, development, gene expression, proliferation, motility and cytoskeletal organization. During cell migration integrin-matrix adhesions are formed in front of the cell while rear-adhesions are released during migration. Integrins are endocytosed from the plasma-membrane into the cytoplasm and partly recycled back to new adhesion sites in a process called integrin trafficking. Also, the cell cytoskeleton and protrusions are important in cell migration. Finger-like actin protrusions called filopodia display an interesting cancer relevant cooperation with integrins that is required for cell migration. The expression and function of integrins changes markedly as cells acquire carcinogenic properties. Changed integrin function is partly responsible for detachment of tumor cells from neighbouring cells and for providing enhanced invasive capabilities for tumor cells to disseminate. Similarly, the formation of filopodia is increased in cancer. High myosin-10 expression is related to poor outcome in breast cancer and increased cell migration. The proper function of myosin-10 induced filopodia needs association with β1 integrins. The importance of integrin trafficking and filopodia formation is becoming increasingly more recognized in cancer. This thesis focusses on the role of integrins, integrin trafficking and myosin-10 induced filopodia cancer cell migration.

Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

Regulation of cell fate by c-FLIP phosphorylation

Programmed cell death is an important physiological cellular process that maintains homeostasis and protects multicellular organisms from diseases. Apoptosis is the principal mode of cell death, which eliminates unwanted cells and an enormous effort has been made to understand the molecular mechanisms of the signaling pathway and its regulatory systems. Irregular apoptosis often has life-threatening consequences to humans, including cancer, autoimmune diseases and degenerative diseases. In cancer for example, cell death is an attractive target to eradicate uncontrollably proliferating cells that have disregard pro-apoptotic signaling. Targeted therapeutic approaches are not as effective as once expected, since now we know that the cell death pathways are not sole entities in cells, but are highly associated with various cellular processes. Proteins that regulate apoptosis can also control non-apoptotic signaling pathways. For example, c-FLIP is a protein that can either inhibit or promote caspase-8 activation, which is required to induce apoptosis. Not only has c-FLIP opposing effects on initiating apoptosis, but it also regulates various pro-survival signaling pathways in the cell. It is well known that protein expression level is a determinant of how c-FLIP can regulate different signaling pathways, but other regulatory mechanisms potentially affecting the role of c-FLIP are less well understood. This work addresses novel insights into the mechanisms of c-FLIP post-translational modifications and their functional consequences. We have identified that phosphorylation is an important inception for subcellular localization of c-FLIP, thereby dictating which apoptotic and non-apoptotic signaling pathways c-FLIP could regulate to promote cell survival. Furthermore, we have constructed mathematical models to unite independent studies to establish more systematic c-FLIP signaling pathways to understand the dynamics of extrinsically-induced apoptosis.

Virulence properties of Aggregatibacter actinomycetemcomitans biofilm and characterisation of its putative cytokine exploitation

Biofilms are surface-attached multispecies microbial communities that are embedded by their self-produced extracellular polymeric substances. This lifestyle enhances the survival of the bacteria and plays a major role in many chronic bacterial infections. For instance, periodontitis is initiated by multispecies biofilms. The phases of active periodontal tissue destruction and notably increased levels of proinflammatory mediators, such as the key inflammatory mediator interleukin (IL)-1beta, are typical of the disease. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans is usually abundant at sites of aggressive periodontitis. Despite potent host immune system responses to subgingival invaders, A. actinomycetemcomitans is able to resist clearance attempts. Moreover, some strains of A. actinomycetemcomitans can generate genetic diversity through natural transformation, which may improve the species’ adjustment tothe subgingival environment in the long term. Some biofilm forming species are known to bind and sense human cytokines. As a response to cytokines, bacteria may increase biofilm formation and alter their expression of virulence genes. Specific outer membrane receptors for interferon-γ or IL-1β have been characterised in two Gram-negative pathogens. Because little is known about periodontal pathogens’ ability to sense cytokines, we used A. actinomycetemcomitans as a model organism to investigate how the species responds to IL-1beta. The main aims of this thesis were to explore cytokine binding on single-species A. actinomycetemcomitans biofilms and to determine the effects of cytokines on the biofilm formation and metabolic activity of the species. Additionally, the cytokine’s putative internalisation and interaction with A. actinomycetemcomitans proteins were studied. The possible impact of biofilm IL-1beta sequestering on the proliferation and apoptosis of gingival keratinocyte cells was evaluated in an organotypic mucosa co-culture model. Finally, the role of the extramembranous domain of the outer membrane protein HofQ (emHofQ) in DNA binding linked to DNA uptake in A. actinomycetemcomitans was examined. Our main finding revealed that viable A. actinomycetemcomitans biofilms can bind and take up the IL-1β produced by gingival cells. At the sites of pathogen-host interaction, the proliferation and apoptosis of gingival keratinocytes decreased slightly. Notably, the exposure of biofilms to IL-1beta caused their metabolic activity to drop, which may be linked to the observed interaction of IL-1beta with the conserved intracellular proteins DNA binding protein HU and the trimeric form of ATP synthase subunit beta. A Pasteurellaceaespecific lipoprotein, which had no previously determined function, was characterized as an IL-1beta interacting membrane protein that was expressed in the biofilm cultures of all tested A. actinomycetemcomitans strains. The use of a subcellular localisation tool combined with experimental analyses suggested that the identified lipoprotein, bacterial interleukin receptor I (BilRI), may be associated with the outer membrane with a portion of the protein oriented towards the external milieu. The results of the emHofQ study indicated that emHofQ has both the structural and functional capability to bind DNA. This result implies that emHofQ plays a role in DNA assimilation. The results from the current study also demonstrate that the Gram-negative oral species appears to sense the central proinflammatory mediator IL-1beta.

Siirtolaisuus, seksityö ja toimijuus : ihmiskauppadiskursseja purkamassa

Kirjallisuusarvostelu

Till försvar av de mänskliga rättigheterna för offren av prostitutionsrelaterad människohandel

Kirjallisuusarvostelu

Tuning cell motility : roles of nestin and vimentin in cancer cell invasion and motility

The cytoskeleton is a key feature of both prokaryotic and eukaryotic cells. Itis comprised of three protein families, one of which is the intermediate filaments (IFs). Of these, the IFs are the largest and most diverse. The IFs are expressed throughout life, and are involved in the regulation of cell differentiation, homeostasis, ageing and pathogenesis. The IFs not only provide structural integrity to the cell, they are also involved in a range of cellular functions from organelle trafficking and cell migration to signalling transduction. The IFs are highly dynamic proteins, able to respond and adapt their network rapidly in response to intra- and extra- cellular cues. Consequently they interact with a whole host of cellular signalling proteins, regulating function, and activity, and cellular localisation. While the function of some of the better-known IFs such as the keratins is well studied, the understanding of the function of two IFs, nestin and vimentin, is poor. Nestin is well known as a marker of differentiation and is expressed in some cancers. In cancer, nestin is primarily described as is a promoter of cell motility, however, how it fulfils this role remains undefined. Vimentin too is expressed in cancer, and is known to promote cell motility and is used as a marker for epithelial to mesenchymal transition (EMT). It is only in the last decade that studies have addressed the role that vimentin plays in cell motility and EMT. This work provides novel insight into how the IFs, nestin and vimentin regulate cell motility and invasion. In particular we show that nestin regulates the cellular localisation and organisation of two key facilitators of cell migration, focal adhesion kinase and integrins. We identify nestin as a regulator of extracellular matrix degradation and integrin-mediated cell invasion. Two further studies address the specific regulation of vimentin by phosphorylation. A detailed characterisation study identified key phosphorylation sites on vimentin, which are critical for proper organisation of the vimentin network. Furthermore, we show that the bioactive sphingolipids are vimentin network regulators. Specifically, the sphingolipids induced RhoA kinasedependent (ROCK) phosphorylation at vimentin S71, which lead to filament reorganisation and inhibition of cell migration. Together these studies shed new light into the regulation of nestin and vimentin during cell motility.

Violations of children's rights in armed conflicts : international treaties, observations by peacekeepers and crisis management training in Finland

Ninety-nine Finnish peacekeepers, who had been serving in 15 different operations around the world, participated in the study (8 women, 27-52 years old, m = 37.4, SD = 8.9; and 91 men, 21-69 years old, m = 41.4, SD = 10.2). Three military crisis management trainers from the Finnish Defence Forces International Centre also participated in the study. The data was collected with two webbased questionnaires. In addition two interviews were made with specialists of civilian crisis management in Finland. The study also provides an overview of international treaties concerning children’s rights in armed conflict. The results show that 48.7 % of dangers for children in conflicts reported by the peacekeepers were related to physical injury (e.g. landmines and traffic), and 27.4 % were related to social problems (e.g. poverty, child soldiers, and trafficking). 24.1 % of the peacekeepers had made observations of children’s rights violations either often or very often during peacekeeping operations. 49.6 % of the observations were related to social problems (e.g. child labour or being forced to beg), and 33.0 % were related to physical injury (e.g. assault). Frequency of observation of children’s rights violations was not associated with either sex or military degree of the peacekeepers; instead it was significantly correlated with the peacekeepers’ degree of knowledge of EU’s child protection guidelines. On the basis of the results, it is recommended that knowledge about children’s rights and protection should be included in the training of Finnish crisis management personnel to a much higher degree than at the present.

Quantitative refinement of reaction-based biomodels

In the field of molecular biology, scientists adopted for decades a reductionist perspective in their inquiries, being predominantly concerned with the intricate mechanistic details of subcellular regulatory systems. However, integrative thinking was still applied at a smaller scale in molecular biology to understand the underlying processes of cellular behaviour for at least half a century. It was not until the genomic revolution at the end of the previous century that we required model building to account for systemic properties of cellular activity. Our system-level understanding of cellular function is to this day hindered by drastic limitations in our capability of predicting cellular behaviour to reflect system dynamics and system structures. To this end, systems biology aims for a system-level understanding of functional intraand inter-cellular activity. Modern biology brings about a high volume of data, whose comprehension we cannot even aim for in the absence of computational support. Computational modelling, hence, bridges modern biology to computer science, enabling a number of assets, which prove to be invaluable in the analysis of complex biological systems, such as: a rigorous characterization of the system structure, simulation techniques, perturbations analysis, etc. Computational biomodels augmented in size considerably in the past years, major contributions being made towards the simulation and analysis of large-scale models, starting with signalling pathways and culminating with whole-cell models, tissue-level models, organ models and full-scale patient models. The simulation and analysis of models of such complexity very often requires, in fact, the integration of various sub-models, entwined at different levels of resolution and whose organization spans over several levels of hierarchy. This thesis revolves around the concept of quantitative model refinement in relation to the process of model building in computational systems biology. The thesis proposes a sound computational framework for the stepwise augmentation of a biomodel. One starts with an abstract, high-level representation of a biological phenomenon, which is materialised into an initial model that is validated against a set of existing data. Consequently, the model is refined to include more details regarding its species and/or reactions. The framework is employed in the development of two models, one for the heat shock response in eukaryotes and the second for the ErbB signalling pathway. The thesis spans over several formalisms used in computational systems biology, inherently quantitative: reaction-network models, rule-based models and Petri net models, as well as a recent formalism intrinsically qualitative: reaction systems. The choice of modelling formalism is, however, determined by the nature of the question the modeler aims to answer. Quantitative model refinement turns out to be not only essential in the model development cycle, but also beneficial for the compilation of large-scale models, whose development requires the integration of several sub-models across various levels of resolution and underlying formal representations.

The Chromatoid body entourage: Molecular characterization of the Chromatoid body‐associated cytoplasmic vesicles

Spermatogenesis is a unique process compared to cell differentiation in somatic tissues. Germ cells undergo a considerable number of metabolic and morphological changes during their differentiation: they initially proliferate by mitosis to increase in number; at some point they scramble their genetic material by meiosis, to create new genetic combinations that are the basis for evolution through natural selection and, finally, they change their shape and produce specialized structures characteristic of the mature sperm. Germ cells display an astonishingly broad transcription of their genome compared to differentiated somatic cells. Moreover, the different RNAs need to be specifically regulated in space and time for sperm production to occur appropriately. Different proteins localized in specific subcellular compartments, along with regulatory small RNAs, have an essential role in the proper execution of the different steps of spermatogenesis. These ribonucleoprotein granules interact with cytoplasmic vesicles and organelles to accomplish their role during sperm development. In this study, we characterized the most prominent ribonucleoprotein granule found in germ cells, the Chromatoid body (CB). For the first time we investigated the interaction of the CB with the cytoplasmic vesicles that surround it. These studies directed us to the description of Retromer proteins in germ cells and their involvement with the CB and the acrosome formation. Moreover, we discovered the interplay between the CB and the lysosome system in haploid round spermatids, and identified FYCO1, a new protein central to this interaction. Our results suggest that the vesicular transport system participates in the CB-mediated RNA regulation during sperm development.

VAP-1 as drug target in inflammation : structural features determining ligand interactions

Vascular adhesion protein-1 (VAP-1), which belongs to the copper amine oxidases (CAOs), is a validated drug target in inflammatory diseases. Inhibition of VAP-1 blocks the leukocyte trafficking to sites of inflammation and alleviates inflammatory reactions. In this study, a novel set of potent pyridazinone inhibitors is presented together with their X-ray structure complexes with VAP-1. The crystal structure of serum VAP-1 (sVAP-1) revealed an imidazole binding site in the active site channel and, analogously, the pyridazinone inhibitors were designed to bind into the channel. This is the first time human VAP-1 has been crystallized with a reversible inhibitor and the structures reveal detailed information of the binding mode on the atomic level. Similarly to some earlier studied inhibitors of human VAP-1, the designed pyridazinone inhibitors bind rodent VAP-1 with a lower affinity than human VAP-1. Therefore, we made homology models of rodent VAP-1 and compared human and rodent enzymes to determine differences that might affect the inhibitor binding. The comparison of the crystal structures of the human VAP-1 and the mouse VAP-1 homology model revealed key differences important for the species specific binding properties. In general, the channel in mouse VAP-1 is more narrow and polar than the channel in human VAP-1, which is wider and more hydrophobic. The differences are located in the channel leading to the active site, as well as, in the entrance to the active site channel. The information obtained from these studies is of great importance for the development and design of drugs blocking the activity of human VAP-1, as rodents are often used for in vivo testing of candidate drugs. In order to gain more insight into the selective binding properties of the different CAOs in one species a comprehensive evolutionary study of mammalian CAOs was performed. We found that CAOs can be classified into sub-families according to the residues X1 and X2 of the Thr/Ser-X1-X2-Asn-Tyr-Asp active site motif. In the phylogenetic tree, CAOs group into diamine oxidase, retina specific amine oxidase and VAP-1/serum amine oxidase clades based on the residue in the position X2. We also found that VAP-1 and SAO can be further differentiated based on the residue in the position X1. This is the first large-scale comparison of CAO sequences, which explains some of the reasons for the unique substrate specificities within the CAO family.

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.