<p>Cellular processes like motility, chemotaxis, phagocytosis and morphogenesis are dependent on the dynamic regulation of the actin cytoskeleton. This cytoskeleton system is tightly controlled by a number of diverse actin-binding proteins (ABPs) by various mechanisms described as nucleation, polymerization, capping, severing, depolymerization and sequestration. The ABPs are grouped based on sequence identity as in the Wiskott-Aldrich Syndrome protein homology domain 2 (WH2), and the calponin homology domain (CH) containing proteins.</p><p>In this work, we elucidate the crystal structures of hybrids of gelsolin domain 1 with thymosin β4, ciboulot domain 2, and the second WH2 domain of N-WASP each bound to actin. We show that the single WH2 motif containing protein thymosin β4 in part sequesters actin by binding its pointed end via a C-terminal helix. This interaction prevents the addition of bound actin protomers to the barbed end of the filament. We propose that sequence variations in some WH2 motifs conferred F-actin binding ability to multiple repeat-containing proteins. These F-actin binding domains interact with the barbed end of a filament and the adjacent WH2 motifs are then freed to add their bound actin to the growing filament end. We demonstrate the binding of ciboulot domains 2 and 3 to both G- and F-actin and that full length ciboulot is capable of binding two actin monomers simultaneously. </p><p>We have also cloned, expressed, purified and crystallized rod domains 14-16 from the actin crosslinking protein a-filamin. Preliminary X-ray crystallography data gives us hope that we shall be able to solve the structure of this triple domain repeat.</p>

Cellular processes like motility, chemotaxis, phagocytosis and morphogenesis are dependent on the dynamic regulation of the actin cytoskeleton. This cytoskeleton system is tightly controlled by a number of diverse actin-binding proteins (ABPs) by various mechanisms described as nucleation, polymerization, capping, severing, depolymerization and sequestration. The ABPs are grouped based on sequence identity as in the Wiskott-Aldrich Syndrome protein homology domain 2 (WH2), and the calponin homology domain (CH) containing proteins. In this work, we elucidate the crystal structures of hybrids of gelsolin domain 1 with thymosin β4, ciboulot domain 2, and the second WH2 domain of N-WASP each bound to actin. We show that the single WH2 motif containing protein thymosin β4 in part sequesters actin by binding its pointed end via a C-terminal helix. This interaction prevents the addition of bound actin protomers to the barbed end of the filament. We propose that sequence variations in some WH2 motifs conferred F-actin binding ability to multiple repeat-containing proteins. These F-actin binding domains interact with the barbed end of a filament and the adjacent WH2 motifs are then freed to add their bound actin to the growing filament end. We demonstrate the binding of ciboulot domains 2 and 3 to both G- and F-actin and that full length ciboulot is capable of binding two actin monomers simultaneously. We have also cloned, expressed, purified and crystallized rod domains 14-16 from the actin crosslinking protein a-filamin. Preliminary X-ray crystallography data gives us hope that we shall be able to solve the structure of this triple domain repeat.

The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex. / Thesis (Ph.D, Biology) -- Queen's University, 2011-07-28 16:20:31.957

Cells use energy to maintain order, as living systems are inherently non-equilibrium. Or- der in the cytoplasm is achieved by compartmentalization. One type of compartment that gained interest in recent years is membraneless organelles (MLOs). Observations of the liquid-like properties of MLOs led to their interpretation in analogy to Liquid-Liquid Phase Separation (LLPS). However, LLPS alone implies a passive closed system that tends towards equilibrium, which is incompatible with the physical nature of the cell. It is unclear then what non-equilibrium interactions give rise to the dynamics of MLOs in the cell. We sought to decipher the regulatory interactions that give rise to active condensation in the actomyosin cortex of C. elegans. The components of the actomyosin cortex, F- actin and its branching nucleation module Arp2/3 and N-WASP (WSP-1 in C. elegans) have been described as a phase separated system in previous reports. In vitro, phase separated N-WASP compartments do not have the non-equilibrium growth and disas- sembly dynamics observed in the multicomponent clusters in vivo. Therefore, our goal is to examine WSP-1, Arp2/3 and F-actin interactions in the endogenous context. We chose the stage in which the quiescent oocyte cortex becomes actively contractile. During the transition out of quiescence, we observed transient WSP-1 Arp2/3 F-actin puncta that assemble and disassemble. To capture growth dynamics for all puncta, we devel- oped a novel phase portrait analysis tool. The phase portrait allows us to simultaneously study puncta growth and disassembly rates as a function of internal composition. The growth rate dependence on internal composition reflects the non-trivial changes to nu- cleation profiles that accompany condensation in active, open, multi-component systems. We observed superlinear WSP-1 growth rates consistent with condensation. Further, we identified the in vivo equivalent of a nucleation barrier for WSP-1 condensation. The in vivo nucleation barrier increases with branching F-actin reaction, which tunes con- densation. Correspondingly, the reactive components WSP-1 and Arp2/3 are important for condensate dynamics. Combining condensation and the branching reaction, we for- mulated a coarse-grained model which captures non-equilibrium condensate dynamics. Altogether, our results showed that WSP-1 grows like condensation, and its growth is steered away from equilibrium by Arp2/3 mediated branching reaction. In summary, combining high-resolution imaging, quantitative analysis and theory, we identified the interactions that could explain non-equilibrium condensation in the acto- myosin cortex. The living dynamics that arise from the interplay between condensation and reaction. The interplay between physical processes (like condensation) and biological regulation (such as reactions) may be a common organizing principle behind MLO for- mation, as well as other non-equilibrium processes in the cell. The methods and concepts developed in this work hold the promise to deepen our understanding of how living cells regulate their dynamic organization, in order to maintain themselves in a non-equilibrium ordered state.:1 Introduction 1 1.1 Evolving concepts of cellular organization 1 1.2 Condensation of biomolecules 3 1.2.1 Terminology for biomolecular condensates 5 1.2.2 Technical considerations for identifying liquid-like properties and LLPS 7 1.2.3 Thermodynamics of condensation 10 1.2.4 The problem of an equilibrium description of living systems 13 1.2.5 Towards active condensation 14 1.3 Actomyosin cortex self-organization 16 1.3.1 F-actin treadmilling and nucleation 17 1.3.2 N-WASP and Arp2/3 regulation 18 1.3.3 Multivalent interactions in condensation of transmembrane receptors and actin regulators 22 1.3.4 Cortex activation in C. elegans 23 2 Aims 25 3 Results 26 3.1 C. elegans cortical activation begins at fertilization 26 3.1.1 C. elegans oocytes as an ex utero model for cortex self-organization 27 3.2 WSP-1, Arp2/3 and F-actin form dynamic multicomponent phases 32 3.2.1 Capping proteins outcompete Formin in WSP-1 Arp2/3 puncta preventing F-actin elongation 32 3.2.2 WSP-1 and Arp2/3 are required for punctate F-actin formation and dynamics 34 3.2.3 Summary of the characterization of cortical activation 34 3.3 Establishment of systematic phase portrait analysis for multicomponent clusters 36 3.3.1 Non-equilibrium features of the multicomponent puncta 36 3.3.2 Recording intensity traces of multicomponent cluster over time 37 3.3.3 Probability flux of composition in the phase portrait show a closed cycle 38 3.3.4 WSP-1 F-actin puncta have a preferred joint concentration 38 3.3.5 The phase portrait is robust to cell-to-cell noise 41 3.3.6 Choosing the appropriate bin size 41 3.4 Existence of a tuned critical size and signatures of active condensation 45 3.4.1 Growth rate dependence on internal composition 45 3.4.2 Stoichiometric growth laws of WSP-1 F-actin clusters 47 3.4.3 Estimation of WSP-1 cluster critical size in vivo 47 3.4.4 Theoretical description of WSP-1 and F-actin interactions in regulating puncta dynamics 48 3.4.5 Summary of 2D phase portrait findings 52 3.5 Towards three dimensional phase portrait analysis of the reaction network 54 3.6 Initial assessment of the compartment’s external environment 54 3.7 Identification of modulators of puncta dynamics 56 3.7.1 CDC-42 controls cortical levels of WSP-1 56 3.7.2 RHO-1 and Formin CYK-1 are not involved in WSP-1 F-actin condensate dynamics 58 3.7.3 WSP-1 and Arp2/3 dynamics are independent of NCK-1 and VAB-1 58 3.7.4 Arp2/3 regulates condensate dynamics 60 3.8 Summary of perturbations 63 4 Conclusions and outlook 64 4.1 Concluding remarks 64 4.2 Discussion 66 4.3 Future directions 67 4.3.1 Realizing the full potential of the phase portraits in identifying biochemical interactions 67 4.3.2 Resolving the ultrastructure of condensates . 70 4.3.3 Further investigation of the biological function 71 4.3.4 Applying full-dynamic data acquisition to other membraneless organelles 71 5 Materials and Methods 72 5.1 C.elegans maintenance and strains 72 5.2 Sample preparation 72 5.2.1 In utero imaging 72 5.2.2 Oocyte imaging 73 5.2.3 C.elegans HaloTag staining 73 5.2.4 Oocyte chemical inhibitor treatments 73 5.3 RNAi Feeding 73 5.4 Microscopy 73 5.4.1 Spinning disk microscopy 73 5.4.2 SIM-TIRF microscopy 74 5.5 TIRF microscopy 74 5.6 Phase portrait analysis pipeline 74 5.7 Kymographs 76 / Zellen verbrauchen Energie, um Ordnung aufrechtzuerhalten, da lebende Systeme von Natur aus ungleichgewichtig sind. Ordnung im Zytoplasma wird durch Kompartimen- tierung erreicht. Eine Art von Kompartiment, das in den letzten Jahren an Interesse gewonnen hat, sind membranlose Organellen (engl.: membraneless organelles, MLOs). Beobachtungen der flu ̈ssigkeits ̈ahnlichen Eigenschaften dieser MLOs fu ̈hrten zu ihrer In- terpretation in Analogie zur Flu ̈ssig-Flu ̈ssig-Phasentrennung (engl.: liquid-liquid phase separation, LLPS). Die LLPS allein impliziert jedoch ein passives geschlossenes System, das zum Gleichgewicht neigt und mit der physikalischen Natur der Zelle nicht kompatibel ist. Es war bisher nicht bekannt, welche Ungleichgewichtswechselwirkungen die Dynamik von MLOs in der Zelle hervorrufen. Wir wollten die regulatorischen Wechselwirkungen entschlu ̈sseln, die zu aktiver Konden- sation im Aktomyosin-Kortex von C. elegans fu ̈hren. Die Komponenten des Aktomyosin- Kortex, F-Aktin und seines verzweigten Nukleationsmoduls Arp2/3 und N-WASP (WSP- 1 in C. elegans) wurden in fru ̈heren Studien als phasengetrenntes System beschrieben. In vitro weisen phasengetrennte N-WASP-Kompartimente allerdings nicht dieselben un- gleichgewichtigen Wachstums- und Zerlegungsdynamiken auf, die in kultivierten Zellen beobachtet werden. Daher wollten wir die Wechselwirkungen zwischen WSP-1, Arp2/3 und F-Aktin im Kontext des Fadenwurms C. elegans untersuchen. Wir haben das C.elegans Lebenstadium gew ̈ahlt, in dem die ruhende Eizellenrinde aktiv kontraktil wird. Wa ̈hrend des U ̈bergangs aus der ruhigen in die aktive Periode konnten wir voru ̈bergehende WSP- 1 Arp2/3 F-Aktin-Puncta beobachten, die sich zusammensetzen und zerlegen. Um die Wachstumsdynamik fu ̈r alle Puncta zu erfassen, haben wir ein neuartiges Tool zur Anal- yse von Phasenportr ̈ats entwickelt. Das Phasenportr ̈at ermo ̈glicht es uns, gleichzeitig die Wachstums- und die Zerlegungsraten von Puncta in Abha ̈ngigkeit der inneren Zusam- mensetzung zu messen. Die Abha ̈ngigkeit der Wachstumsrate von der inneren Zusam- mensetzung spiegelt die nicht trivialen A ̈nderungen der Nukleationsprofile wider, die mit der Kondensation in aktiven, offenen Mehrkomponentensystemen einhergehen. Wir kon- nten superlineare WSP-1-Wachstumsraten beobachten, die mit der Kondensation u ̈bere- instimmen. Ferner konnten wir das In-vivo-A ̈quivalent einer Nukleationsbarriere fu ̈r die WSP-1-Kondensation identifizieren. Die In-vivo-Nukleationsbarriere nimmt mit der verzweigten F-Actin-Reaktion zu, die die Kondensation reguliert. Dementsprechend sind die reaktiven Komponenten WSP-1 und Arp2/3 wichtig fu ̈r die Dynamik des Konden- sats. Wir haben die Kondensations- und Verzweigungsreaktionen kombiniert, um damit ein grobko ̈rniges Modell zu formulieren, das die Ungleichgewichtskondensationsdynamik erfasst. Insgesamt haben unsere Ergebnisse gezeigt, dass WSP-1 kondensiert und diese Kondensation durch Arp2/3-vermittelte Verzweigungsreaktionen aus dem Gleichgewicht gebracht wird. Zusammenfassend konnten wir durch Kombination von hochauflo ̈sender Bildgebung, quan- titativer Analyse und Theorie die Wechselwirkungen identifizieren, die die Ungleichgewicht- skondensation im Aktomyosin-Kortex erkla ̈ren ko ̈nnten. Die Dynamik im lebendem Sys- tem ergibt sich aus dem Zusammenspiel von Kondensation und Reaktion. Die Interaktion zwischen physikalischen Prozessen (wie Kondensation) und biologischen Regulationen (wie Reaktionen) kann ein gemeinsames Organisationsprinzip hinter der MLO-Bildung sowie anderen Ungleichgewichtsprozessen in der Zelle sein. Die in dieser Arbeit entwickel- ten Methoden und Konzepte k ̈onnen daher helfen, unser Versta ̈ndnis daru ̈ber zu vertiefen, wie lebende Zellen ihre r ̈aumlich-zeitlichen Proteinverteilungen dynamisch regulieren, um sich in einem ungleichgewichtigen, geordneten Zustand zu halten.:1 Introduction 1 1.1 Evolving concepts of cellular organization 1 1.2 Condensation of biomolecules 3 1.2.1 Terminology for biomolecular condensates 5 1.2.2 Technical considerations for identifying liquid-like properties and LLPS 7 1.2.3 Thermodynamics of condensation 10 1.2.4 The problem of an equilibrium description of living systems 13 1.2.5 Towards active condensation 14 1.3 Actomyosin cortex self-organization 16 1.3.1 F-actin treadmilling and nucleation 17 1.3.2 N-WASP and Arp2/3 regulation 18 1.3.3 Multivalent interactions in condensation of transmembrane receptors and actin regulators 22 1.3.4 Cortex activation in C. elegans 23 2 Aims 25 3 Results 26 3.1 C. elegans cortical activation begins at fertilization 26 3.1.1 C. elegans oocytes as an ex utero model for cortex self-organization 27 3.2 WSP-1, Arp2/3 and F-actin form dynamic multicomponent phases 32 3.2.1 Capping proteins outcompete Formin in WSP-1 Arp2/3 puncta preventing F-actin elongation 32 3.2.2 WSP-1 and Arp2/3 are required for punctate F-actin formation and dynamics 34 3.2.3 Summary of the characterization of cortical activation 34 3.3 Establishment of systematic phase portrait analysis for multicomponent clusters 36 3.3.1 Non-equilibrium features of the multicomponent puncta 36 3.3.2 Recording intensity traces of multicomponent cluster over time 37 3.3.3 Probability flux of composition in the phase portrait show a closed cycle 38 3.3.4 WSP-1 F-actin puncta have a preferred joint concentration 38 3.3.5 The phase portrait is robust to cell-to-cell noise 41 3.3.6 Choosing the appropriate bin size 41 3.4 Existence of a tuned critical size and signatures of active condensation 45 3.4.1 Growth rate dependence on internal composition 45 3.4.2 Stoichiometric growth laws of WSP-1 F-actin clusters 47 3.4.3 Estimation of WSP-1 cluster critical size in vivo 47 3.4.4 Theoretical description of WSP-1 and F-actin interactions in regulating puncta dynamics 48 3.4.5 Summary of 2D phase portrait findings 52 3.5 Towards three dimensional phase portrait analysis of the reaction network 54 3.6 Initial assessment of the compartment’s external environment 54 3.7 Identification of modulators of puncta dynamics 56 3.7.1 CDC-42 controls cortical levels of WSP-1 56 3.7.2 RHO-1 and Formin CYK-1 are not involved in WSP-1 F-actin condensate dynamics 58 3.7.3 WSP-1 and Arp2/3 dynamics are independent of NCK-1 and VAB-1 58 3.7.4 Arp2/3 regulates condensate dynamics 60 3.8 Summary of perturbations 63 4 Conclusions and outlook 64 4.1 Concluding remarks 64 4.2 Discussion 66 4.3 Future directions 67 4.3.1 Realizing the full potential of the phase portraits in identifying biochemical interactions 67 4.3.2 Resolving the ultrastructure of condensates . 70 4.3.3 Further investigation of the biological function 71 4.3.4 Applying full-dynamic data acquisition to other membraneless organelles 71 5 Materials and Methods 72 5.1 C.elegans maintenance and strains 72 5.2 Sample preparation 72 5.2.1 In utero imaging 72 5.2.2 Oocyte imaging 73 5.2.3 C.elegans HaloTag staining 73 5.2.4 Oocyte chemical inhibitor treatments 73 5.3 RNAi Feeding 73 5.4 Microscopy 73 5.4.1 Spinning disk microscopy 73 5.4.2 SIM-TIRF microscopy 74 5.5 TIRF microscopy 74 5.6 Phase portrait analysis pipeline 74 5.7 Kymographs 76

Yes / The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP ‘hyperactivity’ upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner. / This work was supported by SFB 670 and DFG NO 113/22. K.B. was supported by a fellowship from the NRW International Graduate School “From Embryo to Old Age: the Cell Biology and Genetics of Health and Disease” (IGSDHD), Cologne.

Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.

Η αναδιοργάνωση του κυτταροσκελετού ακτίνης έχει κρίσιμο ρόλο στη διήθηση και τη μετάσταση των καρκινικών κυττάρων. Οι πρωτεΐνες κοφιλίνη και N-WASP συνδέονται με την ακτίνη και ρυθμίζουν τη δυναμική του κυτταροσκελετού, ενώ η πρωτεΐνη β-παρβίνη εντοπίζεται στις εστιακές συνδέσεις και διαμεσολαβεί τη σηματοδότηση διαμέσου των ιντεγκρινών με επίσης σημαντική επίδραση στον κυτταροσκελετό ακτίνης. Μεταβολή της έκφρασης των παραπάνω πρωτεϊνών έχει παρατηρηθεί σε διάφορα νεοπλάσματα στον άνθρωπο. Η παρούσα μελέτη επιχειρεί να προσδιορίσει το ρόλο τους στον καρκίνο του λάρυγγα. Για αυτό το λόγο μελετήθηκε με ανοσοϊστοχημεία η έκφραση των πρωτεϊνών κοφιλίνη, N-WASP β-παρβίνη σε 72 ιστικά δείγματα ασθενών με πλακώδες καρκίνωμα του λάρυγγα και αξιολογήθηκε η συσχέτιση της έκφρασης τους με κλινικές και παθολογοανατομικές παραμέτρους καθώς και με την επιβίωση. Θετική ανοσοϊστοχημικη έκφραση των πρωτεϊνών κοφιλίνη, N-WASP β-παρβίνη παρατηρήθηκε στο 86,1%, 93,1% και 94,4% των περιπτώσεων καρκινώματος του λάρυγγα αντίστοιχα, σε αντίθεση με τον παρακείμενο φυσιολογικό ιστό όπου η έκφραση ήταν ασθενής ή απούσα. Η κυταροπλασματική έκφραση της κοφιλίνης έδειξε τάση συσχέτισης με τα προχωρημένα στάδια της νόσου (p=0,063), ενώ η πυρηνική της έκφραση συσχετίστηκε σε στατιστικά σημαντικό βαθμό (p=0,031) με την προχωρημένη ηλικία (>65 ετών) των ασθενών. Η κυτταροπλασματική έκφραση του N-WASP ήταν μεγαλύτερη στα γλωττιδικά καρκινώματα (p=0,068) και συσχετίστηκε σε στατιστικά σημαντικό βαθμό με τα καλά διαφοροποιημένα καρκινώματα (p=0,031). Η κυτταροπλασματική έκφραση της β-παρβίνης συσχετίστηκε σε στατιστικά σημαντικό βαθμό (p=0,038) με τα προχωρημένα στάδια της νόσου. Παρόλο που οι ασθενείς με υψηλότερη έκφραση του N-WASP εμφάνιζαν καλύτερες επιβιώσεις, δεν επιβεβαιώθηκαν στατιστικά σημαντικές συσχετίσεις της έκφρασης των 3 υπό μελέτη πρωτεϊνών με την επιβίωση. Η αυξημένη έκφραση των πρωτεϊνών κοφιλίνη, N-WASP και β-παρβίνη στον καρκίνο του λάρυγγα μπορεί να υποδεικνύει την πιθανή συμμετοχή τους στην παθογένεση της νόσου. Ενώ η υψηλή έκφραση της κοφιλίνης και της β-παρβίνης φάνηκε να ευνοεί την προαγωγή/εξέλιξη του καρκίνου, τα αυξημένα επίπεδα της πρωτεΐνης N-WASP συσχετιστήκαν με ευνοϊκούς προγνωστικούς παράγοντες. / Actin cytoskeleton dynamics are critically implicated in cancer invasion and metastasis. Actin binding proteins cofilin and N-WASP regulate actin filament turnover and the focal adhesion protein β-parvin mediates integrin signaling to actin cytoskeleton. Altered expression of these proteins has been implicated in human malignancies. This study addresses their role in human laryngeal cancer. Protein expression of cofilin, N-WASP and β-parvin were investigated by immunohistochemistry in 72 FFPE samples of human laryngeal squamous cell carcinoma. Correlations with clinicopathological data and survival were evaluated. Positive immunostaining of cofilin, N-WASP and β-parvin were observed in 86.1%, 93,1% and 94,4% cases respectively, in contrast to the weak or absent staining at the non neoplastic adjacent mucosa. Cytoplasmic cofilin immunoreactivity tended to correlate with advanced disease stage (p=0.063), while its nuclear immunoreactivity correlated significantly with advanced (>65 years) patient age (p=0,031). N-WASP immunoreactivity was higher in glottic laryngeal carcinomas (p=0.068) and significantly correlated with low grade tumors (p=0.031). Expression of β-parvin also correlated significantly with advanced disease stage (p= 0.038). Although patients with high N-WASP expression showed higher survival rates no statistical significant correlation between cofilin, N-WASP or β-parvin immunoreactivity and survival was found. Overexpression of cofilin, N-WASP and β-parvin may be implicated in human laryngeal carcinogenesis. While high expression of cofilin and β-parvin seems to favor tumor progression increased levels of N-WASP associated with favorable prognostic factors.

Cdc42 is a member of the Rho family of small GTPases, which, together with its homologues RhoA and Rac1, controls a multitude of cellular functions via the actin cytoskeleton. Cdc42 exerts its effects on the cytoskeleton via effector proteins of the Wiskott-Aldrich Syndrome (WASP) family and the Transducer of Cdc42-dependent Actin assembly (TOCA) family. The WASP family and their activation by Cdc42 have been thoroughly studied in vitro and are well understood. Conversely, understanding of the TOCA family remains limited by a lack of biochemical, biophysical and structural insight. An investigation of the TOCA1-Cdc42 interaction is described here, revealing a relatively low affinity interaction with a dissociation constant in the micromolar range. This is 10-100x weaker than other Rho-effector interactions and suggests that TOCA1 must first be co-localised with Cdc42 to achieve stable binding in vivo. The solution NMR structure of the Cdc42 binding HR1 domain of TOCA1 provides the first structural data on this protein and reveals some interesting structural features that may relate to binding affinity and specificity. A structural model of the Cdc42-HR1 complex provides further insight into differential specificities and affinities of GTPase-effector interactions. NMR and actin polymerisation assays provide insight into the pathway of Cdc42/TOCA1/WASP-dependent actin assembly, suggesting unidirectional displacement of TOCA1 by N-WASP. A comparison of the Cdc42- TOCA1 model with an NMR structure of Cdc42 in complex with the GTPase binding domain of WASP reveals a possible mechanism by which an ‘effector handover’ from TOCA1 to N-WASP could take place. Small GTPases such as Cdc42 are lipid modified and membrane anchored via their C- termini in vivo, so in vitro studies using truncated, unmodified GTPases are limited in their biological interpretation. This project also aimed to develop methods to study full length and membrane-anchored GTPases in vitro. Lipid modified protein was produced, which showed a weak affinity for liposomes, and so structural studies of membrane anchored protein are within reach. Further method development is now required to achieve stable membrane anchoring of lipid modified GTPases for detailed NMR studies.

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