Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction Normal cerebral cortex development depends on extensive neuronal migration during embryogenesis, permitting the formation of accurate synaptic circuits and a highly ordered laminar neocortex. The motility of a migrating neuron is achieved by a dynamic microtubule cytoskeleton that alternates between states of stabilization/lengthening and destabilization/shortening. This dynamic instability of the microtubule cytoskeleton is controlled by numerous microtubule-stabilizing and -destabilising proteins that bind directly to microtubules. Autophagy (“self-eating”), a major bulk intracellular degradation system, involves the fusion of autophagosomes with lysosomes, followed by proteolysis and recycling of cellular constituents. Like neuronal migration, autophagy is a microtubule-dependent process. The dynamic microtubule network serves as a track for autophagosomes to be transported to the lysosomes. WDR47 is a protein that is expressed in the brain during development, but of which the function is largely unknown. Novel interactions have recently been identified between Reelin and WDR47 and between the microtubule-destabilising protein superior cervical ganglion 10 (SCG10) and WDR47. These findings suggest that WDR47 may be regulating microtubule-dependent processes such as neuronal migration and autophagy. We hypothesize that WDR47 may play a role in regulating neuronal migration and/or autophagy, and that this regulation may be mediated by a tubulin stability-regulating role of WDR47. Aims and Methods Our aims are to assess the cellular localization of WDR47 in GT1-7 cells and to determine whether WDR47 is able to influence neuronal migration, filopodia extension, surface adhesion, ultra-structure, autophagy, tubulin stability, and tau or SCG10 protein levels. GT1-7 neuronal cells were cultured under normal conditions and transfected with WDR47 siRNA for 24 hours, followed by western blot verification of the knock-down. A 36 hour neuronal in vitro cell migration assay was performed and images of the wound were captured every 6 hours; the migration distances and the wound areas for the different time points were measured and analysed. A 24 hour migration assay was performed, capturing images every hour, and the direction of migration was determined. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to analyse neuronal surface morphology and ultra-structure. Western blot analysis of SCG10, acetylated α-tubulin, Tau, LC3, and Sequestosome 1/p62 (SQTM1) protein levels was performed. Super-resolution structured Illumination microscopy (SR-SIM) three-dimensional (3-D) imaging of WDR47-YFP transfected cells, confocal microscopy of LC3 and acetylated tubulin, co-localization analysis of WDR47 and acetylated tubulin, and fluorescence recovery after photo-bleaching (FRAP) analysis were performed. Results WDR47 siRNA treatment significantly reduced the average migration distance and the migration velocity, resulted in fewer filopodia-like extensions as well as perturbed surface adhesion of migrating neurons, and lead to an increased presence of endoplasmic reticulum (ER) structures as well as an expanded nuclear envelope. LC3-II protein levels were significantly lower with WDR47 siRNA treatment, but were significantly increased with WDR47 siRNA treatment in conjunction with Bafilomycin A1 treatment, indicating increased autophagic flux. SCG10 protein levels were significantly decreased with WDR47 siRNA treatment. SR-SIM and confocal microscopy of WDR47 siRNA treated cells revealed a robust presence of highly convoluted acetylated tubulin in the perinuclear region as well as decreased LC3 fluorescence signal. Confocal microscopy revealed co-localization of WDR47 with acetylated tubulin. - Discussion and Conclusion: The results suggest that WDR47 is involved in regulating neuronal migration, neuronal surface adhesion and filopodia formation, microtubule dynamics, and likely also autophagic flux. Taken together, we propose that WDR47 is regulating microtubule dynamics by facilitating assembly of microtubule-regulating proteins such as SCG10, thereby affecting microtubule-dependent processes such as neuronal migration and autophagy. / AFRIKAANSE OPSOMMING: Inleiding Normale serebrale korteks ontwikkeling is hoogs afhanklik van neuronale migrasie tydens embriogenese, en is belanrik vir die vorming van akkurate sinaptiese netwerke en 'n hoogs geordende laminêre neokorteks. Die vermoё van 'n neuron om te migreer berus op 'n hoogs dinamiese mikrotubulien sitoskelet wat verleng/stabiliseer of verkort/destabiliseer soos tubulien-eenhede begevoeg of verwyder word. Hierdie dinamiese onstabiliteit van die mikrotubulien sitoskelet word beheer deur verskeie mikrotubulien-stabiliserende en - destabiliserende proteïene wat direk bind aan mikrotubuliene. Autofagie ("self-eet"), 'n grootmaat intrasellulêre degradasie stelsel, behels die fussie van autofagosome met lisosome, gevolg deur proteolitiese afbraak van sellulêre organelle en proteine. Soos neuronale migrasie is autofagie 'n mikrotubulien-afhanklike proses. Die dinamiese mikrotubulien netwerk dien as 'n spoor vir die vervoer van autofagosome na lisosome. WDR47 is 'n proteïen wat voorkom in die brein tydens ontwikkeling, maar waarvan die funksie grootliks onbekend is. Interaksies was onlangs geïdentifiseer tussen beide Reelin en WDR47 en die mikrotubulien-destabiliserende proteïen SCG10 en WDR47. Hierdie bevindinge dui daarop aan dat WDR47 n rol speel in die regulering van tubulienstabiliteit en sodoende mikrotubulien-afhanklike prosesse. Ons veronderstel dat WDR47 'n rol kan speel in die regulering van neuronale migrasie en/of autofagie en dat hierdie regulasie moontlik afhanklik is van 'n tubulien-stabiliteit-regulerende rol van WDR47. - Doelwitte en Metodes: Ons doelwitte is om die sellulêre lokalisering van WDR47 in GT1-7 neurone te evallueer en om te bepaal of WDR47 n effek het op neuronale migrasie, oppervlak adhesie en filopodia formasie, ultra-struktuur, autofagie, tubulien-netwerke en -stabiliteit, en Tau of SCG10 proteïenvlakke. GT1-7 neuronale selle is gekweek onder normale omstandighede en vir 24 uur getransfekteer met WDR47 siRNA, gevolg deur verifikasie met Western-blot analise. 'n 36 uur neuronale in vitro sel migrasie toets is uitgevoer en fotos van die wond is elke 6 uur geneem. Die migrasie afstande en die wondareas vir die verskillende tydpunte is gemeet en ontleed. 'N 24-uur-migrasie toets is uitgevoer, 'n foto van die wond is elke uur geneem, en die rigting van migrasie is bepaal. Skandering elektronmikroskopie (SEM) en transmissieelektronmikroskopie (TEM) is uitgevoer om neuronale oppervlakmorfologie en ultrastruktuur te observeer. Western blot analise van SCG10, geasetieleerde α-tubulien, Tau, LC3 en Sequestosome 1/p62 (SQTM1) proteïenvlakke is uitgevoer. Super-resolusie gestruktureerde verligting mikroskopie (SR-SIM) driedimensionele (3-D) beelding van WDR47-YFP getransfekteerde selle, konfokale mikroskopie vir visualisering van LC3 en tubulien, co-lokalisering analise van beide WDR47 en LC3 en WDR47 en tubulien, asook fluorescentie hersteling na foto-bleek (FRAP) analise is uitgevoer. Resultate Die gemiddelde migrasie-afstand en die migrasiesnelheid (μm/min) het beduidend afgeneem met WDR47 siRNA behandeling. SEM analise van WD47 siRNA-behandelde neurone het minder filopodia en veranderde oppervlak adhesie vertoon, en TEM analise het 'n verhoogde teenwoordigheid van endoplasmiese retikulum (ER) strukture, en 'n uitgebreide kernmembraan vertoon. LC3-II proteïenvlakke was beduidend laer met slegs WDR47 siRNA behandeling, maar beduidend hoёr met WDR47 siRNA behandeling in samewerking met Bafilomycin A1 behandeling. Hierdie resultate dui aan op toeneemende autofagie met WDR47 siRNA behandeling. Verder, beduidend laer vlakke van SCG10 proteïenvlakke is waargeneem met WDR47 siRNA behandeling. SR-SIM en konfokale mikroskopie van WDR47 siRNA behandelde selle het 'n robuuste teenwoordigheid van hoogs buigende geasetieleerdetubulien in die area rondom die nukleus, 'n afgeneemde LC3 Bespreking en Gevolgtrekking Die resultate dui daarop aan dat WDR47 betrokke is by die regulering van neuronale migrasie, filopodia vormasie, oppervlak adhesie, mikrotubuliendinamika, en waarskynlik ook autofagie. Ons stel voor dat WDR47 mikrotubuliendinamika afekteer deur die regulering van proteïene soos SCG10, en sodoende mikrotubulienafhanklike prosesse soos neuronale migrasie en autofagie fasiliteer.

The Microtubule (MT) network is a central component of the eukaryotic cell cytoskeleton. In the fission yeast S. pombe, a complex of three proteins specifically tracks MT +ends and stabilizes MTs in the cell. It is composed of the proteins Mal3, Tip1 and Tea2. Mal3, the S. pombe homologue of EB1, is a highly conserved ubiquitous protein found to be at the centre of many MT related processes. Tip1 is a CLIP170 homologue and Tea2 a kinesin-like motor protein. The mechanism by which they target the growing end of MTs and stabilize them is still unknown. A combination of biochemistry, electron microscopy and crystallography were used in an attempt to get a more precise understanding of the MT stabilization by this +Tip complex. Protein-A pull-down of the endogenous complex and analysis of its constituents by mass spectrometry revealed that Tea2 and Tip1 form a tight stoichiometric complex, making a much more labile interaction with Mal3. Biochemical experiments, light scattering and DIC microscopy demonstrate that Mal3 stabilizes the MT structure in a stoichiometric fashion by suppressing catastrophe events. 3D helical reconstruction of electron micrographs of Mal3 bound to the MT show that it most probably stabilizes the MT structure by bridging protofilaments together. Deletion mutant analysis suggests that contact with one of the protofilaments is via an interaction between the charged tails of tubulin and Mal3. Mal3 MT binding domain structure was solved by X-ray crystallography so that eventually it may be docked into a higher resolution electron microscopy map to provide a more precise structural insight on how Mal3 stabilizes the MT lattice. The EM analysis also shows that Mal3 regulates MT structure in vitro by restraining their protofilament number to 13, which is the number always found in vivo, and by driving the assembly of MTs with a high proportion of A-lattice. It is the first time that a protein is found to promote formation of A-lattice MTs. The fact that EB1 is such a ubiquitous protein reopens the question of MT structure in cells and has important implications for in vivo MT dynamics.