Elucidating the role of WDR47 in regulating neuronal migration, autophagy and tubulin dynamics

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

Determining mutual challenges faced by opencast mines and their women employees

MBA, North-West University, Potchefstroom Campus, 2015Throughout history various discriminatory inequalities have appeared which are based on traditional beliefs and stereotype principles. As with many other social structures, this has brought with it the challenge of overcoming these inequalities in order to empower those afflicted by unfair treatment and to eradicate both the social and economic effects it has had on society at large. Of the many different groups that have been discriminated against, often for reasons of race and belief, one of the most discriminated against is women. This is clear when one takes into account the numerous struggles over the course of time women faced for the right to take part in the very basic roles of society. The right to vote, the right to freedom of speech and even the simple freedom for women not to have their attire prescribed by what their culture deems socially acceptable. When taking this into account, it comes as no surprise that women have to overcome enormous obstacles when competing for fair employment. Even today the challenges persist in our well-developed and socially advanced labour market. The employment of women in the mining industry serves as the ideal example of current inequalities that need to be overcome if we are to reap both the social and economic rewards of the equal employment of women. The objective of this study is to identify and discuss these challenges, identify how to overcome them, the benefits of doing so and the disadvantages and repercussions of not addressing them. The findings from the empirical study, based on the sample size of 65 women currently employed in the mining sector (n=65), which have been subdivided into various categories, enabled the researcher to draw conclusions and make recommendations. The challenges that were mainly identified included health and safety, recruitment and training, retention strategy and change management. Health and safety challenges refer to women‟s perceived vulnerability in a physically demanding environment. The recruitment and training of women and the challenges that organisations face when recruiting from small skills pools require organisations to develop women‟s skills for the mutual benefit of both women and the organisation. These challenges may be proliferate due to the additional challenges that organisations face when attempting to retain the skills they have developed and the investment they have made. The resistance to change that exists within large mining organisations when women are introduced into environments previously reserved for men needs to be strategically managed. In conclusion, it was found that involving the Human Resource to implement various strategies from the recruitment of women in mining, to the development, retention and the placement of women in senior positions as well as the monitoring and constant evaluation of the progress of these strategies, the current challenges as set out above can be overcome. Furthermore, it has been concluded that it greatly depends on the top and core management of companies in the mining sector to assist in the implementation of various strategies to have these feats succeed.Master

WD40-repeat 47 is essential for brain development via microtubule-mediated processes and autophagy

International audienc

WD40-repeat 47 is essential for brain development via microtubule-mediated processes and autophagy

Erreur dates et n° de conférence dans l'url https://hal.archives-ouvertes.fr/hal-02378786.International audienc

WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy.

The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy