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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96065 |
| Date | 12 1900 |
| Creators | Roos, Marna |
| Contributors | Loos, Benjamin, Kinnear, Craig, Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xxi, 136 p. : ill. |
| Rights | Stellenbosch University |
Page generated in 0.0111 seconds