Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.737862
Title: Analysis of phosphatidylinositol metabolism and ER-mitochondria contact sites in the PINK1/Parkin pathway
Author: Wilson, Emma
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
PINK1 and Parkin are two genes which are commonly mutated in autosomalrecessive juvenile PD (AR-JP). They function in a common mitochondrial quality control pathway to regulate mitochondrial homeostasis. Under mitochondrial depolarization PINK1, a serine/threonine kinase, is stabilized on the outer mitochondrial membrane (OMM) where it recruits Parkin, an E3 ubiquitin ligase, from the cytosol. Parkin then ubiquitinates OMM proteins to aid in the recruitment of the phagophore to the mitochondria and the process of mitophagy. More recently reports have suggested that PINK1 and Parkin are also present at ER-mitochondrial contact sites and may contribute to mitochondrial homeostasis by altering contacts. SYNJ1, a neuronal phosphatidylinositol phosphatase, is also mutated in AR-JP and fwd, a phosphatidylinositol kinase, was reported to alter mitochondrial morphology in Drosophila cells. Together SYNJ1/synj and PI4KB/fwd act to modulate the phosphatidylinositol balance, therefore it was hypothesized that PI metabolism may be a regulatory factor in the PINK1/Parkin pathway and mitochondrial quality control. To examine the effects of manipulating synj or fwd, Drosophila and YFPParkin HeLa cells models were used to investigate genetic interactions with pink1 and parkin. A further aim was to develop a new tool to visualize ER-mitochondrial contact sites in real time, which would allow visualization of dynamic functions of ER-mitochondrial contact sites. This study found that the loss of fwd in vivo caused a significant decrease in climbing but not flight ability in adult Drosophila. This was not thought to be attributed to changes in mitochondrial morphology. However, the overexpression of fwd resulted in a partial rescue of pink1B9 and park25 climbing defect. The silencing of PI4KB and SYNJ1 in YFP-Parkin HeLa cells slowed Parkin translocation and mitophagy however loss of SYNJ2 did not. Furthermore, Bimolecular Fluorescence complementation (BiFC) and dimerization dependent GFP (ddGFP) were tested for visualizing of ER-mitochondrial contact sites. Both systems were functional producing fluorescence when co-expressed. The BiFC probes could report on increases in ER-mitochondrial contact sites, however, changes in mitochondrial morphology were observed and suggested that the BiFC and ddGFP systems has some degree of irreversibility, increasing ER-mitochondrial contact sites. This study reveals an interesting link between phosphoinositide metabolism and mitochondrial quality control and high lights a new tool to potentially investigate this further. PINK1 and Parkin are two genes which are commonly mutated in autosomalrecessive juvenile PD (AR-JP). They function in a common mitochondrial quality control pathway to regulate mitochondrial homeostasis. Under mitochondrial depolarization PINK1, a serine/threonine kinase, is stabilized on the outer mitochondrial membrane (OMM) where it recruits Parkin, an E3 ubiquitin ligase, from the cytosol. Parkin then ubiquitinates OMM proteins to aid in the recruitment of the phagophore to the mitochondria and the process of mitophagy. More recently reports have suggested that PINK1 and Parkin are also present at ER-mitochondrial contact sites and may contribute to mitochondrial homeostasis by altering contacts. SYNJ1, a neuronal phosphatidylinositol phosphatase, is also mutated in AR-JP and fwd, a phosphatidylinositol kinase, was reported to alter mitochondrial morphology in Drosophila cells. Together SYNJ1/synj and PI4KB/fwd act to modulate the phosphatidylinositol balance, therefore it was hypothesized that PI metabolism may be a regulatory factor in the PINK1/Parkin pathway and mitochondrial quality control. To examine the effects of manipulating synj or fwd, Drosophila and YFPParkin HeLa cells models were used to investigate genetic interactions with pink1 and parkin. A further aim was to develop a new tool to visualize ER-mitochondrial contact sites in real time, which would allow visualization of dynamic functions of ER-mitochondrial contact sites. This study found that the loss of fwd in vivo caused a significant decrease in climbing but not flight ability in adult Drosophila. This was not thought to be attributed to changes in mitochondrial morphology. However, the overexpression of fwd resulted in a partial rescue of pink1B9 and park25 climbing defect. The silencing of PI4KB and SYNJ1 in YFP-Parkin HeLa cells slowed Parkin translocation and mitophagy however loss of SYNJ2 did not. Furthermore, Bimolecular Fluorescence complementation (BiFC) and dimerization dependent GFP (ddGFP) were tested for visualizing of ER-mitochondrial contact sites. Both systems were functional producing fluorescence when co-expressed. The BiFC probes could report on increases in ER-mitochondrial contact sites, however, changes in mitochondrial morphology were observed and suggested that the BiFC and ddGFP systems has some degree of irreversibility, increasing ER-mitochondrial contact sites. This study reveals an interesting link between phosphoinositide metabolism and mitochondrial quality control and high lights a new tool to potentially investigate this further.
Supervisor: De Vos, K. J. ; Whithworth, A. J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.737862  DOI: Not available
Share: