Membrane contact sites are regions of proximity between organelles’ membranes involved in the exchange of material and information between them. They represent an important hub for the normal functioning of the cell and ultimately its health. Dysregulation in organelles fuction, especially mitochondria, has been found in many different neurodegenerative conditions among which Parkinson’s disease (PD). Early onset autosomal recessive forms of familiar PD are linked with mutations in genes whose protein products play a key role in the process of mitochondrial quality control. Among them, the gene encoding the mitochondrial serine threonine kinase PINK1 and the one encoding the cytosolic ubiquitin ligase Parkin are the most deeply investigated. While some indirect evidence for the involvement of PINK1 in the control of contact sites between mitochondria and endoplasmatic reticulum is present in the literature, nothing is known about the possibility that it could play a role in the regulation of contact sites between mitochondria and lysosomes, which are involved in the mitophagy pathway that is disfunctional in Parkinson’s disease, and even less is known about the contact sites with the nucleus. The purpose of the study presented in this thesis is to search for a causative effect of the overexpression of PINK1 on the number of contact sites between mitochondria and nucleus, mitochondria and lysosome and mitochondria and endoplasmatic reticulum membranes. A splitGFP palette of sensors (SPLICS S-P2A) developed in the laboratory was employed to monitor contact sites occurring at discrete locations. The SPLICS S-P2A method was used to count the contact sites in HeLa cells overexpressing myc-tagged PINK1 and statistical analysis allowed to assess the difference compared to the control cells that didn’t overexpress PINK1. A decrease in the number of contact sites involving the endoplasmatic reticulum was found, as previously reported, a weaker decrease concerning the number of mitochondria-nucleus contacts with more difference between individual cells, and no detectable change in the number of contacts with the lysosomes. More research is needed to have a better picture of the meaning of this reorganization of the contact sites and to untangle the factors behind it.
I siti di contatto fra membrane sono regioni di prossimità fra le membrane degli organelli coinvolte nello scambio di materiale ed informazioni fra di esse. Rappresentano un centro importante per il normale funzionamento della cellula ed in definitiva la sua salute. La disregolazione nel funzionamento degli organelli, specialmente per quanto concerne i mitocondri, è stata trovata in diverse malattie neurodegenerative fra cui la malattia di Parkinson (PD). Le forme autosomiche recessive familiari di PD ad insorgenza precoce sono state connesse a mutazioni in geni che esprimono per proteine le quali svolgono un ruolo chiave nel controllo di qualità mitocondriale. Fra questi geni, quello che codifica per la serina treonina chinasi mitocondriale PINK1 ed il gene codificante per l'ubiquitina ligasi citosolica Parkin sono i più studiati. Nonostante in letteratura sia presente evidenza indiretta del coinvolgimento di PINK1 per quanto riguarda il controllo dei siti di contatto fra mitocondri e reticolo endoplasmatico, non è noto un suo possibile ruolo nella regolazione dei siti di contatto fra mitocondri e lisosomi, i quali sono coinvolti nel pathway mitofagico che è disfunzionale nel PD, ed ancora meno si sa dei siti di contatto che interessano il nucleo. Lo scopo dello studio presentato in questa tesi è di cercare un effetto causale della sovraespressione di PINK1 sul numero di siti di contatto fra mitocondri e nucleo, mitocondria e lisosomi, mitocondri e reticolo endoplasmatico. Una gamma di sensori splitGFP (SPLICS S-P2A) sviluppata nel laboratorio è stata utilizzata per monitorare i siti di contatto che si verificano in posizioni discrete. Il metodo SPLICS S-P2A è stato usato per contare i siti di contatto in cellule HeLa sovraesprimenti PINK1 taggato myc e un'analisi statistica ha permesso di valutarne la differenza rispetto a cellule di controllo che non sovraesprimevano PINK1. Come già riscontrato, è stata rilevata una diminuzione nel numero di siti di contatto concernenti il reticolo endoplasmatico, una diminuzione più limitata per il numero di siti di contatto fra mitocondri e nucleo con più differenza fra singole cellule, nessun cambiamento significativo nel numero di contatti con i lisosomi. Per sviluppare un'immagine più chiara del significato della riorganizzazione dei siti di contatto riscontrata e per districare i fattori che ne sono all'origine sono necessarie ulteriori ricerche.
Exploring mitochondrial contact sites in PINK1 overexpressing cells
MUNERETTO, GIORGIO
2021/2022
Abstract
Membrane contact sites are regions of proximity between organelles’ membranes involved in the exchange of material and information between them. They represent an important hub for the normal functioning of the cell and ultimately its health. Dysregulation in organelles fuction, especially mitochondria, has been found in many different neurodegenerative conditions among which Parkinson’s disease (PD). Early onset autosomal recessive forms of familiar PD are linked with mutations in genes whose protein products play a key role in the process of mitochondrial quality control. Among them, the gene encoding the mitochondrial serine threonine kinase PINK1 and the one encoding the cytosolic ubiquitin ligase Parkin are the most deeply investigated. While some indirect evidence for the involvement of PINK1 in the control of contact sites between mitochondria and endoplasmatic reticulum is present in the literature, nothing is known about the possibility that it could play a role in the regulation of contact sites between mitochondria and lysosomes, which are involved in the mitophagy pathway that is disfunctional in Parkinson’s disease, and even less is known about the contact sites with the nucleus. The purpose of the study presented in this thesis is to search for a causative effect of the overexpression of PINK1 on the number of contact sites between mitochondria and nucleus, mitochondria and lysosome and mitochondria and endoplasmatic reticulum membranes. A splitGFP palette of sensors (SPLICS S-P2A) developed in the laboratory was employed to monitor contact sites occurring at discrete locations. The SPLICS S-P2A method was used to count the contact sites in HeLa cells overexpressing myc-tagged PINK1 and statistical analysis allowed to assess the difference compared to the control cells that didn’t overexpress PINK1. A decrease in the number of contact sites involving the endoplasmatic reticulum was found, as previously reported, a weaker decrease concerning the number of mitochondria-nucleus contacts with more difference between individual cells, and no detectable change in the number of contacts with the lysosomes. More research is needed to have a better picture of the meaning of this reorganization of the contact sites and to untangle the factors behind it.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/11144