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postgraduate thesis: Role of Atg16L1 in the transport of autophagic vesicles in primary cultured neurons and novel kinases regulating autophagy in fission yeast

TitleRole of Atg16L1 in the transport of autophagic vesicles in primary cultured neurons and novel kinases regulating autophagy in fission yeast
Authors
Issue Date2016
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Zhou, P. [周鵬]. (2016). Role of Atg16L1 in the transport of autophagic vesicles in primary cultured neurons and novel kinases regulating autophagy in fission yeast. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5784849.
AbstractAutophagy is a cellular homeostatic degradation process for the removal of part of the cytoplasm, dysfunctional organelles, and protein aggregates. A large number of studies have reported abnormal accumulation of autophagic vesicles in the patients’ brains with neurodegenerative disorders. However, the cause of such accumulation is incompletely understood. Impairment in the transport of autophagic vesicles along the neurites has been associated with the neurodegenerative disorders. But the molecular mechanisms underlying the transport of autophagic vesicles have remained largely unclear. In this study, Atg16L1 has been discovered as a key protein involved in the transport of autophagic vesicles. Using the tandemly tagged LC3, a protein that is selectively recruited to autophagic vesicles, the effects of Atg16L1 knock-down on the transport of autophagic vesicles were examined by live cell imaging. Analysis of LC3 puncta movements revealed that Atg16L1 knock-down decreased the trafficking velocity of autophagic vesicles, particularly autolysosomes. In addition, quantification of autophagic vesicles revealed that Atg16L1 knock-down significantly increased the number of dendritic autolysosomes. To understand how Atg16L1 is involved in regulating the transport of autophagic vesicles, identification of novel Atg16L1 interacting proteins by mass spectrometry was performed. Among the identified proteins, the kinesin Kif21a is particularly interesting. The interaction between Atg16L1 and Kif21a was detected at the endogenous expression levels in rat brains. In addition, Kif21a co-localized with LC3 puncta. Interestingly, overexpression of Kif21a, but not Kif21a knock-down displayed similar effects on the transport of autophagic vesicles as Atg16L1 knock-down. This evidence, together with the finding that Kif21a knock-down could abrogate the effects caused by Atg16L1, suggests that Atg16L1 regulates autolysosome trafficking and degradation in a Kif21a-dependent manner. To further expand the list of proteins involved in autophagy, a screen using a fission yeast strain library containing 63 kinase deletion mutants was carried out, in which autophagy upon nitrogen starvation was examined. Many of the kinases appeared to be required for proper function of autophagy. Among the kinases, cki3 is intriguing because the absence of cki3 significantly decreased the number of autophagic vesicles upon nitrogen starvation. In addition, inhibition of Tor2 in cki3Δ cells increased the number of GFP-Atg8 puncta, suggesting that cki3 may regulate autophagy in a Tor2-dependent fashion. In summary, this study identified a new interaction between Atg16L1 and Kif21a and demonstrated the role of the interaction in the transport of autophagic vesicles in neurons. In addition, Cki3 has been demonstrated as a potential autophagy activator in S. pombe and it may act in a Tor2-dependent manner to initiate autophagy.
DegreeDoctor of Philosophy
SubjectAutophagic vacuoles
Dept/ProgramBiomedical Sciences
Persistent Identifierhttp://hdl.handle.net/10722/240168
HKU Library Item IDb5784849

 

DC FieldValueLanguage
dc.contributor.authorZhou, Peng-
dc.contributor.author周鵬-
dc.date.accessioned2017-04-14T23:12:26Z-
dc.date.available2017-04-14T23:12:26Z-
dc.date.issued2016-
dc.identifier.citationZhou, P. [周鵬]. (2016). Role of Atg16L1 in the transport of autophagic vesicles in primary cultured neurons and novel kinases regulating autophagy in fission yeast. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5784849.-
dc.identifier.urihttp://hdl.handle.net/10722/240168-
dc.description.abstractAutophagy is a cellular homeostatic degradation process for the removal of part of the cytoplasm, dysfunctional organelles, and protein aggregates. A large number of studies have reported abnormal accumulation of autophagic vesicles in the patients’ brains with neurodegenerative disorders. However, the cause of such accumulation is incompletely understood. Impairment in the transport of autophagic vesicles along the neurites has been associated with the neurodegenerative disorders. But the molecular mechanisms underlying the transport of autophagic vesicles have remained largely unclear. In this study, Atg16L1 has been discovered as a key protein involved in the transport of autophagic vesicles. Using the tandemly tagged LC3, a protein that is selectively recruited to autophagic vesicles, the effects of Atg16L1 knock-down on the transport of autophagic vesicles were examined by live cell imaging. Analysis of LC3 puncta movements revealed that Atg16L1 knock-down decreased the trafficking velocity of autophagic vesicles, particularly autolysosomes. In addition, quantification of autophagic vesicles revealed that Atg16L1 knock-down significantly increased the number of dendritic autolysosomes. To understand how Atg16L1 is involved in regulating the transport of autophagic vesicles, identification of novel Atg16L1 interacting proteins by mass spectrometry was performed. Among the identified proteins, the kinesin Kif21a is particularly interesting. The interaction between Atg16L1 and Kif21a was detected at the endogenous expression levels in rat brains. In addition, Kif21a co-localized with LC3 puncta. Interestingly, overexpression of Kif21a, but not Kif21a knock-down displayed similar effects on the transport of autophagic vesicles as Atg16L1 knock-down. This evidence, together with the finding that Kif21a knock-down could abrogate the effects caused by Atg16L1, suggests that Atg16L1 regulates autolysosome trafficking and degradation in a Kif21a-dependent manner. To further expand the list of proteins involved in autophagy, a screen using a fission yeast strain library containing 63 kinase deletion mutants was carried out, in which autophagy upon nitrogen starvation was examined. Many of the kinases appeared to be required for proper function of autophagy. Among the kinases, cki3 is intriguing because the absence of cki3 significantly decreased the number of autophagic vesicles upon nitrogen starvation. In addition, inhibition of Tor2 in cki3Δ cells increased the number of GFP-Atg8 puncta, suggesting that cki3 may regulate autophagy in a Tor2-dependent fashion. In summary, this study identified a new interaction between Atg16L1 and Kif21a and demonstrated the role of the interaction in the transport of autophagic vesicles in neurons. In addition, Cki3 has been demonstrated as a potential autophagy activator in S. pombe and it may act in a Tor2-dependent manner to initiate autophagy.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshAutophagic vacuoles-
dc.titleRole of Atg16L1 in the transport of autophagic vesicles in primary cultured neurons and novel kinases regulating autophagy in fission yeast-
dc.typePG_Thesis-
dc.identifier.hkulb5784849-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiomedical Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5784849-

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