The long term goal of this project is to understand the regulation and physiological roles of autophagy, a process by which proteins, organelles and bulk cytoplasm are sequestered within autophagic vesicles and delivered to the lysosome for degradation. This process plays several distinct, vital roles in the cell, acting to recycle aged or damaged components, to provide a source of nutrients in response to starvation, and in some cases to initiate cell death. These cellular functions underlie the impact of autophagy on a broad range of human illnesses. Fundamental questions regarding autophagy remain to be addressed, including how autophagy is regulated by nutrients and other signals, how substrates are selectively targeted for degradation, and how autophagy can promote either cell survival or cell death in different contexts. The proposed studies will use genetic, biochemical and imaging-based approaches in the Drosophila system to help define mechanisms of autophagy regulation and its functions in an intact organism. Recent studies in Drosophila and mammalian cells have delineated key factors in a conserved signaling pathway that inhibits autophagy under favorable nutrient conditions. The central component of this nutrient-sensing pathway, the Ser/Thr protein kinase Target of Rapamycin (TOR), was shown to control the activity of a multicomponent complex containing the autophagy related kinase Atg1 and the phosphoprotein Atg13.
Specific aims of this project are to: 1) test potential models of Atg1/13 complex regulation, including the effects of phosphorylation, multimerization, and association with novel inhibitory cofactors. Potential roles of PKA in this regulation as well as the function of the Atg1-related kinase Ulk3 will be characterized. 2) investigate mechanisms and consequences of a novel self-reinforcing feedback signal by which Atg1 inhibits TOR signaling. 3) investigate mechanisms by which Atg1 overexpression leads to autophagy-dependent cell death. As preliminary indicate of a role for Jun kinase (Jnk) signaling, the mechanisms of autophagy induction by Jnk and the roles of autophagy in endogenous Jnk-mediated death will be explored. 4) identify and characterize the functions of novel autophagy regulators and substrates through genetic and proteomic screens. Experiments in these aims were selected for their likelihood of having a high impact on key questions important to the field of autophagy. This proposal makes use of recently developed reagents including knockouts of several autophagy-related (Atg) genes, in vivo markers of autophagic activity, and novel methods of genetic manipulation. Information gained from studies of autophagy in Drosophila will provide an important whole- animal complement to mammalian cell culture-based studies.

Public Health Relevance

Defects in autophagy lead to adverse effects on several areas of human health, including cancer, neurodegeneration, inflammatory disease, myopathies, and ischemic injury. Potential therapeutic approaches that involve inhibiting or potentiating autophagy will benefit from greater understanding of how regulators and effectors of autophagy interact to control this process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062509-13
Application #
8473223
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Maas, Stefan
Project Start
2001-03-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
13
Fiscal Year
2013
Total Cost
$303,523
Indirect Cost
$88,569
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Mauvezin, Caroline; Neisch, Amanda L; Ayala, Carlos I et al. (2016) Coordination of autophagosome-lysosome fusion and transport by a Klp98A-Rab14 complex in Drosophila. J Cell Sci 129:971-82
Braden, Christopher R; Neufeld, Thomas P (2016) Atg1-independent induction of autophagy by the Drosophila Ulk3 homolog, ADUK. FEBS J 283:3889-3897
Mauvezin, Caroline; Nagy, Péter; Juhász, Gábor et al. (2015) Autophagosome-lysosome fusion is independent of V-ATPase-mediated acidification. Nat Commun 6:7007
Kim, Jung; Neufeld, Thomas P (2015) Dietary sugar promotes systemic TOR activation in Drosophila through AKH-dependent selective secretion of Dilp3. Nat Commun 6:6846
Mauvezin, Caroline; Ayala, Carlos; Braden, Christopher R et al. (2014) Assays to monitor autophagy in Drosophila. Methods 68:134-9
Klionsky, Daniel J (author list too long to display - see original citation for additional authors) (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445-544
Neufeld, Thomas P (2012) Autophagy and cell growth--the yin and yang of nutrient responses. J Cell Sci 125:2359-68
Jimenez-Sanchez, Maria; Menzies, Fiona M; Chang, Yu-Yun et al. (2012) The Hedgehog signalling pathway regulates autophagy. Nat Commun 3:1200
Dimitroff, Brian; Howe, Katie; Watson, Adrienne et al. (2012) Diet and energy-sensing inputs affect TorC1-mediated axon misrouting but not TorC2-directed synapse growth in a Drosophila model of tuberous sclerosis. PLoS One 7:e30722
Chang, Yu-Yun; Neufeld, Thomas P (2010) Autophagy takes flight in Drosophila. FEBS Lett 584:1342-9

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