title: Tracking autophagy during proliferation and differentiation ofTrypanosoma brucei.
abstract: Autophagy is a lysosome-dependent degradation mechanism that sequesters target cargo into autophagosomal vesicles. TheTrypanosoma bruceigenome contains apparent orthologues of several autophagy-related proteins including an ATG8 family. These ubiquitin-like proteins are required for autophagosome membrane formation, but our studies show that ATG8.3 is atypical. To investigate the function of other ATG proteins, RNAi compatibleT. bruceiwere modified to function as autophagy reporter lines by expressing only either YFP-ATG8.1 or YFP-ATG8.2. In the insect procyclic lifecycle stage, independent RNAi down-regulation of ATG3 or ATG7 generated autophagy-defective mutants and confirmed a pro-survival role for autophagy in the procyclic form nutrient starvation response. Similarly, RNAi depletion of ATG5 or ATG7 in the bloodstream form disrupted autophagy, but did not impede proliferation. Further characterisation showed bloodstream form autophagy mutants retain the capacity to undergo the complex cellular remodelling that occurs during differentiation to the procyclic form and are equally susceptible to dihydroxyacetone-induced cell death as wild type parasites, not supporting a role for autophagy in this cell death mechanism. The RNAi reporter system developed, which also identified TOR1 as a negative regulator controlling YFP-ATG8.2 but not YFP-ATG8.1 autophagosome formation, will enable further targeted analysis of the mechanisms and function of autophagy in the medically relevant bloodstream form ofT. brucei.
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: William R
authors.initials: WR
authors.lastname: Proto
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Nathaniel G
authors.initials: NG
authors.lastname: Jones
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G4 0RE, UK.
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
keywords: autophagy; cell death; differentiation; protozoan; trypanosome