title: An Automated Image Analysis System to Quantify Endosomal Tubulation.
abstract: Recycling of cargos from early endosomes requires regulation of endosomal tubule formation and fission. This regulation is disrupted in cells depleted of the microtubule severing enzyme spastin, causing elongation of endosomal tubules and mis-trafficking of recycling endosomal cargos such as the transferrin receptor. Spastin is encoded by SPAST, mutations in which are the most frequent cause of autosomal dominant hereditary spastic paraplegia, a condition characterised by a progressive loss of lower limb function resulting from upper motor neuron axonopathy. Investigation of molecular factors involved in endosomal tubule regulation is hindered by the need for manual counting of endosomal tubules. We report here the development of an open source automated system for the quantification of endosomal tubules, using ImageJ and R. We validate the method in cells depleted of spastin and its binding partner IST1. The additional speed and reproducibility of this system compared with manual counting makes feasible screens of candidates to further understand the mechanisms of endosomal tubule formation and fission.
authors.affiliation: Department of Medical Genetics and Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Newton
authors.affiliation: Department of Medical Genetics and Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Evan
authors.identifier: http://orcid.org/0000-0003-1623-7304
authors.initials: E
authors.lastname: Reid
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001331:Automation; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008870:Microtubules / Q000378:metabolism*; D000074181:Spastin
chemical_list: D000251:Adenosine Triphosphatases; D000074181:Spastin; C400283:SPAST protein, human