title: Reconstitution of microtubule-dependent organelle transport.
abstract: Microtubule (MT)-based motor proteins transport many cellular factors to their functionally relevant locations within cells, and defects in transport are linked to human disease. Understanding the mechanism and regulation of this transport process in living cells is difficult because of the complex in vivo environment and limited means to manipulate the system. On the other hand, in vitro motility assays using purified motors attached to beads does not recapitulate the full complexity of cargo transport in vivo. Assaying motility of organelles in cell extracts is therefore attractive, as natural cargoes are being examined, but in an environment that is more amenable to manipulation. Here, we describe the purification and in vitro MT-based motility of phagosomes from Dictyostelium and lipid droplets from rat liver. These assays have the potential to address diverse questions related to endosome/phagosome maturation, fatty acid regulation, and could also serve as a starting point for reconstituting the motility of other types of organelles.
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, Maharashtra, India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Barak
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, Maharashtra, India.
authors.forename: Ashim
authors.initials: A
authors.lastname: Rai
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, Maharashtra, India.
authors.forename: Alok Kumar
authors.initials: AK
authors.lastname: Dubey
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, Maharashtra, India.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Rai
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, Maharashtra, India. Electronic address: roop@tifr.res.in.
authors.forename: Roop
authors.initials: R
authors.lastname: Mallik
mesh_terms: D000818:Animals; D001692:Biological Transport; D004023:Dictyostelium / Q000166:cytology* / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis*; D008099:Liver / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D052898:Optical Tweezers; D015388:Organelles / Q000378:metabolism*; D010588:Phagosomes / Q000378:metabolism*; D051381:Rats
chemical_list: D008055:Lipids
keywords: In vitro motility; Lipid droplets; Optical trap; Organelle motility; Phagosomes