title: Isolation of Latex Bead Phagosomes fromDictyosteliumforin vitroFunctional Assays.
abstract: We describe a protocol to purify latex bead phagosomes (LBPs) fromDictyosteliumcells. These can be later used for variousin vitrofunctional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport onin vitropolymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction forin vitromotility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance.
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
authors.forename: Ashwin
authors.initials: A
authors.lastname: D'Souza
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
authors.forename: Paulomi
authors.initials: P
authors.lastname: Sanghavi
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
authors.forename: Ashim
authors.initials: A
authors.lastname: Rai
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
authors.forename: Divya
authors.initials: D
authors.lastname: Pathak
authors.affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
authors.forename: Roop
authors.initials: R
authors.lastname: Mallik
keywords: Dictyostelium; Dynein; Kinesin; Phagosome maturation; in vitro motility