A protocol for single molecule imaging and tracking of processive myosin motors

Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell’s life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-...

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Main Authors: Lucia Gardini, Claudia Arbore, Marco Capitanio, Francesco Saverio Pavone
Format: Article
Language:English
Published: Elsevier 2019-01-01
Series:MethodsX
Online Access:http://www.sciencedirect.com/science/article/pii/S2215016119302146
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spelling doaj-468786961e25445a95a1de74b5f22b592020-11-25T01:48:50ZengElsevierMethodsX2215-01612019-01-01618541862A protocol for single molecule imaging and tracking of processive myosin motorsLucia Gardini0Claudia Arbore1Marco Capitanio2Francesco Saverio Pavone3LENS - European Laboratory for Non-Linear Spectroscopy, Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy; National Institute of Optics–National Research Council, Largo Fermi 6, 50125, Florence, ItalyLENS - European Laboratory for Non-Linear Spectroscopy, Via Nello Carrara 1, 50019, Sesto Fiorentino, ItalyLENS - European Laboratory for Non-Linear Spectroscopy, Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy; Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019, Sesto Fiorentino, Italy; Corresponding author at: LENS - European Laboratory for Non-Linear Spectroscopy, Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy.LENS - European Laboratory for Non-Linear Spectroscopy, Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy; National Institute of Optics–National Research Council, Largo Fermi 6, 50125, Florence, Italy; Department of Physics and Astronomy, University of Florence, Via Sansone 1, 50019, Sesto Fiorentino, ItalyMyosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell’s life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins. Protocol name: Single molecule motility assay, Keywords: Total internal reflection fluorescence (TIRF) microscopy, Single molecule biophysics, Single particle tracking, Quantum dots, Myosin-5Bhttp://www.sciencedirect.com/science/article/pii/S2215016119302146
collection DOAJ
language English
format Article
sources DOAJ
author Lucia Gardini
Claudia Arbore
Marco Capitanio
Francesco Saverio Pavone
spellingShingle Lucia Gardini
Claudia Arbore
Marco Capitanio
Francesco Saverio Pavone
A protocol for single molecule imaging and tracking of processive myosin motors
MethodsX
author_facet Lucia Gardini
Claudia Arbore
Marco Capitanio
Francesco Saverio Pavone
author_sort Lucia Gardini
title A protocol for single molecule imaging and tracking of processive myosin motors
title_short A protocol for single molecule imaging and tracking of processive myosin motors
title_full A protocol for single molecule imaging and tracking of processive myosin motors
title_fullStr A protocol for single molecule imaging and tracking of processive myosin motors
title_full_unstemmed A protocol for single molecule imaging and tracking of processive myosin motors
title_sort protocol for single molecule imaging and tracking of processive myosin motors
publisher Elsevier
series MethodsX
issn 2215-0161
publishDate 2019-01-01
description Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell’s life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins. Protocol name: Single molecule motility assay, Keywords: Total internal reflection fluorescence (TIRF) microscopy, Single molecule biophysics, Single particle tracking, Quantum dots, Myosin-5B
url http://www.sciencedirect.com/science/article/pii/S2215016119302146
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