In vitro motility system to study the role of motor proteins in receptor-ligand sorting.

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Abstract

This chapter presents fluorescence microscope assays that can be used to study microtubule (MT)-based movement and receptor-ligand sorting in vitro. The strategy is to isolate endosomes in a concentrated active form and store them in frozen aliquots for single use. Glass microchambers are then constructed and coated with fluorescent MTs, and the endosomes are thawed and bound to the MTs. Proteins of interest are then detected and quantified by immunofluorescence. For motility experiments, time-lapse movies are captured using multichannel fluorescence microscopy, and motility is initiated by the addition of ATP. Movies are later categorized and quantified for MT-based motility and other associated events such as endocytic fission. These techniques were developed to assess the role of MTs and MT motor proteins in endocytic processing within liver cells, and we have streamlined a rapid procedure for isolating abundant, highly motile endosomes from rat liver. Cultured cells and other organelles can also be examined, and many important biological questions concerning intracellular traffic and organelle composition can be studied by creative adaptation of the protocols that are presented.

Original languageEnglish (US)
Pages (from-to)143-158
Number of pages16
JournalMethods in molecular biology (Clifton, N.J.)
Volume392
StatePublished - 2007

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Endosomes
Motion Pictures
Ligands
Microtubules
Organelles
Microtubule Proteins
Proteins
Liver
Fluorescence Microscopy
Glass
Fluorescent Antibody Technique
Cultured Cells
Adenosine Triphosphate
Fluorescence
In Vitro Techniques

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

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abstract = "This chapter presents fluorescence microscope assays that can be used to study microtubule (MT)-based movement and receptor-ligand sorting in vitro. The strategy is to isolate endosomes in a concentrated active form and store them in frozen aliquots for single use. Glass microchambers are then constructed and coated with fluorescent MTs, and the endosomes are thawed and bound to the MTs. Proteins of interest are then detected and quantified by immunofluorescence. For motility experiments, time-lapse movies are captured using multichannel fluorescence microscopy, and motility is initiated by the addition of ATP. Movies are later categorized and quantified for MT-based motility and other associated events such as endocytic fission. These techniques were developed to assess the role of MTs and MT motor proteins in endocytic processing within liver cells, and we have streamlined a rapid procedure for isolating abundant, highly motile endosomes from rat liver. Cultured cells and other organelles can also be examined, and many important biological questions concerning intracellular traffic and organelle composition can be studied by creative adaptation of the protocols that are presented.",
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