A Novel Assay to Identify the Trafficking Proteins that Bind to Specific Vesicle Populations

Marvin Bentley1, Gary Banker1

1 Jungers Center for Neurosciences Research, Oregon Health & Science University, Portland, Oregon
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 13.8
DOI:  10.1002/0471143030.cb1308s69
Online Posting Date:  December, 2015
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Abstract

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12‐rapamycin binding domain (FRB)–tagged candidate vesicle‐binding protein that can be inducibly linked to an FKBP‐tagged molecular motor. If the FRB‐tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule‐organizing center and a specialized protocol designed to detect protein‐vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled. © 2015 by John Wiley & Sons, Inc.

Keywords: kinesin; vesicle; Rab; FKBP; FRB; membrane trafficking

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detecting Protein‐Vesicle Interactions Based on an Equilibrium Change in Vesicle Distribution
  • Basic Protocol 2: Detecting Protein‐Vesicle Interactions in Living Neurons Based on Directing Vesicles to the Axon
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detecting Protein‐Vesicle Interactions Based on an Equilibrium Change in Vesicle Distribution

  Materials
  • DMEM (or other appropriate cell culture medium) with and without 10% fetal bovine serum (FBS)
  • DNA construct encoding FKBP‐tagged motor protein (one of the following: GFP‐FLAG‐BicD2594‐FKBP, Addgene ID 63569; tdTM‐FLAG‐BicD2594‐FKBP, Addgene ID 64205; KIF5C559‐tdTM‐FKBP, Addgene ID 64211)
  • DNA constructs encoding FRB‐tagged candidate vesicle‐binding proteins
  • Vesicle label (DNA construct encoding fluorescently tagged protein, fluorescent dye, or immunofluorescence reagents)
  • Fugene 6 (Promega, cat. no. E2691) or other transfection reagent
  • Linker drug stock solution (AP21967, 0.5 mM in ethanol; A/C heterodimerizer, Clontech, cat. no. 635057)
  • Fixative (e.g., 4 % paraformaldehyde in PBS)
  • Mounting medium: Elvanol (Banker and Goslin, ) or Prolong Gold (Life Technologies, cat. no. P10144)
  • 18‐mm circular coverslips (Fisher Scientific, cat. no.12‐545‐84‐1D)
  • 12‐well tissue culture plates
  • Microscope slides
  • Fluorescence microscope equipped with 40× and 60× objectives and a camera
  • NOTE: Here we describe the materials we routinely use in performing this assay in a rat embryonic fibroblast cell line. Different cell cultures and different imaging methods may require some modifications to this list of materials.

Basic Protocol 2: Detecting Protein‐Vesicle Interactions in Living Neurons Based on Directing Vesicles to the Axon

  Materials
  • Isolated hippocampal neurons (Kaech and Banker, )
  • DNA construct encoding FKBP‐tagged Kinesin‐1 motor domain (KIF5C559‐tdTM‐FKBP, Addgene ID 64211)
  • DNA constructs encoding FRB‐tagged candidate vesicle‐binding proteins
  • Vesicle label (DNA construct encoding fluorescently tagged protein or fluorescent dye)
  • Lipofectamine 2000 (Life Technologies, cat. no. 11668027)
  • Imaging medium (Hibernate E Low Fluorescence; BrainBits, SKU: HE‐Lf)
  • Linker drug stock solution (AP21967, 0.5 mM in ethanol; A/C heterodimerizer, Clontech, cat. no. 635057)
  • 18‐mm glass coverslips (Fisher Scientific, cat. no. 12‐545‐84‐1D), poly‐L‐lysine coated (for detailed description, see Kaech and Banker, )
  • Fluorescence microscope: we use a Nikon Ti‐E microscope equipped with a Yokogawa CSU‐W1 spinning disk attachment, hardware‐based drift compensation to maintain focus, and an Andor Zyla sCMOS camera
  • Imaging chamber: Chamlide CMB for 18‐mm coverslips (Quorum Technologies, cat. no. CM‐B18‐1)
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Figures

Videos

Literature Cited

Literature Cited
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