Determination of Membrane Protein Distribution on the Nuclear Envelope by Single‐Point Single‐Molecule FRAP

Krishna C. Mudumbi1, Weidong Yang1

1 Department of Biology, Temple University, Philadelphia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.11
DOI:  10.1002/cpcb.27
Online Posting Date:  September, 2017
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Nuclear envelope transmembrane proteins (NETs) are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM) in eukaryotic cells. The abnormal distribution of NETs has been associated with many human diseases. However, quantitative determination of the spatial distribution and translocation dynamics of NETs on the ONM and INM is still very limited in currently existing approaches. Here we demonstrate a single‐point single‐molecule fluorescence recovery after photobleaching (FRAP) microscopy technique that enables quick determination of distribution and translocation rates for NETs in vivo. © 2017 by John Wiley & Sons, Inc.

Keywords: Cellular imaging; membrane biophysics; super‐resolution; transmembrane protein; FRAP

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Table of Contents

  • Introduction
  • Basic Protocol 1: Tissue Culture and Preparation of Cells For Single‐Molecule and Confocal Microscopy Measurements
  • Basic Protocol 2: Single‐Point Single‐Molecule FRAP (smFRAP) Experiment
  • Basic Protocol 3: Bulk FRAP Imaging with Confocal Microscopy
  • Basic Protocol 4: Single‐Molecule Data Analysis
  • Support Protocol 1: Formulas Used for Diffusion Coefficient, Single‐Molecule Localization Precision, Spatial Concentration Distribution, and Translocation Rate for Nets
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Tissue Culture and Preparation of Cells For Single‐Molecule and Confocal Microscopy Measurements

  • HeLa cells (American Type Culture Collection)
  • Complete DMEM medium (see recipe)
  • TransIT‐LT1 Transfection Reagent (Mirus Bio, see manufacturer's protocol)
  • Plasmid coding for the protein of interest
  • Serum‐free DMEM medium (see recipe)
  • Transport buffer (see recipe)
  • 0.25% trypsin/EDTA
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 25‐cm2 culture flasks
  • 37°C, 5% CO 2 humidified incubator
  • Glass‐bottom dishes (MatTek Corporation)

Basic Protocol 2: Single‐Point Single‐Molecule FRAP (smFRAP) Experiment

  • Immersion oil
  • Glass‐bottom dish containing NETs of interest ( protocol 1)
  • 50‐mW solid‐state coherent 488‐nm laser (Obis)
  • Microscope: Olympus IX81 equipped with a 1.4 NA 100× oil immersion objective (UPLSAPO 100XO, Olympus)
  • 35 mW 633‐nm He‐Ne laser (Melles Griot)
  • Mercury lamp with GFP filter setup
  • Filters:
    • dichroic filter (Semrock, cat. no. Di01‐R405/488/561/635‐25x36)
    • emission filter (Semrock, cat. no. NF01‐405/488/561/635‐25X5.0)
    • two neutral density filters (Newport)
  • Optical chopper (Newport)
  • CCD camera: on‐chip multiplication gain charge‐coupled device camera (Cascade 128+, Roper Scientific)
  • Slidebook software package (Intelligent Imaging Innovations).
  • GLIMPSE software (Gelles lab; see protocol 5Support Protocol)
  • ThunderSTORM software (see protocol 5Support Protocol)

Basic Protocol 3: Bulk FRAP Imaging with Confocal Microscopy

  • Immersion oil
  • Glass‐bottom dish containing NETs of interest ( protocol 1)
  • Leica TCS Sp5 confocal microscope with HCX PL APO CS 100× 1.4 NA oil‐immersion objective equipped with a 100‐W, 488‐nm argon laser
  • ImageJ (NIH)
  • FRAP Profiler ImageJ plugin

Basic Protocol 4: Single‐Molecule Data Analysis

  • smFRAP data generated from protocol 2
  • ThunderSTORM Plugin for ImageJ (Ovesný, Křížek, Borkovec, Švindrych, & Hagen, )
  • GLIMPSE software package (Gelles Lab)
  • Origin 6.1
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Literature Cited

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