Microfluidics‐Assisted TIRF Imaging to Study Single Actin Filament Dynamics

Shashank Shekhar1

1 Department of Biochemistry, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.13
DOI:  10.1002/cpcb.31
Online Posting Date:  December, 2017
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Abstract

Dynamic assembly of actin filaments is essential for many cellular processes. The rates of assembly and disassembly of actin filaments are intricately controlled by regulatory proteins that interact with the ends and the sides of filaments and with actin monomers. TIRF‐based single‐filament imaging techniques have proven instrumental in uncovering mechanisms of actin regulation. In this unit, novel single‐filament approaches using microfluidics‐assisted TIRF imaging are described. These methods can be used to grow anchored actin filaments aligned in a flow, thus making the analysis much easier as compared to open flow cell approaches. The microfluidic nature of the system also enables rapid change of biochemical conditions and allows simultaneous imaging of a large number of actin filaments. Support protocols for preparing microfluidic chambers and purifying spectrin‐actin seeds used for nucleating anchored filaments are also described. © 2017 by John Wiley & Sons, Inc.

Keywords: Actin dynamics; microfluidics; TIRF; single‐filament imaging; actin depolymerization

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of PDMS Microchambers
  • Basic Protocol 2: Growing Anchored Filaments with Free Barbed ENDS
  • Basic Protocol 3: Characterizing the Interaction of Regulatory Proteins at the Barbed Ends
  • Basic Protocol 4: Measuring the Concentration of Steady‐State Actin Monomers
  • Basic Protocol 5: Growing Anchored Filaments with Free Pointed Ends
  • Basic Protocol 6: Studying Filament Depolymerization from Pointed Ends and Severing by ADF
  • Support Protocol 1: Cleaning Microscope Coverslips
  • Support Protocol 2: Purification and Fluorescent Labeling of Skeletal Muscle Actin
  • Support Protocol 3: Purification of Spectrin‐Actin Seeds
  • Support Protocol 4: Cleaning and Maintenance of the Flow System and PDMS Microchambers
  • Support Protocol 5: Image Analysis and Preparation of Kymographs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of PDMS Microchambers

  Materials
  • Sylgard 184 Silicone Elastomer Kit (PDMS base & curing agent)
  • Molds for PDMS casting (commercially ordered)
  • 100% ethanol
  • 100‐ml glass beaker
  • Stirring spatula
  • Desiccator (BEL‐ART)
  • Vacuum pump
  • Plastic Petri dish
  • Oven (70°C)
  • Scalpel
  • PDMS puncher (Elveflow, France)
  • Kimwipe tissues
  • Parafilm

Basic Protocol 2: Growing Anchored Filaments with Free Barbed ENDS

  Materials
  • 1 mg/ml PLL‐PEG solution (see recipe)
  • TIRF buffer (see recipe)
  • Spectrin‐actin seeds (see protocol 9)
  • 10% (w/v) BSA solution (see recipe)
  • Purified G‐actin, 10% Alexa 488‐labeled (See protocol 8)
  • Purified profilin
  • Pre‐cleaned coverslips (see protocol 7)
  • Plasma cleaner (oxygen plasma)
  • PDMS microchambers ( protocol 1)
  • Compressed nitrogen/air
  • 20‐µl pipette
  • Microscrew reservoir tubes
  • Maesflow microfluidic flow‐control system (Fluigent, France)

Basic Protocol 3: Characterizing the Interaction of Regulatory Proteins at the Barbed Ends

  Materials (also see protocol 2)
  • Purified capping protein
The association rate constant (k on) of capping protein for barbed ends is determined as follows.

Basic Protocol 4: Measuring the Concentration of Steady‐State Actin Monomers

  Materials (also see protocol 2)
  • F‐actin solution (10% Alexa 488‐labeled)
  • Profilin
  • Regulatory proteins of interest (e.g., ADF, CP), purified as described in Shekhar and Carlier ( ) or purchased from Cytoskeleton Inc.

Basic Protocol 5: Growing Anchored Filaments with Free Pointed Ends

  Materials (also see protocol 2)
  • Purified gelsolin (for details, see Shekhar and Carlier, )
  • Modified TIRF buffer
  • G‐actin ( protocol 8)
  • 1 M magnesium chloride solution
  • 250 mM EGTA solution

Basic Protocol 6: Studying Filament Depolymerization from Pointed Ends and Severing by ADF

  Materials (also see protocol 5)
  • Purified actin depolymerizing factor (ADF; for details, see Shekhar and Carlier, )

Support Protocol 1: Cleaning Microscope Coverslips

  Materials
  • Deionized distilled water
  • Acetone
  • Ethanol
  • 2 M potassium hydroxide solution
  • Glass coverslips (40 × 24‐mm #1.5, Fisher Scientific)
  • Multi‐coverslip holder
  • Water bath sonicator

Support Protocol 2: Purification and Fluorescent Labeling of Skeletal Muscle Actin

  Materials
  • Rabbit muscle acetone powder (Pel‐Freez Biologicals/Cytoskeleton Inc.)
  • Extraction X buffer (see recipe)
  • Dialysis buffer D1 (see recipe)
  • Potassium chloride (KCl), powder and 4 M solution
  • G‐buffer (see recipe)
  • Modified F‐buffer (see recipe)
  • Alexa 488 succinimidyl ester (Thermo Fisher Scientific)
  • Glass beakers
  • Glass rod
  • Glass wool
  • Glass funnel
  • Water bath, 20° to 25°C
  • Thermometer
  • Dialysis membrane (12 to 14,000 kDa)
  • Potters (5‐ and 30‐ml)
  • Tip sonicator
  • 2.5 × 100‐cm gel filtration column (GE Life Sciences)

Support Protocol 3: Purification of Spectrin‐Actin Seeds

  Materials
  • Packed human red blood cells, ∼100 ml (acquired from a blood bank)
  • Washing buffers A and B (see reciperecipes)
  • Lysis buffer (see recipe)
  • Spectrin extraction buffer (see recipe)
  • 10% pyrene‐labeled CaATP‐G‐actin
  • G‐buffer (see recipe)
  • 20× KME solution (see recipe)
  • MgCl 2
  • EGTA
  • F‐buffer
  • DTT
  • Protease inhibitors
  • Glycerol
  • 50‐ml conical tubes
  • Measuring cylinders
  • Fluorimeter

Support Protocol 4: Cleaning and Maintenance of the Flow System and PDMS Microchambers

  Materials
  • 0.5 M sodium hydroxide (NaOH)
  • Deionized, distilled water
  • 100% ethanol
  • Compressed air/N 2

Support Protocol 5: Image Analysis and Preparation of Kymographs

  Materials
  • ImageJ image analysis software
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Figures

Videos

Literature Cited

 
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Internet Resources
  https://rsb.info.nih.gov/ij/
  ImageJ website.
  https://imagej.net/Multi_Kymograph
  ImageJ kymograph plug‐in.
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