Functional Characterization of Proteins Regulating Actin Assembly

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

A very large, ever?increasing repertoire of actin?binding proteins regulates the assembly dynamics and the spatial organization of actin filaments, thus orchestrating the motile behavior of the cell. The authors describe a series of biochemical functional assays that allow one to characterize the function of a putative actin?binding protein in actin filament dynamics. These tests allow the characterization of three types of actin?binding proteins: G?actin?sequestering proteins, profilin?like proteins, and barbed?end capping proteins. Biochemical tests include the use of sedimentation of actin filaments, polymerization assays at the barbed or pointed end of actin filaments derived from fluorescently labeled actin, thermodynamic measurements of actin assembly at steady state and during turnover of actin filaments, measurements of nucleotide exchange on G?actin, and the use of the intrinsic or extrinsic fluorescence of actin to measure direct binding of different protein ligands to G?actin.

Keywords: actin; polymerization; actin?binding proteins; ADF/cofilin; profilin; actin?sequestering ??thymosins; capping proteins

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Basic Protocol 1: Co‐sedimentation Assay for Measuring Binding of a Protein to F‐actin (or G‐actin)
Basic Protocol 2: Direct Binding to G‐actin: Fluorescence Measurements
Basic Protocol 3: Binding to Actin Derived from a Change in the Rate of Nucleotide Dissociation From G‐actin
Basic Protocol 4: Steady‐State Measurements of Actin Assembly: Critical Concentration Plots
Basic Protocol 5: Initial Rate of Filament Growth at Barbed or Pointed Ends
Basic Protocol 6: Measurement of Dilution‐Induced Depolymerization of Filaments
Basic Protocol 7: Measurements of the Treadmilling of Actin Filaments
Support Protocol 1: Actin Purification from Rabbit Muscle
Support Protocol 2: Preparation of Pyrenyl‐Labeled Actin
Support Protocol 3: Preparation of 7‐Chloro‐4‐Nitrobenzeno‐2‐Oxa‐1,3‐Diazole‐Labeled Actin
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Co‐sedimentation Assay for Measuring Binding of a Protein to F‐actin (or G‐actin)

Materials

50 µM G‐actin in G‐buffer (see protocol 8 ), store on ice
2 M KCl
20 mM MgCl₂ ( appendix 2A )
100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT)
10 mM Tris·Cl (pH 7.5)/1 mM DTT
F‐buffer (see recipe )
G‐buffer (see recipe )

0.5‐ml polycarbonate centrifuge tubes
Beckman TL100 tabletop ultracentrifuge (or equivalent)
Densitometer

Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (unit 6.1 )

Basic Protocol 2: Direct Binding to G‐actin: Fluorescence Measurements

Materials

1.5 µM 100% fluorescently labeled G‐actin in G‐buffer (see Support Protocols protocol 92 and protocol 103 )
Actin‐binding protein of interest in G‐buffer (see recipe )
Spectrofluorometer (time base mode) at wavelengths appropriate for label.

Basic Protocol 3: Binding to Actin Derived from a Change in the Rate of Nucleotide Dissociation From G‐actin

Materials

50% suspension of Dowex‐1‐X8 (Sigma) or AG‐1‐X8 (Bio‐Rad) strong anion exchange resin in G₀ ‐buffer
50 µM G‐actin in G‐buffer (see protocol 8 )
G₀ ‐buffer: G‐buffer (see recipe ) without ATP
1 mM etheno‐ATP (ɛ‐ATP), pH adjusted to 7.2 with NaOH, stock solution (Sigma)
100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT)

Microcentrifuge, 4°C
Spectrophotometer and quartz cuvettes
Spectrofluorometer (time base mode): λ_ex = 350 nm, λ_em = 410 nm, slits = 5‐ to 10‐nm band width

Basic Protocol 4: Steady‐State Measurements of Actin Assembly: Critical Concentration Plots

Materials

50 µM pyrenyl‐labeled G‐actin (see protocol 9 ), 10% labeled
G‐buffer (see recipe )
Gelsolin solution: 100 µM gelsolin (Sigma) in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT), for assaying actin with capped barbed ends only
Protein of interest
KM solution: 500 µl of 4 M KCl/20 µl of 1 M MgCl₂ ( appendix 2A )
Spectrofluorometer (time base mode): λ_ex = 360 nm, λ_em = 407 nm, slits = 5‐ to 10‐nm band width

Basic Protocol 5: Initial Rate of Filament Growth at Barbed or Pointed Ends

Materials

Actin seeds (choose one):
- 100 nM gelsolin‐actin (for pointed‐end growth): add 2.5 molar equiv G‐actin to gelsolin in G‐buffer
- 5 nM spectrin‐actin isolated from human blood (Casella et al., ; for barbed‐end growth) in G‐buffer
- 5 µM F‐actin (with or without 20 nM gelsolin)
KME solution (see recipe )
5 µM pyrenyl‐labeled (10%) G‐actin (see protocol 9 )
G‐buffer (see recipe )
100 µM protein of interest in 10 mM Tris⋅Cl (pH 7.5)/1 mM dithiothreitol (DTT)

Microcuvettes suitable for spectrofluorometer
Spectrofluorometer (time base mode): λ_ex = 366 nm, λ_em = 407 nm, slits = 5‐ to 10‐nm band width

Additional reagents and equipment to generate a calibration curve (see protocol 4 )

Basic Protocol 6: Measurement of Dilution‐Induced Depolymerization of Filaments

Materials

G‐buffer (see recipe )
4 M KCl
100 mM MgCl₂ ( appendix 2A )
50 µM G‐actin in G‐buffer (see protocol 8 )
50 µM G‐actin, 50% to 100% pyrenyl labeled in G‐buffer (see protocol 9 )
Gelsolin solution: 100 µM gelsolin in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT), for assaying depolymerization from pointed ends only
100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM DTT
F‐buffer (see recipe )

Microcuvettes, suitable for spectrofluorometer
Spectrofluorometer (time base mode): λ_ex = 366 nm, λ_em = 407 nm, slits = 5‐ to 10‐nm band width

Basic Protocol 7: Measurements of the Treadmilling of Actin Filaments

Materials

50% (w/v) suspension of Dowex‐1‐X8 (Sigma) or AG‐1‐X8 (Bio‐Rad) strong anion exchange resin in G₀ ‐buffer
50 µM G‐actin in G‐ buffer (see protocol 8 ), store on ice
5 mM etheno‐ATP (ɛ‐ATP), pH adjusted to 7.2 with NaOH, stock solution (Sigma)
KME solution (see recipe )
G‐buffer (see recipe )
100 µM protein of interest in 10 mM Tris·Cl (pH 7.5)/1 mM dithiothreitol (DTT)
200 mM ATP, pH adjusted to 7.2 with NaOH, stock solution (Sigma)

Microcentrifuge, 4°C
Spectrophotometer microcuvette, suitable for spectrofluorometer
Spectrofluorometer (time base mode; PM voltage adequate): λ_ex = 350 nm, λ_em = 410 nm, slits = 5‐ to 10‐nm band width

Support Protocol 1: Actin Purification from Rabbit Muscle

Materials

Acetone powder of rabbit muscle (Cytoskeleton)
Extraction buffer (see recipe ), 4°C
Solid KCl and 4 M KCl
Buffer D1 (see recipe )
1 M MgCl₂ ( appendix 2A )
G‐buffer (see recipe )

500‐ml beaker
Sorvall centrifuge and A641 rotor (or equivalent), 4°C
Glass wool
20°C water bath
Large (3‐cm wide) dialysis bags (MWCO 14,000)
Dounce homogenizer (glass‐Teflon; 20‐ml working volume)
Sonicator with microtip (e.g., Vibra‐cell; Sonics & Materials)
10‐ml ultracentrifuge tubes
Beckman ultracentrifuge and 70.1 Ti rotor (or equivalent), 4°C
2.5 × 100–cm gel filtration column (Superdex‐200 prep grade; Amersham Biosciences)
Spectrophotometer (290 nm) and quartz cuvettes

Support Protocol 2: Preparation of Pyrenyl‐Labeled Actin

4 mg/ml (∼95 µM) G‐actin in G‐buffer (see protocol 8 )
Buffer A (see recipe )
N ‐pyrenyliodoacetamide (NPI; Sigma)
Dimethylformamide
0.2 M dithiothreitol (DTT; appendix 2A )
Rotating wheel, 4°C

Support Protocol 3: Preparation of 7‐Chloro‐4‐Nitrobenzeno‐2‐Oxa‐1,3‐Diazole‐Labeled Actin

0.1 M KCl ( appendix 2A )
50 µM G‐actin in G‐buffer (see protocol 8 )
100 mM N ‐ethylmaleimide (NEM), prepare fresh
20 mM dithiothreitol (DTT; appendix 2A )
G‐buffer (see recipe ) with and without DTT and NaN₃
F‐buffer (see recipe ) without DTT and NaN₃
10 mM 7‐chloro‐4‐nitrobenzeno‐2‐oxa‐1,3‐diazole (NBD‐Cl) in dimethylformamide
15°C water bath

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Figures

Figure 13.6.1 Treadmilling of the actin filament. This diagram emphasizes that the actin filament has a polar structure, with a barbed end and a pointed end. Under physiological ionic conditions, F‐actin is at a steady state with ATP‐G‐actin monomers. An energetic imbalance occurs between the two ends, because of ATP hydrolysis linked to F‐actin assembly. Monomer‐polymer exchanges at the two ends do not sum up to zero at each end; instead the barbed ends undergo net growth, balanced by equal depolymerization at the pointed end. This flux of subunits through the filament is called treadmilling. C _ss , steady‐state (critical) concentration; D, ADP‐actin; DPi, ADP‐P_i ‐actin; T, ATP‐actin.

View Image

Figure 13.6.2 Functional assays of a G‐actin‐sequestering protein. (A ) Steady state measurements of F‐actin in the presence of increasing amounts of the sequestering protein (see ). F‐actin (1.5 µM, 10% pyrenyl labeled) with free barbed ends (solid line) or gelsolin‐capped barbed ends (dashed line) was supplemented by the protein as indicated. Linear depolymerization was consistent with a K _d of 2 µM. (B ) Steady‐state measurements of F‐actin in the presence of the sequestering protein. Experiments were conducted as in A at 2, 1.5, and 1 µM F‐actin with free barbed ends. The parallel straight lines are consistent with a K _d of 2 µM. (C ) Critical‐concentration ( C _ss ) measurements for actin alone or in the presence of 2 µM sequestering protein. The sequestering protein shifts C _ss for both free and capped barbed ends, in agreement with the K _d obtained in B. Solid line, free barbed ends alone; short‐dashed line, capped barbed ends alone; dotted line, free barbed ends with sequestering protein; long‐dashed line, capped barbed ends with sequestering protein. (D ) Depolymerization rate measurements (see ). The solution of 2.5 µM F‐actin (50% pyrenyl labeled) with free barbed ends was diluted 20‐fold in F‐buffer at time zero in the absence (solid line) and presence (dotted line) of 10 µM sequestering protein. The sequestering protein has no effect on actin depolymerization. (E ) The sequestering protein inhibits pointed‐end growth of actin filaments (see ). The rate of pointed‐end growth was measured in the presence of 2.5 µM Mg‐ATP‐G‐actin, 2 nM gelsolin‐actin seeds, and the sequestering protein at the indicated concentration. The inhibition observed is consistent with a K _d of 2 µM. (F ) The sequestering protein inhibits barbed‐end growth of actin filaments. The rate of barbed‐end growth was measured in the presence of 2.5 µM Mg‐ATP‐G‐actin and 0.16 nM spectrin‐actin seeds. The inhibition observed is consistent with a K _d of 2 µM, as in E. (G ) Co‐sedimentation assay (see ). F‐actin (10 µM) was sedimented alone or in the presence of increasing amounts of the sequestering protein. The sequestering protein depolymerizes F‐actin, leading to an increasing amount of G‐actin in the supernatant (S) and less G‐actin in the pellet (P). Lines shown in A‐F represent typical experimental curves, derived from known proteins (e.g., thymosin β₄ ).

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Figure 13.6.3 Functional assays of profilin‐like proteins (barbed‐end assembly‐promoting factors). (A ) Steady‐state measurements of F‐actin in the presence of increasing amounts of profilin‐like protein. The experiment was conducted as in Figure A. When barbed ends are capped (dashed line), the profilin‐like protein depolymerizes F‐actin. Linear depolymerization was consistent with a K _d of 2 µM. When the barbed ends are free (solid line), the profilin‐like protein fails to depolymerize F‐actin. (B ) Critical‐concentration ( C _ss ) measurements for actin alone and in the presence of 2 µM profilin‐like protein. The experiment was conducted as in Figure C. Solid line, free barbed ends alone; short‐dashed line, capped barbed ends alone; dotted line, free barbed ends with sequestering protein; long‐dashed line, capped barbed ends with sequestering protein. (C ) Profilin‐like protein inhibits pointed growth of actin filaments. The inhibition is consistent with a K _d of 2 µM. The experiment was conducted as in Figure E. (D ) The profilin‐like protein fails to inhibit barbed‐end growth of actin filaments. The experiment was conducted as in Figure F. (E ) Co‐sedimentation assay in the absence or presence of profilin‐like protein. The experiment was conducted as in Figure G. (F ) Depolymerization rate measurements in the absence (solid line) and presence (dotted line) of 10 µM profilin‐like protein. The experiment was conducted as in Figure D. The profilin‐like proteins have no effect on actin depolymerization. Lines shown in A‐D and F represent typical experimental curves derived from known proteins (e.g., profilin).

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Figure 13.6.4 Functional assays of capping proteins. (A ) Steady state measurements of F‐actin in the presence of increasing amounts of capping protein. The experiment was conducted as in Figure A. When barbed ends are free (solid line), capping proteins depolymerize F‐actin, partially blocking all barbed ends; the maximum amount of unpolymerized actin equals the critical concentration of the pointed end ( C _c = 0.6 µM, under physiological conditions). When barbed ends are capped (dashed line), addition of another capping protein has no effect on the steady‐state amount of F‐actin; all barbed ends remain capped and C _ss remains equal to the C _c at the pointed ends. (B ) C _ss measurements for actin alone or in the presence of capping proteins. The experiment was conducted as in Figure C. When barbed ends are capped, capping proteins have no effect on C _c . When barbed ends are free, capping proteins shift C _c to 0.6 µM, in physiological conditions. Solid line, free barbed ends alone; dotted line, free barbed ends with capping protein; dashed line, capped barbed ends alone or with capping protein. (C ) Capping proteins do not inhibit pointed‐end growth of actin filaments. The experiment was conducted as in Figure E. (D ) Capping proteins inhibit barbed‐end growth of actin filaments. The experiment was conducted as in Figure F. (E ) Depolymerization rate measurements. The experiment was conducted as in Figure D. Capping proteins block depolymerization at the barbed end. Dotted line, no capping protein; solid lines, capping protein present at indicated concentrations. (F ) Co‐sedimentation assay. The experiment was conducted as in Figure G. At a saturating amount of capping protein, the amount of G‐actin in the supernatant corresponds to the C _c at the pointed end. Lines shown in A‐E represent theoretical lines in each case.

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Literature Cited

Literature Cited
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Functional Characterization of Proteins Regulating Actin Assembly

Abstract

Table of Contents

Materials

Basic Protocol 1: Co‐sedimentation Assay for Measuring Binding of a Protein to F‐actin (or G‐actin)

Basic Protocol 2: Direct Binding to G‐actin: Fluorescence Measurements

Basic Protocol 3: Binding to Actin Derived from a Change in the Rate of Nucleotide Dissociation From G‐actin

Basic Protocol 4: Steady‐State Measurements of Actin Assembly: Critical Concentration Plots

Basic Protocol 5: Initial Rate of Filament Growth at Barbed or Pointed Ends

Basic Protocol 6: Measurement of Dilution‐Induced Depolymerization of Filaments

Basic Protocol 7: Measurements of the Treadmilling of Actin Filaments

Support Protocol 1: Actin Purification from Rabbit Muscle

Support Protocol 2: Preparation of Pyrenyl‐Labeled Actin

Support Protocol 3: Preparation of 7‐Chloro‐4‐Nitrobenzeno‐2‐Oxa‐1,3‐Diazole‐Labeled Actin

Figures

Videos

Literature Cited