Methods in Enzymology

Volume 134

Structural and ContractileProteins

Part C

The Contractile Apparatus and the Cytoskeleton

Richard B . Vallee

CONTRIBUTORS TO VOLUME 134 Xi

PREFACE XVii

VOLUMES IN SERIES X1X

Section I. Actin-Associated Proteins

1 . Preparation and Assay of Cytoplasmic and Se- H . L . YIN

3

creted Gelsoli n

2. Purification of Severin

SUSAN S . BROWN

9

3 . Isolation of Fascin, an Actin-Bundling Protein, JOSEPH BRYAN

1 3

and SU45, an Actin-Severing/Capping Proteinfrom Sea Urchin Egg s

4 . Purification of the Intestinal Microvillus Cyto- ANTHONY BRETSCHER

24

skeletal Proteins Villin, Fimbrin, and Ezrin

5. Purification of Caldesmon

WILLIAM LYNCH AN D

ANTHONY BRETSCHER

3 7

6 . Purification of Fodrin from Mammalian Brain

RICHARD CHENEY ,

JOEL LEVINE ,AND MARK WILLARD

42

7 . Purification of Brain Analogs of Red Blood Cell VANN BENNETT ,

Membrane Skeletal Proteins : Ankyrin, Protein ANTHONY J . BAINES, AND

4 .1 (Synapsin), Spectrin, and Spectrin Subunits JONATHAN DAVIS

5 5

8 . Purification and Assay of Vinculin, Metavinculin, T . O'HALLORAN, L . MOLONY ,

and Talin

AND K . BURRIDGE

69

9 . Isolation of Metavinculin from Chicken Smooth JANET D ' ANGELO SILICIANO

Muscle

AND SUSAN W . CRAIG

78

Section II . Microtubules

A. Tubulin and Microtubule-Associated Protein s

10 . Reversible Assembly Purification of Microtubules RICHARD B . VALLEE

89

without Assembly-Promoting Agents and Fur-ther Purification of Tubulin, Microtubule-Asso-ciated Proteins, and MAP Fragments

11 . Purification of Brain Microtubules and Microtu- RICHARD B . VALLEE

104

bule-Associated Protein 1 Using Taxo l

12 . Purification of Microtubules and Microtubule- RICHARD B . VALLEE AN D

Associated Proteins from Sea Urchin Eggs and CHRISTINE A . COLLINS

11 6

Cultured Mammalian Cells Using Taxol, an dUse of Exogenous Taxol-Stabilized Brain Mi-crotubules for Purifying Microtubule-Associ-ated Protein s

13. Isolation of Sea Urchin Egg Tubulin

H . WILLIAM DETRICH, III

12 8

14 . Direct Identification of Microtubule-Associated FRANK SOLOMON

13 9Proteins by Selective Extraction of Culture dCell s

15 . Purification and Assay of Primate 210K Microtu- JEANNETTE CHLOE BULINSKI 14 7

bule-Associated Protei n

16. Purification of Tau Protein from Brain

DAVID DRUBIN AN D

MARC KIRSCHNER

15 6

17 . Purification and Assay of Cold-Stable Microtu- ROBERT L . MARGOLIS ,

bules and STOP Protein

CHARLES T . RAUCH, AN D

DIDIER JOB

16 0

18 . Isolation and Purification of Tubulin Tyrosine JÜRGEN WEHLAND ,

Ligase

HEINZ C . SCHRÖDER, AN D

KLAUS WEBER

17 0

19. Proteolytic Modification of Tubulin

LUIS SERRANO ,

FRANCISCO WANDOSEIL ,

JAVIER DE LA TORRE, AN D

JESUS AVILA

179

B . Microtubule-Containing and -Organizing Organelle s

20. Isolation of Sea Urchin Spindles and Cytasters

RYOxo KURIYAMA

190

21 . Isolation of Native, Membrane-Containing Mitotic ROBERT B . SILVER

200Apparatus from Sea Urchin Embryo s

22 . Isolation of Mitotic Spindles from Mammalian R . C . BRADY, M. J . SCHIBLER ,Cells

AND F . CABRAL

21 7

23 . Isolation of Mitotic Spindles and Spindle Pole JEREMY S . HYAMS AN DBodies from Yeast

STEPHEN M . KING

22 5

24. Isolation of the Erythrocyte Marginal Band

WILLIAM D. COHEN AN DMARY F . GINSBURG

23 2

25. Isolation of Basal Bodies from Chlarnydomonas WILLIAM J . SNELL

25 2

26. Isolation of Mammalian Centrosomes

T . J . MI I CHISON AN DM . W . KIRSCHNER

261

27 . Isolation of a Kinetochore-Centromere Fraction MANUEL M . VALDIVIA AND

from HeLa Metaphase Chromosomes

B . R . BRINKLEY

26 8

28 . Isolation of Chlamydomonas Flagella and Flagel- GEORGE B . WITMAN

28 0lar Axonemes

C . Dynein

29 . Purification and Characterization of Chlamydomo- STEPHEN M . KING ,

nas Flagellar Dyneins

TIM OTTER ,

AND GEORGE B . WITMAN

29 1

30 . Preparation and Properties of Dynein from Tetra- KENNETH A . JOHNSON

30 6hymena Cilia

31 . Extraction and Properties of Dyneins from Bull CLAUDE GAGNON

31 8Spermatozo a

32 . Purification of Cytoplasmic Dynein from Strongy- M. M . PRATT

32 5locentrotus Sea Urchin Eggs

33 . Purification of Cytoplasmic Dynein from Sea Ur- SHIN-ICHI HISANAGA AND

chin Eggs

HIKOICHI SAKAI

33 7

Section III . Intermediate Filaments and Other Cytoskeletal Element s

34 . Separation of Cytokeratin Polypeptides by Gel THOMAS ACHTSTAETTER,

Electrophoretic and Chromatographic Tech- MECHTHILD HATZFELD ,

niques and Their Identification by Immunoblot- RoY A . QUINLAN ,

ting

DAVID C . PARMELEE, AN D

WERNER W . FRANKE

355

35 . Isolation of Intermediate Filaments from Neuro- ROBERT V . ZACKROFF

37 1nal Tissues by Cycles of Assembly/Disassembl y

36 . Purification of Neurofilaments and Their Constitu- RONALD K. H . LIEM

380ent Polypeptide s

37 . Isolation of Paired Helical Filaments and Amyloid DENNIS J . SELKOE AN D

Fibers from Human Brain

CARMELA R . ABRAHAM

38 8

38. Methods for Studying Spasmin

W . BRAD Amos

404

39 . Isolation of the Calcium-Modulated Contractile J . L. SALISBURY ,

Protein of Striated Flagellar Roots

K . W . AEBIG ,

AND D. E . COLING

408

40. Isolation of Nematode Major Sperm Proteins

SAMUEL WARD AND

MICHAEL R . KLASS

414

Section IV. Immunological, Pharmacological, and Fluorescence Probe s

41 . Antibody Inhibitors of Nonmuscle Myosin Func- DANIEL P . KIEHART ,

tion and Assembly

DONALD A . KAISER, AN D

THOMAS D . POLLARD

42 3

42 . Preparation of Antibodies Reactive with Specific JEANNETTE CHLOE BULINSK I

Regions of Cytoskeletal Proteins

AND GREGG G . GUNDERSEN 45 3

43 . Purification of Isoform-Selective Actin Antibody SUSAN W . CRAIG ,

from Polyclonal Antiserum

SHEILA K . SANDERS, AN D

JOSE V . PARDO

460

44 . Analysis of Cytoskeletal Structures Using Blot- J . B . OLMSTED

467Purified Monospecific Antibodie s

45 . Preparation of N-Ethylmaleimide-Moditied Heavy W . ZACHEUS CANDE

473Meromyosin and Its Use as a Functional Probeof Actomyosin-Based Motility

46 . Erythro-9-13-(2-hydroxynonyl)]adenine and Vana- STEPHEN M . PENNINGROTH

477date as Probes for Microtubule-Based Cytoske-letal Mechanochemistry

47 . Preparation of a Fluorescent Analog : Acetamido- JOHN R . SIMON AN Dfluoresceinyl-Labeled Dictyostelium discoid- D . LANSING TAYLOR

487eum a-Actinin

48 . Preparation, Assay, and Microinjection of THOMAS E . KREIS

507Fluorescently Labeled Cytoskeletal Proteins :Actin, a-Actinin, and Vinculin

49. Preparation and Characterization of Fluorescent PATRICIA WADSWORTH AN D

Analogs of Tubulin

E . D. SALMON

51 9

Section V. Analysis of Function, Distribution, and Structure o fCytoskeletal Proteins

50 . Myosin Movement in Vitro : A Quantitative Assay MICHAEL P . SHEETZ ,

Using Oriented Actin Cables from Nitella

STEVEN M . BLOCK, AND

JAMES A . SPUDICH

53 1

51 . Methods to Study the Microtubule-Neurofilament ERIC J . AAMODT AN D

Network in Vitro

ROBLEY C . WILLIAMS, JR .

544

52 . Gel Overlay Methods for Detecting Specific Pro- JOANN J . OTTO

55 5tein-Protein Interactions

53 . Viewing Single Microtubules by Video Light Mi- BRUCE J . SCHNAPP

56 1croscopy

54. Agar Overlay Method : High-Resolution Immuno- YOSHIO FuKul ,fluorescence for the Study of the Contractile SHIGEHIKO YUMURA ,Apparatus

TOSHIKO K . YUMURA ,AND HIROSHI Mm«

57 3

55 . Polyethylene Glycol Embedding Protocol for Im- JOHN J . WoLosEwicK

58 0munofluorescence Microscopy

56 . Preembedding for Localization by Electron Mi- J . DE MEY, G . LANGANGER ,

croscopy of Cytoskeletal Antigens in Cultured G . GEUENS, R . NUYDENS ,Cell Monolayers Using Gold-Labeled Anti- AND M . DE BRABANDER

592bodies

57. Quick-Freeze, Deep Etch of the Cytoskeleton

NOBUTAKA HIROKAWA

598

58 . Cryo-Electron Microscopy of Unstained Frozen- EVA-MARIA MANDELKO WHydrated Microtubules

AND ECKHARD MANDELKOW 61 2

59. X-Ray Diffraction of Cytoskeletal Fibers

ECKHARD MANDELKOW

63 3

60 . Time-Resolved X-Ray Scattering of Microtubule ECKHARD MANDELKOW AN DAssembly Using Synchrotron Radiation

JOAN BORDAS

657

61 . Rapid Kinetic Analysis of Mechanochemical KENNETH A . JOHNSON

677Adenosinetriphosphatase s

AUTHOR INDEX 70 7

SUBJECT INDEX 729