Centrosomes and Spindle Pole Bodies

366 Pages·2001·17.005 MB·1-375\366

by  Robert E. Palazzo, Trisha N. Davis (Eds.)

CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors' contributions begin.
David A. Agard (141), Department of Biochemistry and Biophysics and HHMI,
University of California-San Francisco, naS Francisco, California 94143
Bruce M. Alberts (141), National Academy of Sciences, Washington, DC 20418
Ron Balczon (257), Department of Cell Biology and Neuroscience, The University of
South Alabama, Mobile, Alabama 36688
Gary Borisy (43), Department of Cell Molecular and Structural Biology, Northwestern
University Medical School, Chicago, Illinois 60611
Michel Bornens (213, 289), Institut Curie, Section de Recherche, 75248 Paris Cedex
05, France
Michael B. Braunfeld (141), Department of Biochemistry and Biophysics and HHMI,
University of California-San Francisco, naS Francisco, California 94143
W. Zacheus Cande (167), Department of Molecular and Cell Biology, University of
California-Berkeley, Berkeley, California 94720
Duane A. Compton (225), Department of Biochemistry, Dartmouth Medical School,
Hanover, New Hampshire 03755
Trisha N. Davis (95), Department of Biochemistry, University of Washington, Seattle,
Washington 98195
Alain Debec (113), Universite Pierre et Marie Curie, Observatoire Oceanologique de
Villefranche-sur-Mer, Biologie du Developpement, UMR 7009, BP 28, ehcHarfelliV
sur Mer 06234, France
Stephen J. Doxsey (53), Department of Pathology, University of Massachusetts Medical
School, Worcester, Massachusetts 01605
Alison J. Faragher (305), Department of Biochemistry, University of Leicester, Leicester
1EL 7RH, United Kingdom
Andrew M. Fry (305), Department of Biochemistry, University of Leicester, Leicester
1EL 7RH, United Kingdom
Thomas H. Giddings, Jr. (27), Department of Molecular, Cellular and Dew, lop-
mental Biology, Porter Biosciences, University of Colorado-Boulder, Boulder,
Colorado 80309
Ralph Gr/if (337), Adolf-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-
ti~tisrevinU Miinchen, D-80336 Miinchen, Germany
Ruwanthi N. Gunawardane (1), Department of Embryology, Howard Hughes Medical
Institute, Carnegie Institution of Washington, Baltimore, Maryland 21210; and Biology
Department, Johns Hopkins University, Baltimore, Maryland 21218
Rebecca Heald (241), Department of Molecular and Cell Biology, University of
California-Berkeley, Berkeley, California 94720
xi
Contributors
Edward H. Hinchcliffe ,)962( Department of Cell Biology, University of sttesuhcassaM
Medical School, Worcester, sttesuhcassaM 50610
Yasushi Hiraoka ,)761( Cell Biology Group, Kansai Advanced Research Center, Com-
munications Research Laboratory, Kobe 651-2492, Japan
C. Janke ,)17( The Beatson Institute for Cancer Research, Bearsden, Glasgow 16G ,DB1
United Kingdom
Harish C. Joshi (179), Department of Cell Biology, Emory University School of
Medicine, Atlanta, Georgia 22303
Carol Koyama (113), Department of Biology, University of California, Santa Cruz,
Sinshiemer Laboratories, Santa Cruz, California 46059
Ryoko Kuriyama (125), Department of Genetics, Cell Biology, and Development,
University of Minnesota, Minneapolis, Minnesota 55455
Wilma L. Lingle ,)523( Experimental Pathology and Tumor Biology Program, Mayo
Clinic, Rochester, Minnesota 50955
David N. Mastronarde (27), Boulder Laboratory for D3 Fine Structure, Porter
,secneicsoiB University of Colorado-Boulder, Boulder, Colorado 90308
Hirohisa Masuda ,)761( Cell Biology Group, Kansai Advanced Research Center, Com-
munications Research Laboratory, Kobe 651-2492, Japan
J. Richard Mclntosh (27), Department of Molecular, Cellular and Developmental
Biology and Boulder Laboratory for D3 Fine Structure, Porter ,secneicsoiB University
of Colorado-Boulder, Boulder, Colorado 90308
Michelle Moritz (141), Department of Biochemistry and Biophysics, University of
California-San Francisco, naS Francisco, California 34149
Mary Morphew (27), Boulder Laboratory for D3 Fine Structure, Porter ,secneicsoiB
University of Colorado-Boulder, Boulder, Colorado 90308
Elena Nadezhdina (43), Institute for Protein Research, Russian Academy of ,secneicS
Poushchino, Moscow Region ,292241 Russia
Berl R. Oakley ,)591( Departments of Molecular Genetics and Plant Biology, The Ohio
State University, Columbus, Ohio 01234
Karen Oegema (l), European Molecular Biology Laboratory, Cell Biology Programme,
D-69117 Heidelberg, Germany
Toshiro Ohta ,)521( Department of Genetics, Cell Biology, and Development, Univer-
sity of Minnesota, Minneapolis, Minnesota 55455
Eileen T. O'Toole ,)72( Boulder Laboratory for D3 Fine Structure, Porter ,secneicsoiB
University of Colorado-Boulder, Boulder, Colorado 90308
Yulia Ovechkina 1 ,)591( Department of Molecular Genetics, The Ohio State ,ytisrevinU
Columbus, Ohio 01234
Robert E. Palazzo ,)941( Department of Molecular Biosciences, University of ,sasnaK
Lawrence, Kansas 66045; and The Marine Biological Laboratory, Woods Hole,
sttesuhcassaM 34520
John Peloquin ,)34( Department of Cell Molecular and Structural Biology, Northwest-
ern University Medical School, Chicago, Illinois 11606
tni eserP :sserdda tnemtrapeD fo ygoloisyhP dna ,scisyhpoiB ytisrevinU fo ,notgnihsaW ,elttaeS notgnihsaW
59189
Contributors iii~.
Gang Peng (125), Department of Molecular Biosciences, University of ,sasnaK Lawrence,
sasnaK 66045
Matthieu Piel (289), Institute Curie, Section de Recherche, 75248 Paris Cedex 05,
France
German A. Pihan (53), Program in Molecular Medicine, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
Aruna Purohit (53), Department of Pathology, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
Blake Riggs (113), Department of Biology, University of California, Santa Cruz,
Sinshiemer Laboratories, Santa Cruz, California 95064
Vladimir Rodionov (43), Department of Physiology and Center for Biomedical Imag-
ing Technology, University of Connecticut Health Center, Farmington, Connecticut
06032
Jeffrey L. Salisbury (325), Tumor Biology Program, Mayo Clinic, Rochester,
Minnesota 55905
E. Schiebel (71), The Beatson Institute for Cancer Research, Bearsden, Glasgow G61
1BD, United Kingdom
Bradley J. Schnaekenberg (149), Department of Molecular Biosciences, University of
,sasnaK Lawrence, Kansas 66045
C. Schramm (71), The Beatson Institute for Cancer Research, Bearsden, Glasgow 16G
1BD, United Kingdom
Takehiko Shibata (167), Cellular and Molecular Biology Laboratory, RIKEN (The
Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-01~8,
Japan
Greenfield Sluder (269), Department of Cell Biology, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
William Sullivan (113), Department of Biology, University of California, Santa Cruz,
Sinshiemer Laboratories, Santa Cruz, California 95064
Saeko Takada (167), Cellular and Molecular Biology Laboratory, RIKEN (The Insti-
tute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama ,8)q10-153
Japan
Fr6d~ric Tournier (213), Laboratoire de Cytophysiologie et Toxicologie ,erialuHeC
Universit6 Paris ,7 75251 Paris Cedex 05, France
Uyen Tram (113), Department of Biology, University of California, Santa Cruz,
Sinshiemer Laboratories, Santa Cruz, California 95064
Jacalyn Vogel (125), Department of Molecular Biosciences, University of ,sasnaK
Lawrence, Kansas 66045
Christiane Wiese (1), Howard Hughes Medical Institute, Department of Embryology,
Carnegie Institution of Washington, Baltimore, Maryland 21210
Sarah M. Wignall (241), Department of Molecular and Cell Biology, University of
California-Berkeley, Berkeley, California 94720
Mark Winey (27), Department of Molecular, Cellular and Developmental Biology, Porter
Biosciences, University of Colorado-Boulder, Boulder, Colorado 80309
Yixian Zheng (1), Howard Hughes Medical Institute, Department of ,¢qgoloyrbmE
Contributors
Carnegie Institution of Washington, Baltimore, Maryland, 21210; and Biology
Department, Johns Hopkins University, Baltimore, Maryland 21218
Jun Zhou (179), Department of Cell Biology, Emory University School of Medicine,
Atlanta, Georgia 30322
PREFACE
Centrosomes and spindle pole bodies are two representatives of a morphologically
diverse set of organelles that serve to organize the cytoplasm of eukaryotic cells. These
organelles are known to organize microtubules and have been implicated in diverse cell
processes including meiosis, fertilization, mitosis, cell morphogenesis, and diseases such
as cancer. In spite of their significance, and over one hundred years of research, we are
only now beginning to make significant progress in our understanding of the molecular
composition, mechanisms of assembly, and regulation of these important organelles.
Since cells typically contain only one such organelle, which precisely replicates once
and only once during the cell cycle, the low titer of material contained within cells has
posed a major barrier to progress. However, the past decade has lead to the establishment
of a number of experimental model systems to overcome this barrier, providing the ge-
netic and biochemical approaches outlined in this book. The following chapters describe
numerous methods for exploration using a diverse set of experimental model systems,
offering an introduction to those entering this exciting field, and hopefully enhancing the
progress of those already immersed in it. The assembly of this book was made possible
by the dedication, hard work, and generosity of the investigators involved. This book is
one testimony to the indomitable dedication of these researchers who continue to show
considerable courage in facing one of the most challenging problems in cell and develop-
mental biology; an understanding of the composition, replication, and regulation of the
function of centrosomes and spindle pole bodies and related structures in eukaryotic ceils.
XV
CHAPTER 1
Purification dna 1Keconstitution
alihposorD
of y-Tubulin Complexes
Ruwanthi N. Gunawardane, *,t Yixian Zheng,*4
Karen Oegema,* and Christiane Wiese*
drawoH* Hughes Medical Institute
Department of ygoloyrbmE
Carnegie Institution of notgnihsaW
Baltimore, Maryland 01212
t Biology Department
snhoJ Hopkins ytisrevinU
,eromitlaB Maryland 81212
naeporuE Molecular Biology Laboratory
Cell Biology emmargorP
D-69117 Heidelberg, Germany
I. Introduction
II. Isolation of v-Tubulin-Containing Complexes from alihposorD Embryo Extracts
A. Preparation of Embryo Extracts
B. Isolation of y-Tubulin-Containing Complexes from Embryo Extracts
III. yTuSC Reconstitution in Sf9 Cells
A. Sf9 Cell Culture
B. Virus Preparation (Molecular Biology)
C. Expression and Purification of yTuSC Components in Sf9 Cells
.VI Functional Assays for Isolated yTuSC or yTuRC
A. Buffers, Solutions, and Special Equipment
B. Microtubule Nucleation Assays
C. Capping Assay
D. Direct Labeling ofyTuRC or yTuSC
.V Concluding Remarks
References
METHODS IN CELL BIOLOGY, VOL. 67
1
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
0091-679X/01 $35.00
2 Ruwanthi N. Gunawardane et al.
I. Introduction
y-Tubulin was the first new member of the growing "tubulin superfamily" of edfl-
tubulin homologous proteins. It was originally identified as an extragenic suppressor of
a fl-tubulin mutation in Aspergillus nidulans (Oakley and Oakley, 1989; Oakley et al.,
1990). Since then, it has become clear that y-tubulin is a ubiquitous eukaryotic protein
that plays an essential role in microtubule (MT) nucleation (Zheng et al., 1991; Stearns
et al., 1991; reviewed in Wiese and Zheng, 1999).
y-Tubulin is found at MT organizing centers and, to a lesser extent, is associated
with spindle MTs (reviewed by Pereira and Schiebel, 1997). In addition, soluble cy-
toplasmic complexes containing y-tubulin have been characterized in several systems
(reviewed in Wiese and Zheng, 1999). The "basic" unit of y-tubulin assembly (Oegema
et al., 1999) appears to be a heterotetramer containing two y-tubulin subunits and one
each of two additional proteins (Knop and Schiebel, 1997). The proteins that asso-
ciate with y-tubulin in this small complex are homologous to each other and vary in
molecular mass between ~80 and 011 kDa, depending on the organism (reviewed in
Jeng and Stearns, 1999; Wiese and Zheng, 1999). The "prototype" core y-tubulin in-
teracting proteins were identified in yeast as the spindle pole components (Spc) 97
and 98, named according to their calculated molecular mass (Geissler et al., 1996;
Knop et al., 1997; Knop and Schiebel, 1997; reviewed in Pereira and Schiebel, 1997).
Together with y-tubulin, these proteins form the "Tub4p complex" (Tub4 is the Sac-
charomyces cerevisiae gene for y-tubulin; Sobel and Snyder, 1995; Marschall et al.,
1996; Spang et al., 1996). Biochemical characterization of y-tubulin-containing com-
plexes in higher eukaryotes revealed that a similar "small" complex exists (Oegema
et al., 1999), but, unlike yeast, the metazoan small complex appears to be a stable
structural subunit of a larger (~36S) complex that contains ~6 small complexes and
various stoichiometries of at least five additional proteins (Zheng et al., 1995; Martin
et al., 1998). The large y-tubulin complex was first purified from Xenopus egg ex-
tracts (Zheng et al., 1995), and subsequently from Drosophila embryos (Oegema et al.,
1999). Complexes of similar dimensions have been reported to exist also in surf clam,
mouse, human, and sheep (Vogel et al., 1997; Murphy et al., 1998; Tassin et al., 1998;
Detraves et al., 1997; Akashi et al., 1997). The ~2.2-MDa y-tubulin-containing pro-
tein complex isolated from Xenopus eggs or Drosophila embryos has a striking 25- to
28-nm diameter lock-washer shape, which prompted the name "y-tubulin ring complex,"
or yTuRC (Zheng et al., 1995). Interestingly, the shape and size of the yTuRC are very
reminiscent of the first turn of the MT helix. In keeping with our nomenclature, we refer
to the smaller subcomplex as yTuSC (y-tubulin small complex; Oegema et al., 1999).
One model for the relationship between the yTuSC and the yTuRC is shown in Fig. .1
The yTuRC is located mainly at the major MT organizing center, the centrosome,
where it is found at the minus ends of MTs (Moritz et al., 1995). The yTuRC is re-
cruited and tethered to the centrosome by a(n) unknown factor(s) (Moritz et al., 1998;
Schnackenberg et al., 1998). Consistent with a role in MT nucleation, yTuRC puri-
fied from Xenopus eggs (Zheng et al., 1995) or Drosophila embryos (Moritz et al.,
1998; Oegema et al., 1999) nucleates MTs in vitro (Zheng et al., 1995; Oegema et al.,
1999). yTuSC purified from Drosophila embryo extracts (Oegema et al., 1999) or from
.1 Purification of Complexes F-Tubulin 3
CRuT7 CSuT~
nilubut-y
spirgD ,48 19
~ spirgD ,821 ,361 s57
Fig. 1 lacitehtopyH pihsnoitaler neewteb CSuTF dna .CRuTF desaB no setamitse of eht yrtemoihciots
of stinubus eht gnehZ( te ,.la amegeO ;5991 te ,.la eht ni )9991 CSuTF dna ,CRuTF 5-7 CSuTF stinubus
elbmessa htiw eht lanoitidda spirg ot hcae mrof .CRuTF ehT tcaxe tnemegnarra of nilubut-y stinubus nihtiw
si CRuTF eht yltneserp .nwonknu evitanretlA stnemegnarra era elbissop ,.g.e( ees ,noskcirE .)0002
baculovirus-infected insect cells (Gunawardane et al., 2000) also nucleates MTs in ~itro,
although much less efficiently than the FTuRC. We have discovered that both the Xeno-
pus (Zheng et al., 1995; Wiese and Zheng, 2000) and the Drosophila FTuRC (C. Wiese,
K. Oegema, and .Y Zheng, unpublished observation), in addition to their nucleating ac-
tivity, also play a nucleation-independent role in capping the minus ends of MTs. This
capping activity has a profound effecto n MT stability, as it prevents both growth and
shrinkage from the MT minus end (Wiese and Zheng, 2000).
To clone the individual Xenopus and Drosophila FTuRC subunits, we took two
approaches: (1) we raised mouse polyclonal antibodies against purified FTuRCs and
(2) we obtained protein sequences for individual FTuRC subunits. To date, we have
cloned, sequenced, and characterized four of the Drosophila FTuRC subunits (which
we refer to as Drosophila gamma-ring proteins, or Dgrips; manuscript in preparation),
whereas only two of the Xenopus grips (Xgrips) have been cloned and sequenced
(Martin et al., 1998; Zhang et al., 2000). Thus, the reconstitution experiments de-
scribed in this chapter focus on the work we have done with the Drosophila F-tubulin
complexes.
II. Isolation of'y-Tubulin-Containing Complexes
from Drosophila Embryo Extracts
A protocol for isolating FTuRC from Xenopus egg extracts has been described
(Zheng et al., 1998). Here, we describe protocols for isolating F-tubulin-containing
complexes from Drosophila embryo extracts. Like the purification scheme for Xenopus
FTnRC, isolating FTuRC from Drosophila embryos involves an affipnuirtiyf ication step
using antipeptide antibodies specific for the C-terminal 71 amino acids of Drosophila
F-tubulin (Oegema et al., 1999; see also Field et al., 1998).
An advantage of using Drosophila extracts is that once fly population cages are estab-
lished, embryos are readily available in large quantities, and flies, unlike frogs, do not
require hormone injections. Another advantage is that Drosophila embryo extracts are
less variable in quality than Xenopus egg extracts.
4 Ruwanthi N. Gunawardane et al.
A. Preparation of Embryo Extracts
The protocol for preparing fly embryo extracts for yTuRC/yTuSC purification is sim-
ilar to the protocol used to prepare extracts for the purification of centrosomes from
alihposorD embryos (Moritz and Alberts, 1999). An overview over the procedure is
diagrammed in Fig. 2a.
.1 Buffers, Solutions, and Special Equipment
Population cages of "wild-type" flies
Molasses agar trays for embryo collections: For ~-. 01 trays, combine in a 6-liter flask:
2225 ml dH20, 360 ml unsulfhured molasses, and 88 g agar; autoclave for 30 min,
allow to cool, then add 74 ml of 5% Tegasept (mold inhibitor; p-hydroxybenzoic
acid methyl ester in 95% ethanol; Sigma #H-5501); pour into styrofoam meat trays
(Holt Paper Co., catalog #12S) and allow to solidify; store in plastic bags at 4°C;
trays are covered with a thin layer of yeast paste for collecting embryos
Yeast paste (dry baker's yeast): Hydrate dry yeast --q:l (mUg) in dH20 overnight and
stir; the consistency of this paste should be smooth and spreadable, but not too thin.
Sieves for emb D o collections and washing (VWR, Standard testing sieves, sizes #18,
40, and 100; catalog numbers 57334-450, 57334-460, and 57334-472, respectively)
Soft brush for scraping embryos from the molasses trays
Dounce homogenizer with 55-ml Wheaton jar
Wheaton overhead stirrer
5-ml screw-cap tubes with O rings (VWR, catalog number 66008-400)
Liquid nitrogen for snap-freezing the extract
Embryo wash: 0.03% Triton X-100, 0.04% NaC1
Freshly prepared 50% bleach
Homogenization buffer: 50 mM HEPES, pH 7.6, 100 mM NaC1, 1 mM ,21CgM 1 mM
EGTA, 10% glycerol (v/v); add 1 mM fl-mercaptoethanol (r-ME)just before use
Phenylmethylsulfonyl fluoride (PMSF): 100 mM in isopropanol; store at -20°C
wrapped in foil; heat to 37°C to redissolve before use
Protease inhibitor stock (200 x): 01 mMbenzamidine-HC1, 0.1 mg/ml phenanthroline,
1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin A in 100% ethanol;
store at -20°C; vortex briefly before use
2. Method
.1 Collect embryos for 3.5 to 4.5 h. We typically collect 0- to 4.5-h embryos from
two population cages (dimensions: 91 x 91 x 61 in.) on molasses trays covered with
a thin layer of yeast paste. A 4.5-h collection typically yields 01 to 51 g of embryos.
We place the trays with the embryos in a container with a tight-fitting lid and introduce
2OC to anesthetize any adult flies to minimize the impact of loose flies on neighboring
laboratories.
@-
)a(
Fly Embryo Extract Prep
.1 collect 0 to 3.5-h 2. collect embryos on 3. dechorionate 4. blot dry and
embryos on stacked sieves; wash in 50% bleach weigh; record
molasses plates with embrywoa sh for 2 min; weight
solution rinse extensively
with water to remove
bleach
J
..... i v
..... i
..... i
@ ..... i
..... i
5. place weighed 6. add equal amount 7. homogenize with a 8. aliquot into
embryos in (weight/volume) of tight-fitting teflon pestle, 5 ml aliquots;
prechilled 55 ml homogenization buffer; setting 4-5, 4 strokes freeze in screw-
Wheaton jar add 1/50 volume each capf reezer vials
of PMSF and PIs
(b) embryo or
SF9 cell clarify
I
extract
II I
discard PEG precipitate
I
II I
clarify ~ resuspend discard
I
II I
discard immunoprecipitate
I
II I
collect precipitate on beads discard
wash beads
leoend:
elute complex
I
II I
pellet supernatant
analyze
Fig. 2 (a) Flow chart of the fly embryo collection. (b) Flow chart of the purification scheme for FTuRC
and FTuSC, starting from embryo extract or cell lysate.