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?????
???. 13: 399?434 (1997)
Review: An Overview of the Saccharomyces cerevisiae
Microtubule and Microfilament Cytoskeleton
BARBARA WINSOR1* AND ELMAR SCHIEBEL2*
1
Institut de Biologie Mole?culaire et Cellulaire, UPR 9005 du CNRS, 15 Rue Rene? Descartes,
67084 Strasbourg-cedex, France
2
Max-Planck Institut fu?r Biochemie, Genzentrum, Am Klopferspitz 18a, 82152 Martinsried, Germany
Received 2 October 1996; accepted 17 December 1996
Yeast 13: 399?434. 1997.
No. of Figures: 4. No. of Tables: 9.
No. of References: 240.
??? ????? ? S. cerevisiae; tubulin; MAPS; SPB; motor proteins; actin; ABP; ARP
CONTENTS
Introduction
The microtubule system
Tubulin
Microtubule-associated proteins
Motor proteins associated with yeast
microtubules
Non-motor proteins associated with
yeast microtubules
Spindle pole body
Components of the SPB
Microtubule nucleation
The microtubule system and the cell cycle
SPB duplication in G1 of the cell cycle
SPB separation and spindle formation
in S/G2 phase
Mitosis, chromosome segregation and
exiting from mitosis
The microfilament system
Actin
Proteins associated with microfilaments
Actin-binding proteins
Myosins and myosin-like proteins
Other proteins interacting with actin
Actin-related proteins
Distantly related ARPs
Regulation of the actin cytoskeleton
Cell-cycle dependence
Regulation by Rho and signalling
Interactions between microtubules and
microfilaments
References
*Correspondence to: Barbara Winsor or Elmar Schiebel.
CCC 0749?503X/97/050399?36 $17.50
? 1997 by John Wiley & Sons Ltd
INTRODUCTION
399
400
401
402
405
407
407
407
409
409
409
410
410
412
412
414
414
416
419
422
423
423
423
424
424
425
Microtubules and microfilaments are ubiquitous
in eukaryotic cells. Along with the intermediate
filaments and perhaps septins, they constitute the
principal components of the cytoskeleton. The
yeast cytoskeleton is involved in a variety of
essential cellular processes including cell division,
chromosome segregation, organelle movement
and positioning, bud formation, polarized growth,
secretion and endocytosis. Yeast is especially
suitable for the analysis of microfilament and
microtubule systems since the function and interrelationship of gene products can be studied by a
broad variety of biochemical, cell biology and
sophisticated genetic techniques. The availability
for Saccharomyces cerevisiae of the first complete
eukaryotic genome sequence, in addition to the
genetic methods for searching out interacting
proteins, stimulates the search for defining and
understanding all components of a particular system; a far-off perspective until very recently. The
perspective of a complete collection of the essential
genes in the cytoskeleton (and in other systems)
should allow identification and characterization of
new targets for drugs and toxic compounds.
Members of the family of proteins called the
septins are essential for neck filament assembly
and localize to the 10 nm neck filaments. They are
considered as elements of the cytoskeleton but will
not be described here as comprehensive reviews on
septins have appeared recently.46,133
Little is known about possible intermediate
filaments in yeast. However, putative homologues
?. ?????? ??? ?. ????????
400
Figure 1. Cell-cycle-dependent changes in spindle pole body. Shown is the morphology of yeast cells relative
to the spindle. The spindle is enlarged about three times. Details are given in the text. B, bridge; Bu, bud; C,
central plaque; CM, cytoplasmic microtubules; G1, G1 phase of the cell cycle; G2, G2 phase; H, half bridge;
I, inner plaque; M, M phase; Mo, mother cell; N, nuclear envelope; NM, nuclear microtubules; Nu, nucleus;
O, outer plaque; S, S phase; Sa, satellite.
of lamins A and B and the Erg24 protein which
has sequence similarity to the chicken lamin B
receptor (LBR) might be considered as candidates.74 In addition, the heterologous expression in
S. cerevisiae of human lamin B with the LBR,
showed co-localization to the nuclear envelope.
However, the chicken LBR gene did not
complement a Фerg24 mutation.202
Microfilaments and microtubules have characteristic cell-cycle-dependent localizations in yeast
cells.6,111 Therefore, co-localization of a protein
with actin or tubulin structures is good evidence
to suggest that it is a component of the cytoskeleton or a closely interacting protein. In this
respect, epitope tagging and green fluorescent
protein (GFP)36,49 fusion localizations have been
extremely useful. The phenotypes of conditional
lethal mutants as well as depletion and overexpression mutants provide insight into the function of a
protein.
THE MICROTUBULE SYSTEM
The yeast microtubule system consists of microtubules, microtubule-associated proteins (MAPs)
and the microtubule organizing centre (MTOC),
known as the spindle pole body (SPB; Figures 1
and 2). The function and cell-cycle-dependent
behaviour of the yeast microtubule system has
been deduced from electron microscopic analysis,29,30,32 immunofluorescence studies using
??????????? ??? ????????????? ????????????
401
Figure 2. Schematic diagram showing localization of the spindle pole body (SPB)
components Cdc31p,205 CaM,208,213 Kar1p,206 Spc42p,54 Spc98p,72,183 Spc110p208
and Tub4p207 relative to SPB substructures. The cytoplasmic (c-half bridge) and the
nuclear (n-half bridge) sides of the SPB half bridge are most likely different in
composition.205,206
anti-tubulin antibodies6,111 and the phenotype of
conditional lethal mutants.98 These studies
identified two arrays of microtubules which function in different cellular processes. The cytoplasmic
microtubules start at the SPB and are directed
toward the cytoplasm. They are needed for nuclear
positioning, the migration of the nucleus into
the bud cell during mitosis (Figure 3A) and the
movement of the two nuclei during karyogamy
(Figure 3C). The nuclear microtubules are also
organized by the SPB and are involved in chromosome segregation in mitosis and in meiosis
(Figure 3B).
Additional aspects of the microtubule system
that attract more and more attention are proteins
involved in tubulin assembly, the identification
and function of MAPs, composition, force
production and cellular functions of microtubule-
based motor proteins, microtubule nucleation
by the SPB and cell-cycle-dependent processes
like SPB duplication, spindle assembly and
disassembly.
Tubulin
Microtubules are composed of tubulin, a heterodimer of с- and т-tubulin. In S. cerevisiae
с-tubulin is encoded by two highly related isogenes
TUB1 and TUB3 having 90% identity.191,192 Yeast
strains containing a disrupted TUB3 gene are
viable with minor growth defects, while the disruption of TUB1, is lethal. This difference can be
explained by a five-fold higher expression of
TUB1, since overexpression of TUB3 rescues a
deletion of TUB1 indicating that the с-tubulin
genes are functionally interchangeable.191 TUB2
encodes the essential т-tubulin.158 The mutant
?. ?????? ??? ?. ????????
402
Figure 3. Functions of nuclear and cytoplasmic microtubules in yeast. (A) Cytoplasmic microtubules
function in nuclear positioning and the migration of the nucleus into the bud. (B) Nuclear microtubules are
involved in chromosome segregation. (C) During mating fusion of the two cell bodies of a and с cells is
followed by fusion of the nuclei. This requires cytoplasmic microtubules and the kinesin-like motor Kar3p.
Also the two spindle pole bodies (SPBs) fuse resulting in one double-sized SPB. CM, cytoplasmic
microtubules; NM, nuclear microtubules.
phenotypes of tub1 and tub2 cells have been reviewed extensively98,204 and will not be discussed
here. Furthermore, the potential GTP-binding site
of Tub2p has been mutagenized to study the effect
of GTP hydrolysis on microtubule dynamics.52,187
This aspect of т-tubulin function has been covered
in a recent review by Burns and Farrell.28
Tubulin formation from с- and т-tubulin requires the assistance of cytosolic chaperonins like
Tcp1p38 and cofactors such as Rbl2p, which has
been identified as a suppressor of the toxic defect
of TUB2 overexpression.11,26 Rbl2p is homologous
to the mammalian cofactor A found in a т-tubulin
folding assay.216 Additional cofactors of yeast
tubulin formation may be CIN1209 and PAC2,
both of which show similarity to mammalian
cofactors D and E.216
The yeast genome sequencing project identified
TUB4, which is 31% identical to Tub1p and
Tub3p, and 27% identical to Tub2p. Subsequent
analysis revealed that Tub4p is associated with the
SPB and is the у-tubulin of S. cerevisiae.137,203,207
Its function is discussed together with other SPB
components (see below).
Microtubule-associated proteins
Microtubules contain associated proteins
(MAPs) which influence the properties of microtubules. One group of MAPs are motor proteins
that translocate along microtubules upon ATP
hydrolysis (Tables 1 and 2). Non-motor MAPs
bind to microtubules and regulate their dynamic
behaviour (Table 3). In addition, MAPs may
Non-essential,
temperature-sensitive
growth defect
Non-essential
Non-essential
cin8Фkip1Ф lethal
Non-essential
Non-essential
Non-essential
CIN8
KAR3
KIP1
KIP2
RRC805/
YGL216W
SMY1
No obvious defect
No obvious defect
No obvious defect
Growth defect, accumulates
large-budded cells with a
single nucleus and a short
mitotic spindle
Defect in SPB separation at
37)C which is suppressed by
overproduced Kip1p
Karyogamy defect,
chromosome instability,
accumulates large-budded
cells at the permissive
temperature, many of which
lack a detectable spindle
Phenotype of null
mutant
p.c., personal communication; n.d., not determined; SPB, spindle pole body.
Non-essential
temperature-sensitive
growth defect
Gene disruption
CIK1
Gene
name
Localization
Localization does not
depend on microtubules;
localizes to caps at bud site
and tip of small buds, bud
neck during cytokinesis
n.d.
n.d.
Colocalizes with mitotic
spindles
Kar3p localizes to the SPB
region and cytoplasmic
microtubules, localization is
dependent on Cik1p
Colocalizes with
microtubules between poles
of a mitotic spindle
Cik1p localizes to the SPB
region and cytoplasmic
microtubules, its localization
is dependent on Kar3p
Table 1. Kinesin-related motor proteins in Saccharomyces cerevisiae.
M. A. Hoyt, p.c.
129, 130
Unknown
181
181, 189
63, 94, 143, 144,
166
94, 188, 189
165, 166
Reference
Unknown
Functionally redundant with
Cin8p
Minus-end directed motor,
spindle integrity,
chromosome segregation,
nuclear fusion, possible role
in kinetochore function
Spindle formation, spindle
maintenance, chromosome
segregation
Interacts with Kar3p,
putative light chain
Function
??????????? ??? ????????????? ????????????
403
Essential in the absence of
CIN8
Non-essential
Non-essential
cin8Фslc1Ф are
viable
PAC13
SLC1
n.d.
n.d.
n.d.
p.c., personal communication; n.d., not determined; SPB, spindle pole body.
n.d.
Essential in the absence of
CIN8
PAC11/
Non-essential
YDR488C
Jnm1p localizes to astral
microtubules and near the
SPB
Non-essential
JNM1
Phenotype similar to
DYN1/DHC1
Non-essential
Defective in spindle orientation GFP-Dhc1p fusion protein
cin8Фdyn1Ф lethal and nuclear migration
decorates the SPB and
cytoplasmic microtubules
Localization
DYN1/
DHC1
Higher frequency of abnormally n.d.
positioned nuclei and spindles,
multiple multi- or anucleated
cells
Phenotype of null mutant
Non-essential
Gene disruption
ACT5/
ARP1
Gene
name
Table 2. Subunits of dynein-related motor protein complexes in Saccharomyces cerevisiae.
141
64, 128, 188
41, 151
Reference
Putative dynein light chain
Putative p150Glued homologue of
the dynactin complex
53
M. A. Hoyt, p.c.
Putative dynein intermediate chain M. A. Hoyt, p.c.
Dynein light chain or accessory
protein, proper nuclear migration
Dynein heavy chain, nuclear
migration, spindle elongation,
chromosome segregation
Component of the dynactin
complex, nuclear migration and
positioning
Function
404
?. ?????? ??? ?. ????????
??????????? ??? ????????????? ????????????
crosslink microtubules and therefore stabilize
spindle structures. Many non-motor MAPs and
all motor proteins are non-essential for viability,
suggesting that they are redundant in their function or are not absolutely required in the growth
conditions examined.
Motor proteins associated with yeast microtubules
Similar to other organisms, two classes of potential
microtubule-based motor proteins, kinesin (Table
1) and dynein (Table 2), have been identified in
S. cerevisiae. Motor proteins have a defined
direction of movement along the inherently polar
microtubules. Most kinesins move to the plus-end
of microtubules, which polymerizes tubulin with
a higher rate than the minus-end. In contrast,
dynein is a minus-end-directed motor protein
(reviewed by Gibbons,75 Goldstein80 and Roof
et al.180).
CIN8, KIP1, KIP2, SMY1 and KAR3 encode
proteins with homology to the motor domain of
the protein kinesin. These genes have been found
by homology or genetic screens.43,93,129,143,181
RRC805/YGL216W is the only additional gene
coding for a kinesin-like protein which was found
by the yeast genome sequencing project (A. M. A.
Hoyt, personal communication). Altogether, the
S. cerevisiae genome encodes a total of six proteins
with homology to kinesin.
An important question is whether all of these
proteins function as microtubule-based motors.
Microtubule-based motor function has been established only for Kar3p using an in vitro mobility
assay. In contrast to most kinesins, it is a minusend microtubule motor protein with the motor
domain at the carboxy-terminal end of the protein.63 Smy1p was identified as a dosage-dependent
suppressor of a mutation in the myosin-like MYO2
gene. Smy1p localizes with Myo2p to regions of
polarized growth and this localization is independent of microtubules, making a microtubuledependent motor function unlikely.129,130 The
localization of Cin8p and Kip1p along microtubules and their involvement in spindle formation
and chromosome segregation are consistent with
a function as microtubule motor proteins.93,181
For Kip2p and RCC805/YGL216W it is unclear
whether they have motor activity.
What is the role of the kinesin-like motor proteins in S. cerevisiae? Kar3p was found in a screen
for genes involved in karyogamy.43 It most likely
moves the two nuclei of a zygote together via
cytoplasmic microtubules. Additional functions of
405
Kar3p in spindle formation and chromosome segregation are suggested by the mitotic defects of
kar3 cells.143 Kar3p fulfils these functions together
with Cik1p, which binds to Kar3p and may be
the kinesin light chain. The functional interrelationship of these proteins is also indicated by
the observation that the localization of Kar3p and
Cik1p to astral microtubules is dependent on both
proteins.165,166
CIN8 and KIP1 were identified in a screen for
genes required for chromosome stability.93,95 In
addition KIP1 and KIP2 have been cloned based
on their homology to kinesins.181 CIN8 and KIP1
are functionally redundant genes such that the
double mutants arrest in mitosis.94,181 The
function of CIN8, KIP1 and KAR3 in spindle
formation is discussed under the section on the
microtubule system and the cell cycle.
The DYN1/DHC1 gene of S. cerevisiae encodes
the single dynein heavy chain. Disruption of
DYN1/DHC1 causes misalignment of the spindle
relative to the bud neck and results in abnormal
distribution of the dividing nuclei between the
mother cell and the bud.64,128 Based on these
studies, it has been suggested that dynein exerts a
pulling force from outside the nucleus to assist in
nuclear positioning and spindle pole separation.
Dynein, together with the kinesin-like proteins
Kip1p and Cin8p, is also required for anaphase
chromosome segregation.188
As in mammalian cells, yeast dynein seems to
be part larger complex (for reviews see Mullins
et al.153 and Schroer et al.194). Possible components of the dynein complex are Pac11p, which has
similarity to the intermediate chain of rat cytoplasmic dynein (M. A. Hoyt, personal communication)
and Slc1p, a putative homologue of the metazoan
cytoplasmic dynein light chain 1.53
The motor activity of vertebrate and Drosophila
dynein is regulated by the dynactin complex, which
is a heteroligomer of ten different polypeptides (see
Mullins et al.153 and Schroer et al.194). Therefore,
similarly to dynein, the dynactin complex is
important for spindle orientation and nuclear
migration.41,151 Components of the yeast dynactin
complex are most likely Jnm1p,141 Pac13p
(Nip80p; M. A. Hoyt, personal communication)
and Act5p/Act3p/Arp1p.41,151 PAC13 as well as
PAC11 (perishes in the absence of CIN8) were
identified in a screen for synthetic lethal mutations
with CIN8. Pac13p encodes a version of the
p150Glued component of the dynactin complex that
may bind to microtubules and cytoplasmic dynein
Non-essential Null mutant has bilateral
karyogamy defect,
supersensitive to benomyl,
short cytoplasmic
microtubules
Essential
BIK1
STU1
SPB region, particulate dots in
short spindles
c.l., conditional lethal; p.c., personal communication; n.d., not determined; SPB, spindle pole body.
80-kDa Essential
protein
n.d.
Unknown
c.l. mutant arrests with large SPB region, nuclear microtubules Mitotic spindle formation
bud and bipolar shortened
mitotic spindle, nucleus in
bud neck
Spindle elongation
NUF2
Essential
19
Reference
183, J. V. Kilmartin,
p.c.
164, E.S., unpublished
T. C. Huffaker, p.c.
Required for spindle assembly,
168
may mediate interaction between
antiparallel microtubules
See Ase1p
c.l. mutant assembles
SPB and weakly microtubules
normal-looking short
spindles, spindle elongation
and chromosome segregation
are defective
Spindle microtubules
Mitotic spindle and SPB
Cell-cycle dependent localization, Overlapping function with Bik1p 170
not detected in G1, nuclear
in mitotic spindle formation
microtubules in G2/M, spindle
midzone in mitose
Function
STU2/ Essential
YLR045C
c.l. mutant lacks short and
long mitotic spindles
Non-essential Null mutant is supersensitive
to benomyl, ase1Фbik1Ф are
defective in anaphase B
spindle elongation
ASE1
Localization
Gene
disruption
Gene
name
Phenotype of mutant
Non-motor microtubule-associated proteins in Saccharomyces cerevisiae.
Table 3.
406
?. ?????? ??? ?. ????????
??????????? ??? ????????????? ????????????
(M. A. Hoyt, personal communication). Interestingly, Act5p/Act3p/Arp1p is an actin-related protein and represents a link between the microtubule
and microfilament systems (for further discussion
see the section on actin-related proteins).
Non-motor proteins associated with yeast microtubules The classical approach to identify MAPs
is their copurification with microtubules. Due to
the low yield, this was at first not very fruitful for
yeast. More recently this biochemical approach
identified about 25 potential MAPs, of which
three are known metabolic enzymes, two are
GTP-binding proteins and two colocalize with
microtubules.13
More successful were genetic strategies based on
suppressors of tubulin mutations and defects in
karyogamy. STU1 and STU2 were obtained in a
genetic screen for suppressors of cold-sensitive
т-tubulin mutants. STU1 is essential for growth
and an epitope-tagged Stu1p colocalizes with
microtubules in the mitotic spindle of yeast cells. A
series of in vitro and in vivo results suggest that
Stu1p specifically interacts with microtubules168
(T. C. Huffacker, personal communication). First,
genetic interactions between mutant stu1 alleles
and alleles of tub1 and tub2 have been found.
Second, Stu1p binds directly to microtubules
in vitro. Stu1p is required for spindle assembly,
in particular for the integrity of long spindles,
suggesting that Stu1p may mediate the interaction
between antiparallel polar microtubules (T. C.
Huffacker, personal communication). Also STU2
is essential for growth. It codes for a protein of
101 kDa that binds to microtubules in vitro. An
epitope-tagged or a GFP-tagged Stu2p stained the
SPB of yeast cells as well as microtubules (weakly).
Temperature-sensitive stu2 mutant cells are unable
to assemble a mitotic spindle, suggesting a function of Stu2p in this process. Stu2p interacts with a
novel protein of 72 kDa in the two-hybrid system,65 which has been named Sip6p. Sip6p is
located close to the SPB (T. C. Huffacker, personal
communication).
The product of the BIK1 gene is required for
microtubule function during mating and mitosis.
The Bik1p protein colocalizes with tubulin to the
SPB and the mitotic spindle.19 The structure of
Bik1p is similar to that of the human MAP CLIP170 and the dynactin component p150Glued all
containing a 40-residue microtubule-binding
motif, coiled-coil regions and a basic head domain
(reviewed by Rickard and Kreis176). A genetic
407
screen for mutations that are synthetically lethal
with the bik1 null mutation identified ASE1. Its
gene product is localized to the mid-zone of the
anaphase spindle. In addition, Ase1p co-sediments
with stabilized microtubules revealing a direct
interaction with microtubules (D. Pellman, personal communication). Interestingly, yeast cells
with defects in BIK1 and ASE1 are impaired
for anaphase spindle elongation, suggesting that
both proteins have overlapping function in this
process.170
Nuf2p is localized near the SPB164 and along
nuclear microtubules (E. Schiebel, unpublished). It
is an essential 53 kDa protein with a high content
of predicted coiled-coil structure. Conditional
lethal nuf2 mutant cells are defective in mitotic
spindle formation.164 The localization of Nuf2p
by indirect immunofluorescence is similar to
the 80 kDa protein described by Rout and
Kilmartin,183 who raised monoclonal antibodies
against spindle and SPB proteins. The 80 kDa
protein is located on the spindle microtubules, in
particular the region close to the SPB.183 The gene
coding for the 80 kDa spindle component has
been cloned and shown to be essential for growth
(J. V. Kilmartin, personal communication).
Spindle pole body
The SPB is a multi-laminated organelle that is
embedded into the nuclear envelope (Figure 2).
Electron microscopic analyses revealed SPB substructures, some of which could be correlated with
specific functions.32 The outer and inner plaques of
the SPB organize the cytoplasmic and nuclear
microtubules, respectively. The central plaque is
embedded in the nuclear envelope. The half bridge
appears as a darkly stained strip along the cytoplasmic margin of the nuclear envelope on one side
of the SPB. It has important functions in the
cell-cycle-dependent duplication of the SPB. A
structure called the satellite forms in early G1
phase of the cell cycle on the cytoplasmic side of
the half bridge. The satellite may be a precursor
of the newly formed SPB29,30,32 but component
proteins have not been identified directly.
Components of the SPB Although the SPB is 50
times larger than the ribosome, hitherto only seven
SPB components have been described (Figure 2;
Table 4). This deficiency makes the identification
of additional SPB components and their functional
analysis an important task. Unfortunately SPB
Essential
Essential
Essential
Essential
Essential
CDC31
CMD1
KAR1
SPC42
SPC98
Essential
Tub4p
Unknown
Cdc3p
Spc110p
Kar1p
Interaction
at the SPB
Defect in microtubule nucleation
and mitotic spindle formation,
Cdc-phenotype*
Spc98p
21, 43, 182,
206, 219
71, 163, 208,
211, 213
16, 21, 30,
205, 219
Reference
Inner and outer plaques Microtubule organization
137, 203, 207
71, 113, 208,
211, 213
72, 183
Forms polymeric layer at the 54
central plaque. May facilitate
SPB attachment to the nuclear
membrane
Karyogamy and initiation of
SPB duplication
Interacts with the C-domain
of Spc110p; function of this
interaction is unknown
Initiation of SPB duplication
Function
Inner and outer plaques Microtubule organization by
the SPB
Central plaque
Half bridge
Central plaque
Half bridge
Localization
at the SPB
Failure of nuclear DNA separation. Calmodulin Between the cental and Connects the central with the
the inner plaque with its inner plaque
Occurrence of an unusual
amino-terminus towards
microtubule organizer in the nucleus
the inner plaque
(IMO), connection of nuclear
microtubules to the SPB,
Cdc-phenotype*
Defect in mitotic spindle formation,
Cdc-phenotype*
Defect in SPB duplication,
Cdc-phenotype*
SPB duplication defect,
Cdc-phenotype*
cmd1-101 has a defect in SPB
duplication, Cdc-phenotype*?
SPB-duplication defect,
Cdc-phenotype*
Phenotype of conditional
lethal mutant
*Mutant cells arrest in the cell cycle with large bud and replicated DNA.
?Only a subset of cmd1 mutants affect the SPB.163
TUB4
SPC110/ Essential
NUF1
Gene
disruption
Gene
name
Table 4. Spindle pole body (SPB) components.
408
?. ?????? ??? ?. ????????
??????????? ??? ????????????? ????????????
components do not exhibit common motifs.
Consequently, the sequence of the yeast genome
did not facilitate the identification of new SPB
components.
Cdc31p and Kar1p are components of the SPB
half bridge,205,206 which is the site where cell-cycledependent SPB duplication is initiated.29,30 CDC31
codes for a small Ca2+ -binding protein which
belongs to the centrin family of EF-hand proteins
(reviewed by Schiebel and Bornens193). KAR1 was
originally identified by the karyogamy defect of
certain kar1 mutants.43 Further analysis revealed
that KAR1, like CDC31, has a function in the
cell-cycle-dependent duplication of the SPB.29,182
In particular, temperature-sensitive cdc31(ts) and
kar1(ts) mutants fail to duplicate the SPB,
although DNA replication and bud formation
are normal.30,182 Genetic219 and biochemical evidence21,206 suggests that Cdc31p and Kar1p function together in SPB duplication. This is achieved
by the binding of Cdc31p to about 20 amino acids
in the central part of Kar1p. This interaction is
regulated by physiologically relevant changes in
Ca2+ in vitro, raising the possibility that Cdc31p
function is influenced by Ca2+ in vivo. In agreement with this hypothesis, Ca2+ -binding to
Cdc31p is essential for its function.70
Rout and Kilmartin raised monoclonal antibodies against enriched SPBs.183 These antibodies
recognized three SPB components with apparent
molecular weights of 42, 90 and 100 kDa. The
110 kDa SPB component is encoded by
SPC110112,147 (Spindle Pole Body Component of
110 kDa). Spc110p is a filamentous protein that
connects the central with the inner plaque of the
SPB,112,183 thereby determining the distance
between these plaque structures. Spc110p contains
a calmodulin-binding site at its carboxy-terminal
end71,211 directing calmodulin to the central
plaque of the SPB.208,213 This finding defines the
orientation of Spc110p in the SPB with the
carboxy-terminal end near the central, and
the amino-terminus close to the inner plaque. The
function of the Spc110p?calmodulin interaction is
not understood, since deletion of the calmodulinbinding site of Spc110-p does not affect viability71
nor Spc110p incorporation into the SPB.208 In
contrast, mutations in the calmodulin-binding site
of Spc110p that affect calmodulin-binding in vitro
are lethal or conditional lethal in vivo.71,113,211,213
Spc42p is associated with the central plaque of
the SPB and has an essential function during SPB
duplication54,184 (Figure 2). The 90 kDa SPB
409
component is associated with the outer and inner
plaques of the SPB.183 The gene coding for the
90-kDa SPB, SPC98, has been cloned as a dosagedependent suppressor of the temperature-sensitive
tub4-1(ts) mutant.72 Its function in microtubule
nucleation is discussed later. TUB4 encodes the
у-tubulin of S. cerevisiae. Its identity to у-tubulins
from other organisms is only about 40% whereas
other у-tubulins show 70% identity to each other.27
Microtubule nucleation One of the main functions of MTOCs is the organization of microtubules. у-Tubulin as a universal component of
MTOC seems to be a key component in this
process.161,162,210 Therefore it was not surprising to
find Tub4p associated with the SPB substructures
that organize the nuclear and cytoplasmic microtubules, respectively.207 The function of Tub4p in
microtubule organization is further supported by
the phenotype of conditional lethal tub4 mutants.
Depending on the tub4 allele, tub4 cells show either
defects in microtubule nucleation137 or mitotic
spindle formation.207
As in higher eukaryotic cells,210,239 Tub4p seems
to be part of a large protein complex containing
Tub4p, Spc98p and at least one additional SPB
component72 (E. Schiebel, manuscript submitted).
A number of results suggest that Tub4p and
Spc98p physically interact and that both proteins
are required for microtubule organization.72,207
Most interestingly, the ends of yeast microtubules
next to the SPB are sealed by a terminal component of unknown composition.33 It is tempting
to speculate that this cap structure is formed by
the Tub4p/Spc98p complex. However, whether
this cap functions similarly to the у-tubulin ring
complex from Xenopus oocytes remains to be
determined.239
The microtubule system and the cell cycle
SPB duplication in G1 of the cell cycle SPB duplication and separation, spindle formation and elongation, and finally chromosome segregation occur
at defined cell-cycle stages and their completion is
a prerequisite for exiting mitosis.30,32 The latter is
achieved by mitotic checkpoint control96,126,156
(Figure 1). This cell-cycle-dependent behaviour
raises the question of how the microtubule system
is regulated during the cell cycle.
In early G1 phase of the cell cycle, each yeast cell
contains one SPB which is associated with nuclear
and cytoplasmic microtubules. SPB duplication is
410
initiated in G1 phase before START of the cell
cycle with the formation of the satellite on the
cytoplasmic side of the half bridge.30,32 The SPB
components Kar1p and Cdc31p182,205,206 are
required for this step. An additional protein
involved in the early steps of SPB duplication may
be Dsk2p (Table 5). A DSK2-1 mutant allele
is able to suppress the deletion of the normally
essential KAR1.219 Furthermore, deletions in
DSK2 and RAD23, both encoding proteins with
amino-terminal domains homologous to ubiquitin,
cause a moderate temperature-dependent SPBduplication defect.20 The role of Cdc31p, Kar1p,
Dsk2p and Rad23p in satellite formation and
especially the cell cycle signals that initiate SPB
duplication remain a matter of debate. Cdc31p
may sense cell-cycle-dependent changes in [Ca2+ ],
which have been described for the time of SPB
duplication.99 Kar1p function in SPB duplication
is unclear. However, it goes beyond concentrating
Cdc31p at the SPB.70 This conclusion is based on
the finding that kar1-Ф17219 encoding a mutant
Kar1p protein without the Cdc31p-binding site
supports growth at lower temperatures.70 Since
Dsk2p and Rad23p are enriched in the nucleus but
not associated with the SPB,20,224 their role in SPB
duplication may be rather indirect.
Yeast cells arrested at START of the cell cycle
by pheromone, starvation or mutations in cdc28
contain a single, satellite-bearing SPB.30,32 As cells
progress through START, the second phase of
SPB duplication is initiated resulting in a fully
duplicated SPB. This duplication step depends on
activation of Cdc28p kinase by G1 cyclins. However, whether the Cdc28p/Cln kinase complex
directly phosphorylates a SPB component or
transmits its signal via additional kinases is not
known. A link between Cdc28p/Cln and the SPB
may be the kinase Mps1p/Rpk1p119,171,234 since
mutants in MPS1 fail to duplicate the SPB after
START.
How the SPB duplicates is not fully understood,
since no duplication intermediates have been
described in wild-type cells. However, mutants
in NDC1215,235 or MPS2234 arrest in the cell cycle
with a partially duplicated SPB containing the
outer and central plaques sitting on the cytoplasmic side of the nuclear envelope. Based on these
mutant phenotypes, it has been proposed that the
outer and central plaques are assembled in thew
cytoplasm followed by their insertion into the
nuclear envelope.233 The latter step may require
the Ndc1p and Mps2p proteins which are compo-
?. ?????? ??? ?. ????????
nents of the nuclear envelope235 (M. Winey,
personal communication). The inner plaque may
assemble in the nucleus by the binding of its
components to the inserted central and outer
plaques. This is suggested by the finding that
Spc98p, a component of the inner plaque, is
directed into the nucleus by a nuclear localization
sequence.72 The second phase of SPB duplication
results in a fully duplicated SPB which lies
adjacent to the existing SPB, both embedded in the
nuclear envelope. The two SPBs are connected by
the two half bridges (named the bridge) which is
later severed as the SPBs undergo separation to
form the poles of the spindle.30,32
SPB separation and spindle formation in S/G2 phase
SPB separation and spindle formation require
microtubules,101 motor proteins189 and the
Cdc28p/Clb kinase complex.66 A substrate for the
Cdc28p/Clb kinase may be the kinesin-like motor
protein Cin8p, which together with Kip1p is
needed for spindle assembly93,181 (M. A. Hoyt,
personal communication). Formation of a short
spindle is already complete at the end of S or the
beginning of G2 phase.29,30 Cin8p and Kip1p are
also required to maintain spindle integrity after
spindle formation. Loss of Cin8p and Kip1p functions at this cell cycle stage is accompanied by
spindle collapse which is suppressed by deletion of
KAR3. This finding suggests that the structure of
the pre-anaphase spindle is maintained by counteracting forces created by Cin8p and Kip1p on one
hand and Kar3p on the other.189 In contrast,
anaphase spindles are resistant to loss of Cin8p
and Kip1p functions,189 indicating that the spindle
is now balanced differently.
Mitosis, chromosome segregation, and exiting from
mitosis The three-dimensional structure of the
mitotic spindle of S. cerevisiae has been reconstructed from electron micrographs. The SPBs are
connected by about eight interpolar microtubules.
The remaining 32 microtubules of a haploid yeast
cell are in contact with the kinetochores, meaning
that each kinetochore is associated with one
microtubule.236
The microtubule system has two main functions
in mitosis. It is required for the migration of
the nucleus into the bud and for segregation of the
replicated chromosomes. Before anaphase, the
spindle is positioned through the bud neck, which
ensures that one set of chromosomes is driven to
the mother and the other to the bud. This nuclear
Non-essential
Essential
Essential
Essential
None-essential
UV sensitive
DSK2
MPS1
MPS2
NDC31
RAD23
p.c., personal communication.
Gene
disruption
Gene
name
40% of rad23Фdsk2Ф mutants
show a SPB duplication defect
Defect in SPB insertion
Defect in SPB insertion
Defect in SPB duplication
See RAD23
Phenotype of conditional
lethal mutant
Unknown
Unknown
Unknown
Unknown
Unknown
Interaction
with SPB
components
Nucleus
Nuclear envelope
Nuclear envelope
Unknown
Nucleus
Localization
Table 5. Non-spindle pole body (SPB) components involved in SPB duplication.
119, 234
20, 219
Reference
Unknown
20, 224
Insertion of the new SPB into the 215, 235
nuclear envelope
Insertion of the new SPB into the 234
nuclear envelope
M. Winey p.c.
Kinase, unknown
Unknown
Function in SPB duplication
??????????? ??? ????????????? ????????????
411
?. ?????? ??? ?. ????????
412
positioning and migration is accomplished in part
by dynein, which may create a pulling force from
the bud along cytoplasmic microtubules onto the
nucleus.64,128 Since dynein is non-essential and its
deletion causes only a moderate defect in nuclear
migration,64,128 kinesin-like or other motor
proteins may also function in nuclear migration.
In anaphase A sister chromosomes move toward
opposite poles of the spindle, while in anaphase B,
wherein chromosomes are segregated in yeast,
the spindle elongates from about 1?1З5 ьm to
8?10 ьm.31,236 Anaphase chromosome segregation
requires the kinesins Cin8p and Kip1p together
with dynein.188 Cytoplasmic microtubules are not
required for anaphase B, suggesting that the two
SPBs are pushed apart by the anti-parallel sliding
of the interpolar microtubules.212 Beside motor
proteins, non-motor MAPs like Bik1p and Ase1p
fulfil essential functions in anaphase spindle
formation.170 They may mediate the interaction
between antiparallel polar microtubules.
A mitotic checkpoint requiring the MAD and
BUB genes makes exiting from mitosis dependent
on formation of a functional spindle.96,125,178
This checkpoint control explains why most yeast
mutants affecting spindle structure show cell-cycle
arrest in mitosis. Exiting from mitosis requires the
destruction of the B-type cyclins by the anaphasepromoting complex containing Cdc16p, Cdc23p
and Cdc27p.114,118 Interestingly, homologous proteins are associated with the spindle and spindle
pole in the fungus Aspergillus nidulans146 and
human cells.217 However, the localization of these
proteins in yeast is unknown. Exiting from mitosis
is also accompanied by the disassembly of the
mitotic spindle. Ase1p, a protein localized on the
anaphase spindle, is degraded by ubiquitindependent proteolysis at the end of mitosis (D.
Pellman, personal communication). Thus, degradation or inactivation of spindle proteins may
facilitate this process.
THE MICROFILAMENT SYSTEM
The major component of microfilaments is actin,
whose interaction with a large number of actinbinding and other associated proteins composes
and controls the actin cytoskeleton. Microfilaments of yeast can be visualized in whole cells
by fluorescent phalloidin staining as two types of
polymerized structures. Cytoplasmic cables and
cortical patches form characteristic polarized
arrays which are redistributed during veg-
etative growth of both haploid and diploid cells
(Figure 4). Upon entry into the cell cycle after G0,
cortical patches are evenly distributed at the cell
surface and cables are randomly oriented in the
cytoplasm. Before bud emergence patches are seen
at the incipient bud site and cables are oriented to
this area, patches are concentrated in buds at areas
of cell surface growth until the bud reaches a
critical size. At this time patches and cables again
show an isometric distribution for a short time
period, after which patches concentrate in a ring in
the bud neck area until cytokinesis with cables
appearing to radiate from the bud neck.67,111
During the formation of hormone-induced mating
projections in haploid cells, patches are localized
to the growing tip.69,88 As such, the formation of
a ?shmoo? is equivalent to a ?mating bud? and
requires an intact polarized cytoskeleton, although
fewer studies of cytoskeletal components have
reported on these structures.
It has been proposed that cytoplasmic cables
represent longitudinal bundles of actin filaments6
while patches appear to be coils of filaments
around a membrane invagination.152 Neither biochemical separation nor isolation of the two types
of structures has yet been reported. Exciting recent
results demonstrate in vivo monitoring of fusion
proteins between the naturally fluorescent protein,
GFP, and cytoskeletal components, actin, Sac6p
(fimbrin), Abp1p56 or Cap2p.223 While decoration
with antibodies had shown actin and fimbrin in
both cables and patches, all four GFP fusions
localized essentially to actin patches showing rapid
movement (less than a second) of patches and a
refinement of patch distribution in growing cells.
For example, patches concentrate at the incipient
bud site before all the patches at the site of the
preceding cytokinesis have disappeared. Changes
from polarized to non-polarized distribution of
patches occurred within a few minutes. Representative films demonstrating these results are
available at http sites (http://genome-www.
stanford.edu/group/botlab/index.html; see ref. 56;
http://www.cooperlab.wustl.edu; see ref. 223).
Actin
Actin is an abundant, ubiquitous protein of 375
amino acids with a well-characterized ATPase
activity. Most actins are acetylated at the amino
terminus and methylated on His73. Actin is present
in cells as monomers of 43 kDa, G-actin, and as
polymers or filaments of F-actin, in varying ratios
??????????? ??? ????????????? ????????????
413
Figure 4. Changing polarity throughout the cell cycle; based on the model proposed by Lew and
Reed.123 (A) Indication of cell-cycle controls. (B) Cortical actin distribution in morphogenic
transitions of the cell cycle. (C) Cytoskeletal rearrangements as revealed by phalloidin staining of
actin filaments in fixed whole cells during cell cycle progression (photo V. Moreau). (D) Polarity
of secretion in morphogenic transitions of the cell cycle.
depending on the species and cell type. Actin
filaments spontaneously assemble via non-covalent
interactions between monomeric subunits in vitro
and in vivo. Actin filaments are dynamic structures
with subunit turn-over at both ends, defined as
either barbed (faster growing end) or pointed,
relative to their association at an angle with
myosin heads. Both end-binding and side-binding
proteins can nucleate actin assembly. In vivo actin
assembly is tightly controlled in time and
space.172?174,190 The three-dimensional structure of
globular т-actin has been determined by X-ray
crystallography in complex with DNaseI107 as well
as with profilin.196 The core of the protein consists
of four subdomains composed of с helices and
т sheets comprising a structure now referred to as
the actin fold which constitutes the binding site for
ATP and a divalent cation106. Regions of interaction between actin subunits and between actin
and myosin are situated essentially in loops on the
surface of the molecule.
Holmes and co-workers have proposed a model
of the F-actin filament as a ?genetic helix?,89 which
is consistent with electron microscope reconstruction data.23,145 Accordingly, results obtained with
a profilin-т-actin complex reveal molecules intertwined in a ribbon structure held together through
intermolecular contacts consistent with the monomeric structure.196 The assembly of actin into
microfilaments and its polarized localization in the
cell is regulated by a large number of proteins
called actin-binding proteins (ABPs) which bind
either the globular (monomeric) or filamentous
(polymeric) form. The relative proportions of
actin found in polymerized structures differ in
different species. In contrast to animal cells, yeast
cells have most of their actin in the polymerized
form.109
414
Actin is encoded by a single essential gene,
ACT1, in budding yeast. Its extreme conservation
across species allows the extrapolation to other
eukaryotic cells (with the exception of specialized
muscle cells) of results obtained from the facile
yeast genetics of a single protein. In vivo functions
of yeast microfilaments have been elucidated principally by the study of actin mutants. Analysis of
the first conditional mutants of actin obtained
by chemical mutagenesis showed defects in the
morphological development of cells and polarized
growth, in asymmetrical actin distribution, in the
transport of membrane vesicles, in the secretion
of invertase159 and in the internalization step of
endocytosis.116 Different strategies of mutagenesis,
whether generally targetted to overall surface areas
by systematic ?charge to alanine? substitution231 or
directed to specific residues37,44,104 (precisely targetted by the availability of the three-dimensional
structure of actin107) have been complementary in
defining additional cellular functions and interactions and in testing the molecular model of actin.
Studies of this larger collection of temperaturesensitive mutants revealed roles for actin in the
organization of mitochondria in the cell, and
identified the residue necessary for binding the
toxin phalloidin.59,230,231 Other studies of act1
mutants showed that the actin cytoskeleton, in
addition to microtubules, plays a role in spindle
orientation.167 Combined in vitro and in vivo
studies of mutated actins showed that loss of the
amino-terminal residues interfered with interaction
between actin filaments and myosin,44,45 while a
L266D substitution revealed the site of important
contacts between the two strands of actin filaments.37 An interesting in vivo strategy to identify
which amino acids of actin were required for
functional interactions with ABPs is based on the
assumption that a mutation in actin which specifically impairs interaction with an ABP will cause a
phenotype similar to a null mutation in that ABP.
Mutations in specific regions of actin subdomain 1
were implicated in interaction with the filamentbundling protein, fimbrin, because the subdomain
1 mutants and Фsac6 were both synthetically lethal
with null alleles of ABP1 and SLA2 genes.90
Independently, Honts et al.92 have also identified
a likely fimbrin-binding site by analysing act1
mutant alleles that are capable of suppressing sac6
mutant alleles. The mutations cluster in subdomains 1 and 2 of actin and these mutant actins
are defective in their in vitro interaction with
Sac6p.
?. ?????? ??? ?. ????????
The large number of ACT1 conditional mutants
studied have allowed dissection of some of
its interactions necessary for formation of
filamentous actin structures and revealed actin?s
multiple roles in complex cellular functions, while
genetic screens have identified numerous actinbinding and associated proteins which are
described in the following section.
Proteins associated with microfilaments
In a 1994 yeast cytoskeleton review, 28 genes
involved in function of the S. cerevisiae actin
cytoskeleton were tabulated.228 A number of new
genes can now be added to the actin cytoskeleton
list (see Tables 6?8). In addition, available
sequence from the S. cerevisiae genome suggests
the presence of a number of new genes ?related? to
known participating proteins, especially actin (see
Table 9) and functional analyses should soon be
forthcoming. Known ABPs and other associated
proteins interacting with microfilaments are considered separately here but some ?associated? proteins are likely to become ABPs as biochemical
and cellular functions of these proteins become
more clearly defined.
Actin-binding proteins Actin-binding proteins
have been classified and grouped into categories
such as monomer-binding proteins, barbed end
capping/severing proteins, cross-linking proteins,
lateral binding proteins and miscellaneous.173 Certain myosins are also intimate ABPs; we consider
them separately only to emphasize possible motor
functions. At least one representative of each
category of eukaryotic ABPs is found in yeast.
However, homologues for many ABPs of higher
eukaryotic cells have not been found. т-Thymosin
is one example of a small monomer-binding protein with an important regulatory role,34,35 where
inspection of the S. cerevisiae genomic sequence
does not reveal any obvious homologue (O. Poch
and B. Winsor, unpublished). Functional characterization of known yeast ABPs are summarized
in Table 6.
Profilin is an essential multifunctional
monomer-binding protein present in both S.
cerevisiae84,136 and Schizosaccharomyces pombe.12
In the molecular model of profilin with т-actin,
profilin is seen as a molecular wedge in the filament
ribbon, apparently binding two actin molecules.196
In vivo profilin has been implicated in the sequestration of actin monomers since overexpression
Gene disruption
No visible phenotype,
tpm1Ф tpm2Ф lethal
Overexpression alters axial budding
?Loss of actin cables, random
patches, vesicle accumulation,
impaired mating, abnormal chitin
*Round, enlarged cells, depolarized
actin, deficient endocytosis, vesicle
accumulation
ts, temperature-sensitive; SU, sub-unit.
Miscellaneous
ABP1 No phenotype, abp1Ф sac6Ф Overexpression causes ts growth,
lethal, abp1Ф sla1Ф lethal,
alters cytoskeleton
abp1Ф sla2Ф lethal
TPM2
Lateral binding
TPM1 ts, slow growth, tpm1Ф
tpm2Ф lethal
Cross linking
SAC6 ts, sac6Ф cap1Ф lethal,
sac6Ф cap2Ф lethal, sac6Ф
abp1Ф lethal
?Depolarized patches, absence of
cables, degradation of Cap1p
?Depolarized patches, absence of
cables, degradation of Cap2p
Barbed end capping
CAP1 Slight growth defect, cap1Ф
sac6Ф lethal
Slight growth defect, cap2Ф
sac6Ф lethal
?
Severing
COF1 Lethal
CAP2
Cellular
localization
Protein function or sequence
similarity
Capping protein SUb; addition of
SU to barbed end enucleates
filaments in vitro
Capping protein SUa; addition of
SU to barbed end enucleates
filaments in vitro
F-actin fragmentation, actophorin
and depactin similarity, EF hand
Ca2+ -binding domain
Patches
?
Cables
2, 3, 4, 60, 116
4, 7, 8, 108
4, 7, 8, 9
100, 148, 149, 214
84, 86, 135, 222
Reference
SH3 domain, cofilin-like domain
4, 60, 61, 221
Minor form of tropomyosin, spans 57
four actin monomers, 64З5% identity
to Tpm1p
Major form of tropomyosin, spans 131, 132
five actin monomers, saturable
cation-dependent binding to F-actin
in vitro
Patches and Suppresses act1-1 ts, fimbrin, actin
cables
filament bundling protein
Patches
Patches
Patches
?Round, enlarged cells, disorganized Cytoplasmic Sequesters actin monomers,
actin, abnormal chitin
diffuse
regulates actin polymerization,
suppresses loss of CAP function,
EF hand Ca2+ -binding domain
Phenotype of ?null or
*conditional mutant
Actin-binding proteins of Saccharomyces cerevisiae.
Monomer binding
PFY1 Almost inviable, severely ts
Gene
name
Table 6.
??????????? ??? ????????????? ????????????
415
416
relieves the lethality of actin overexpression,
albeit without restoring normal microfilaments.135
Profilin also activates nucleotide exchange on
actin, inhibits the actin ATPase activity, and
participates in assembly at the barbed end of
filaments. Site-directed mutations aimed at actin
and PIP2 binding sites on profilin78,79 confirmed
that actin-binding is important for in vivo function
but the importance of PIP2 binding is less clear.86
In Sz. pombe, Cdc3 (profilin) localizes to the
F-actin contractile ring and overproducing
cells arrest at cytokinesis without visible actin
staining.12
A homologue of the filament-severing family of
proteins referred to as cofilin/ADF (actin depolymerizating factor), coded by the gene COF1, was
identified independently by two groups.100,149 The
protein is essential, has actin-severing activity
in vitro and localizes essentially to cortical patch
structures. According to the model proposed
by Moon and Drubin,148 cofilin binds, with
greater affinity, monomers in the filament which
have already hydrolysed ATP. This would create
a curvature of the filament exposing other
surfaces with higher affinity for cofilin, allowing
intercalation and severing of the filament.
Capping protein (CP), which binds the barbed
end of actin filaments and nucleates actin polymerization in vitro, is an ст heterodimer encoded
by two non-homologous genes whose protein
products appear to have completely overlapping
functions. Since disruption of either CAP1 or
CAP2 genes gives rise to a rapid loss of the other
protein, single or double null mutants result in
depletion of CP.7?9 Capping protein co-localizes
with actin in cortical patches8 and actin filaments
are unstable in the absence of capping protein or
fimbrin, suggesting that CP stabilizes the actin
filament.109 Most loss-of-function mutants and
deletions result in the same phenotype, slow
growth and variable cell size, loss of actin cables
and depolarization of cortical patches. Together
with the fact that several of the mutant proteins no
longer co-localize with actin, these experiments
argue strongly that the biochemical function of CP
in vivo is also to bind actin filaments.201 A new
phenotype, pseudohyphal growth, was found in
mutants of two genes, SCL1 (synthetically lethal
with Cap) and SLC2, which rendered loss of
capping protein essential.108 This raises the
fascinating question of whether the regulation of
actin filament formation might have a role in the
switch to pseudohyphal, invasive growth.
?. ?????? ??? ?. ????????
A major (TPM1) and more recently, a minor
(TPM2) form of tropomyosin have been identified
and studied. Tpm1p was shown to stabilize actin
cables and loss of the protein resulted in vesicle
accumulation apparently independent of other
secretory defects.131,132 While cells are seemingly
unaffected by loss of only Tpm2p, both proteins fix
F-actin and loss of both forms is lethal. Their
functions are thus partially overlapping but the
fact that overexpression of either form alone does
not completely compensate for the double loss of
function suggests that they may be important for
different functions.57
An orphan ABP called Abp1p was isolated on
an F-actin column.60 The protein is non-essential
and contains a cofilin-like domain and an SH3
region (Src homology 3) found in certain signal
transmission proteins, cytoskeletal-associated
proteins and class I myosins.169 No equivalent has
been found in mammalian cells.58,61 The Apb1
protein localizes to actin patches and, based on
genetic results, is important for cortical cytoskeleton interactions.4,91,221 abp1Ф is synthetically
lethal with mutations in SAC6 (fimbrin) and
two other genes, SLA1 and SLA2,91 involved in
assembly of the cortical cytoskeleton, although the
precise role of Abp1p remains to be elucidated.
The possibility of functional redundancy between non-essential ABPs in vivo has been investigated by searching for lethal interactions between
null mutations in genes for CP, fimbrin, tropomyosin and ABP (Abp1p).4 Mutants lacking fimbrin
and either sub-unit of CP were inviable. Mutants
lacking fimbrin and Abp1p were either dead or
very sick. All other combinations of double
mutants were viable, as was an abp1 cap2 tpm1
triple mutant, although some double mutants
grew quite slowly, suggesting varying degrees of
functional overlap. Thus, functional redundancy
does not seem very great among these nonessential ABP proteins and different mechanisms
of action are implied in spite of some functional
homologies. This may be a consequence of the
small number of these proteins relative to higher
eukaryotic cells.
Myosins and myosin-like proteins The transport
and maintenance of cytoplasmic organelles and
cell division depend on actin-based molecular
motors belonging to the myosin family. The
conserved N-terminal motor domain (myosin head
or S1) contains the ATPase function necessary for
force generation as well as the actin-binding site.
No visible phenotype,
myo3Ф, myo5Ф, very sick
*(Myo3, myo5Ф, myo5 ts),
disorganized actin,
receptor-mediated
internalization defect
?(myo3Ф, myo5Ф), random
budding, osmosensitive,
disorganized actin, vesicle
accumulation
MYO5
No visible phenotype,
myo3Ф, myo5Ф, very sick
MYO3
*Delocalized actin, abnormal
actin bars, delocalized chitin,
vesicle accumulation
Overexpression causes
unseparated diploids and
clusters of haploids
Lethal
MYO2
?Altered budding, cell
separation defect, abnormal
chitin and cell wall deposition
Phenotype of ?null or
*conditional mutant
MYO4/ No visible phenotype
SHE1
Altered growth
Gene disruption
Myosin proteins of Saccharomyces cerevisiae.
MYO1
Gene
name
Table 7.
Actin patches
Accumulates in growing buds
Bud tips and areas of
polarized growth, colocalizes
with suppressor SMY1
Bud neck
Cellular localization
73, 81, 82
25, 39, 83,
105, 130
179, 225
Reference
Class I, N-ter motor domain,
high homology to Myo3p,
3#IQ motif, proline-rich
region and SH3 domain
73, 81
Class V, minimyosin transport 22, 85, 102
of Ash1p to bud
Class I, N-ter motor domain,
2#IQ motif, proline rich
region and SH3 domain, high
homology to Myo5p
Class V, minimyosin binds
calmodulin , 6#IQ motif,
SMY1 suppresses myo2-66
Class II, myosin heavy chain
Protein function or sequence
similarity
??????????? ??? ????????????? ????????????
417
418
Calmodulins bind to myosins through a regulatory
domain of one or several IQ motifs for which the
consensus sequence is Ile-Gln-x-x-x-Arg-Gly-x-xx-Arg. The C-terminal domain of myosins is much
more variable and probably accounts for their
diverse cellular functions.39,47,237 In S. cerevisiae,
five genes, MYO1?MYO5, coding for proteins
belonging to three of the 13 proposed classes of
myosins have been investigated (Table 7).
Class I classical myosins, encoded by the MYO3
and MYO5 genes, have recently been identified
and found to have overlapping functions. Neither
null mutation by itself results in an observable
phenotype whereas the double mutant is dead, or
severely affected, depending on the strain background.73,81,82 Surviving double deletion strains
have severe growth defects and display multiple
actin cytoskeleton defects, including round cells,
random bud site selection, sensitivity to high
osmotic strength and low pH as well as defects
in chitin and cell wall deposition, invertase
secretion and fluid phase endocytosis.81 When a
temperature-sensitive allele myo5-1 was used
to partially complement an almost-lethal
Фmyo3Фmyo5 double mutant, a strong defect in
с-factor-receptor-mediated internalization was
shown, while invertase secretion and carboxypeptidase Y maturation were apparently unaffected.73
These results suggest a more specific role in endocytosis than the effects on cytoskeleton observed
previously.
The MYO1 gene codes for a heavy chain myosin
which is similar to conventional class II myosins
found in both muscle and non-muscle cells. Myo1p
was first described as being necessary for cell
division because growing deficient cells formed
chains.225 It has since been shown that deposition
of chitin and cell wall components necessary for
cell separation are affected and that Myo1p is
necessary for the maintenance of cell-type-specific
budding patterns.179
The unconventional class V myosin, Myo2p,
was identified by the myo2-66 mutation which
causes vesicles to accumulate in the cytoplasm and
thus it was suggested as a possible motor linking
actin and secretory vesicles.105 Further analyses
of the temperature-sensitive mutation myo2-66,
showed that it is involved in a post-Golgi stage of
a secretory pathway distinct from bulk secretion.83
It has been shown through genetic interactions and
several direct protein-binding methods that
Myo2p is a Ca2+ -independent target of calmodulin
and that it is localized at sites of cell growth. In
?. ?????? ??? ?. ????????
support of this, mutations in CMD1 and MYO2
show allele-specific synthetic lethality and Myo2p
and calmodulin distributions are indistinguishable
at sites of cell growth throughout the cell cycle.24,25
Results are consistent with a model in which
Myo2p is implicated in polarized growth and
suggest that transport of vesicles from the mother
cell into the bud occurs in a distinct secretory
pathway.
Surprisingly, a dosage-dependent suppressor of
the temperature-sensitive mutation myo2-66 is
Smy1p, a kinesin-related protein of the superfamily of microtubule motor proteins.129 Although
Myo2p localization does not require Smy1p, overexpression of SMY1 enhances Myo2p staining.
Neither distribution colocalizes with actin spots
but both correlate temporally with the presence of
actin patches. This correlation was observed after
temperature or osmotic shift and when patch distribution was perturbed due to the presence of
actin and secretory gene mutations. Since Smy1p
co-localizes with Myo2p in areas of active cell
growth which are the same regions as actin
patches, it is unlikely to be a specific microtubule
motor.130
The other class V myosin identified in yeast,85
Myo4p (also called She1p), has recently been
shown to be involved in mating-type-specific
switching. HO expression depends on at least five
proteins including the Myo4p/She1p minimyosin.
It accumulates preferentially in growing buds
where it is necessary for the preferential accumulation of Ash1p, a repressor of HO.22,102 Thus,
Myo4p/She1p is probably responsible for the
transport of proteins from the mother to the
bud.
A role for myosins in mitochondrial movement
has been suggested by work from Lisa Pon?s group
who showed that the binding of F-actin to isolated
mitochondria could be competed with myosin subfragment 1.120 The motor activity on the mitochondrial surface has been further characterized
in vitro, where F-actin sliding on isolated mitochondria shows kinetic characteristics similar to
members of the myosin family.199 However, while
actin mutants were shown to be defective in transport of mitochondria from the mother to the bud
(confirming that the actin cytoskeleton is important for normal mitochondrial movement during
inheritance), mutants lacking myo1, myo2, myo3
and myo4 or both myo2 and myo4 were not
affected in mitochondrial spatial arrangement or
inheritance, suggesting that the protein responsible
??????????? ??? ????????????? ????????????
for this actin-based motor activity remains to be
identified.120,199
An S. cerevisiae gene, MLP1, was found by
screening with an antibody to human heavy chain
myosin. The Mlp1 protein resembles myosin in
that it contains multiple heptad repeats similar to
myosin coiled-coil regions but shows no head
domain similarity. Antibodies to a bacterial Mlp1
fusion protein stained a region adjacent to the
nucleus in strains overexpressing MLP1. Null
alleles were sensitive to UV radiation, suggesting a
possible role in DNA repair.115
Other proteins interacting with actin The list of
proteins associated with actin is long. Many of
these proteins have been identified by model
genetic screens and have been reviewed by Welch
et al.228 Table 8 contains a summary of results
concerning actin ?associated? or interacting proteins. Several genes whose mutant alleles disrupt
the actin cytoskeleton or which react with actin in a
two-hybrid system have been documented recently.
SAC and RAH genes (see Table 8) are two
categories of previously described suppressors,1,40,62,160 each of which compensates one
phenotype of actin mutations. That is, the SAC
genes were selected as suppressors of the temperature sensitivity of actin mutations while the RAH
genes suppress only the osmosensitivity. Recent
investigation has shown that Sac3p, like another
previously described actin interactant important
for morphogenesis and actin cytoskeleton function, Anc1p,227 is located in the nucleus.14 Mutant
sac3 strains display a high proportion of large
budded cells. Some of these cells had a single
nucleus at the mother-bud neck and a short intranuclear spindle indicating a G2/M cell cycle delay.
Increased chromosome loss was also observed.
Thus, Sac3p, which was identified as a suppressor
of actin, is required for normal progression
of mitosis and indirectly affects microtubule
function.14
The RVS (for recovery from starvation) genes
are an interesting example of genes which were
selected for their possible role in nutrient signalling. In starvation conditions rvs161 and rvs167
mutants had abnormal morphologies and died
rapidly. Further characterization revealed that in
either nutrient starvation or salt stress conditions,
mutants were osmosensitive and the actin cytoskeleton was disturbed.15,48,200 Although mutations in the genes display indistinguishable
phenotypes, they are different in that Rvs167p
419
contains an SH3 domain and binds to actin in the
two-hybrid system.10 Rvs167p and Rvs161p also
interact with each other (M. Aigle, personal communication). Localization of Rvs161p as a functional GFP fusion revealed punctate staining at the
plasma membrane (M. Aigle, personal communication). This is especially notable in light of the
fact that an rvs161/end7 mutation was isolated in a
screen for endocytosis-deficient mutants. In fact,
mutants in both RVS genes are defective in the
internalization step of receptor-mediated endocytosis.154 While it appears logical that endocytosis
and nutrient sensing should be linked, the precise
intersection of these functions with each other and
with the cytoskeleton promises to involve a
number of other, as yet unidentified, liaisons.
A number of selection screens and investigations
by the Riezman group have identified genes
involved in fluid phase and receptor-mediated
endocytosis. Both actin and fimbrin were shown to
be necessary for the initial internalization step
while subsequent traffic to the vacuole was unaffected,175 suggesting that the actin cytoskeleton
was necessary for endocytosis. The finding that the
endocytosis mutant, end3-1, also showed delocalization of actin patches at the non-permissive
temperature17 suggested an interdependence of
possible structures necessary for endocytosis and
the cortical cytoskeleton. In a recent screen for
endocytosis-deficient mutants, END4, END5,
END6 and END7 genes were found to correspond
to four previously known genes, SLA2/MOP2,
VRP1, ACT1 and RVS161, all of which are part
of, or interact with, the cortical cytoskeleton.154 As
previously mentioned, the class I myosins have
also been implicated in endocytosis.73,81
A two-hybrid screen reported for actin cytoskeletal components used actin as bait and identified four new actin-interacting proteins as well as
seven known proteins: actin, profilin, Rvs167p,
Srv2p (suppressor of activated Ras), OYE2 (old
yellow enzyme=NADPH oxidoreductase), GLK1
(glucokinase) and RPL45 (a ribosomal protein).10
Differential interactions with cloned act1 alleles
revealed possible actin surfaces involved in these
interactions. Profilin and the SH3 domain containing protein; Rvs167p showed a similar pattern of
differential interactions. Other SH3-containing
proteins were tested and Fus3p identified as a
protein with essentially the same pattern of differential interactions. After recalling that profilin and
SH3 domains have similar crystal structures, and
that both bind polyproline, the suggestion was
ts
Lethal
?
?
?
?
Lethal
ts
Lethal
?
?
?
ts, rvs161Ф act1-1
lethal
ts, rvs167Ф act1-1
lethal
ANC2
ANC3
ANC4
AIP1
AIP3/BUD6
ARP2/ACT2
END3
PAN1
RAH1
RAH2
RAH3
RVS161/END6
RVS167
Gene disruption
Reduced viability upon starvation,
disorganized actin in salt, defective
endocytosis
Reduced viability upon starvation,
disorganized actin in salt, defective
endocytosis
*ts, delocalized patches
*ts and cs for growth
*ts for growth
?
Karyokinesis, cytokinesis defects
*Osmosensitive, mitochondrial
clumping, disorganized actin,
random budding, endocytosis
defect, vesicles accumulation
?Defective endocytosis,
disorganized actin
*Delocalized large patches,
abnormal buds, cytokinesis defect,
random budding
*ts and cs for growth, randomly
oriented cables, delocalized
patches, osmosensitive
*ts for growth, defective
morphogenesis, disorganized actin
Phenotype of ?null or
*conditional mutant
Actin-associated proteins of Saccharomyces cerevisiae.
ANC1/TFG3
Gene
name
Table 8.
Plasma membrane Two-hybrid reaction with actin,
GPA domain, SH3 domain
Cortical patches
221, 229
221, 229
10
10
150, V.
Moreau et al.,
submitted
38, 221, 229
221, 227, 229
Reference
48, 154, 200,
M. Aigle,
unpublished
10, 15, 48, 154
40
40
40
185, 186, 214,
240
EF hand Ca2+ domain, PIP2 motif 17, 175
Actin non-complementing, MF
and MT chaperonin, TCP-1
related
Actin non-complementing
Actin non-complementing
Two-hybrid reaction with actin
Two-hybrid reaction with actin
46% identity to actin
Actin non-complementing
Protein function or
sequence homology
EF hand Ca2+ domain, 2#EH
domain, SH3 domain, similarity
Sla1p, PIP2 motif
?
Suppresses osmosensitivity, not ts
act1-1
?
Suppresses osmosensitivity, not ts
act1-1
?
Suppresses osmosensitivity, not ts
act1-1, may be ABP in osmotic
stress recovery
Plasma membrane GPA domain, similarity to
amphiphysin
?
?
?
?
?
Cortical patches
?
Nucleus
Cellular
localization
420
?. ?????? ??? ?. ????????
*Osmosensitive, delocalized
patches, randomly oriented cables,
inositol auxotrophy
*Osmosensitive, delocalized
patches, random cables
*Osmosensitive, delocalized
patches, randomly oriented cables,
large-budded cells (G2/M delay),
chromosome loss
*Osmosensitive, delocalized
patches, random cables
*Osmosensitive, delocalized
patches, random cables
*Osmosensitive, delocalized
patches, random cables
?Large, delocalized patches
ts, sla1Ф abp1Ф
lethal
ts, sla2Ф abp1Ф
?Endocytosis deficient,
lethal sla2Ф sac6Ф disorganized actin
lethal
slc1Ф cap2Ф lethal *SD ts at 39)C, cell wall fragility,
osmosensitive, disorganized actin,
pseudomycelial growth
slc2Ф cap2Ф lethal *SD ts at 39)C, cell wall fragility,
osmosensitive, disorganized actin,
pseudomycelial growth
ts
Aberrant morphology, aberrant
chitin deposition, disorganized
actin, defective endocytosis
c old-sensitive
?
?
Nucleus
Golgi and ER
Proline-rich regions
?
?
C-terminus similarity to talin
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses act1-4 ts, but not
osmosensitivity
3#SH3 domain
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses ts, but not
osmosensitivity of act1-1
SD, semi-dominant; ts, temperature-sensitive; MF, microfilament; MT, microtubule; ER, endoplasmic reticulum.
VRP1/END5
SLC2
SLC1
SLA2/END4/
MOP2
SLA1
SAC7
SAC5
SAC4
SAC3
SAC2
SAC1
55, 154
108
108
91, 154, 157,
175
91
62
160
160
14, 160
160
42, 160, 232
??????????? ??? ????????????? ????????????
421
?. ?????? ??? ?. ????????
422
Table 9.
Proposed names of S. cerevisiae actin-related proteins (ARPs) according to actin relatedness.
ORF no.
YHR129c
YDL029w
YJR065c
YJL081c
YNL059c
YLR085c
YP9367.14
YOR3348c
YM9973.07
YD9727.02
Chr no.
% Identity
% Similarity
Proposed
gene name
Published
gene name
VIII
IV
X
X
XIV
XII
XVI
XV
XIII
IV
45З1&1З9
45З7&1З5
38З5&1З0
30З4&1З4
25З7&0З9
24З1&1З2
21З9&0З8
20З8&0З8
17З0&0З7
17З3&0З7
68З5&1З4
69З3&1З2
60З3&0З9
52З5&1З4
51З2&1З1
45З8&0З7
43З6&0З8
43З5&0З8
40З2&0З8
37З6&0З9
ARP1
ARP2
ARP3
ARP4
ARP5
ARP6
ARP7
ARP8
ARP9
ARP10
ACT3/ACT5
ACT2
ACT4
ACT3
?
?
?
?
?
?
made that SH3 domains may be actin-binding
motifs with somehow similar consequences to
profilin-actin binding.10
Very recently, an essential gene, PAN1, originally thought to code for poly(A) ribonuclease,185,186 has instead been shown to be a factor
necessary for normal organization of the actin
cytoskeleton.214 A pan1-4 mutant was unable to
reorganize the actin cytoskeleton during the cell
cycle, showed random budding patterns and
abnormal bud growth, and was defective in cytokinesis. In addition to containing an EF-hand
Ca2+ -binding domain and an SH3-binding
domain, Pan1p localizes to cortical actin patches.
It thus appears to be a new ABP.214
Actin-related proteins
Since the serendipitous finding of the first protein similar to actin, yet clearly removed from the
family of classical actins,197,198 many actin-related
proteins (ARPs) have been found in yeast, flies,
frogs and mammals. The finding that an Hsp70
protein with only an 11 amino acid conserved
homology in the ATPase binding pocket, as well as
sugar kinases and bacterial cell cycle proteins had
similar crystal structures to actin around what has
been called the ?actin fold? constitutes the creation
of a superfamily of actins with a wide variety of
functions.106 Actin-related proteins are part of the
superfamily of actins and have been reviewed
recently.68,153 Three classes of ARPs most closely
related to actin are widely present across species
and a nomenclature has been suggested.195 Which
members belong to Arp1p, Arp2p or Arp3p classes
is evident by virtue of their higher intra-class
homology. They show, respectively, 50?55%, 45?
Reference
41, 151
150, 198
97
87, 103, 226
50% and 35?40% identity with actin. The unique
Arp1p (actin-RVP121 or centractin41 homologue in
S. cerevisiae, chromosome VIII), named both
ACT5151 and ACT3,41 is less homologous to other
members of the Arp1p group (see Table 9). Functions for the closely related ARPs have been
suggested in various organisms and are beginning
to emerge in S. cerevisiae.
Arp1p is the best characterized of the ARPs. As
mentioned in the section on microtubule motors,
in mammalian cells Arp1p has been shown to
be part of a multi-protein dynactin complex
which activates dynein to move membrane vesicles
along microtubules.121 Part of this complex is a
37 nm mini-filament structure observed by electron
microscopy, which is estimated to contain one
molecule of actin for nine molecules of Arp1p and
is thought to be involved in the association of
cargo with the complex.194 In S. cerevisiae, loss
of Arp1p function results in mis-orientation of
the mitotic spindle and defective nuclear
migration.41,151 These phenotypes are identical to
those caused by deletion of the DHC1 gene coding
for the dynein heavy chain; thus a similar role is
proposed. What is more, an arp1Фdhc1Ф strain
does not appear to have any additional defects,
suggesting that the two proteins act in the same
process.151 No direct interaction with the actin
cytoskeleton has been established.
Arp2p was first reported197 as an essential protein whose deletion gave rise to spores which
produced uniformly arrested cells with a single
large bud. This suggested a possible role in cytokinesis.198 Since then, biochemical studies in
Acanthamoeba from the Pollard group isolated
Arp2p, in complex with Arp3p and five other
??????????? ??? ????????????? ????????????
proteins, on a profilin?agarose column.134 Threedimensional modelling suggested that Arp2p had
a profilin-binding site, while antibody staining
revealed the protein in the cortex just under the
plasma membrane.110 In S. cerevisiae, the Arp2
protein (as revealed by an anti-peptide antibody)
localized essentially in actin cortical patches at
sites of membrane growth.150 Analysis of one
strain with a temperature-sensitive mutation, act2H330L, in a loop region non-homologous to actin,
revealed multiple phenotypes resembling those
found for act1 mutants. Random budding, osmosensitivity, delocalized actin after prolonged incubation at the non-permissive temperature, and lack
of Lucifer Yellow uptake at all temperatures were
observed.150 It has also been found by analysis of a
membrane transporter, uracil permease, that the
internalization step of endocytosis is severely
blocked in the temperature-sensitive mutant act2H330L, while secretion of invertase and carboxypeptidase Y appeared normal (V. Moreau, J. M.
Galan, G. Devilliers, R. Tsapsis-Haguenaur and
B. Winsor, submitted). Arp2p may have additional
functions, as an ARP2 temperature-sensitive
mutant (act2-1) was isolated in a screen for
deficient nuclear import. Nuclear pores appeared
to be affected at early times. By indirect immunofluorescence, an HA-tagged Arp2p is associated
with the nucleus (C. Yan and T. Mele?se, in press).
The differences between these localization results
have not yet been resolved, but multiple roles and
interactions seem to be part of the explanation.
The function of Arp3p is unknown. The protein
has been found as part of the same seven-protein
complex as Arp2p, isolated on a profilin-agarose
from Acanthamoeba, and localized to the cortex.110,134 In contrast to these results, Murgia et al.
found the Dictyostelium Arp3 protein in mitochondria.155 In S. cerevisiae, its function is essential and
null mutant cells stop growth after gemination
without a uniform arrest phenotype97 in several
different strains (A. Madania and B. Winsor,
unpublished). Its localization in yeast should be
informative.
Distantly related ARPs ARPs are a good
example of a family of proteins where genomic
sequencing has greatly increased the number of
family members. Inspection of the S. cerevisiae
complete genomic sequence revealed the presence
of at least ten actin-related sequences, five new
genes for distantly related ARPs in addition to
Arp1 (ACT5/ACT3) on chromosome VIII, Arp2
423
(ACT2) on chromosome IV, Arp3 (ACT4) on
chromosome X, and the more distant ARPs on
chromosome X87 (described below, also called
ACT3) and on chromosome XIV,18 show substantial blocks of conserved residues when the primary
sequences are aligned with a group of 29 disparate
and representative actins (O. Poch and B. Winsor,
in press). However, the existing nomenclature is
somewhat confusing. We therefore analysed and
ordered the amino acid sequences of the new ARPs
in order to put forth a nomenclature for the newly
found S. cerevisiae open reading frames. The distantly related ARPs were named according to their
relatedness to representative actins (Table 9).
Functional studies on only one of the more
distantly related ARP genes, ARP4 (ACT3) in the
proposed nomenclature, have been reported. The
gene is essential,87 and the protein has been localized in the nucleus (without any apparent connection to actin filaments).226 In a transcription assay,
genetically identical mutant cells gave rise to positive or negative sectoring phenotypes for a reporter
gene. These epigenetic effects of the mutant protein
apparently result from effects on chromatin structure.103 The question remains, for many of the
ARPs, whether they are directly implicated in the
actin cytoskeleton or whether they carry out other
diverse functions.
Regulation of the actin cytoskeleton
Cell-cycle dependence The question of what initiates polarization of the actin cytoskeleton to
a specialized pre-bud site has been addressed
in elegant S. cerevisiae studies by Lew and
Reed.123,124 Various cyclin/Cdc28 kinase complexes control cell cycle progression including the
commitment point called START. Using both
kinase and cyclin mutants affected in G1 or G2,
polarization of the actin cortical cytoskeleton in
unbudded G1 cells was shown to depend on activation of Cdc28p by the G1 Cln cyclins at START
of the cell cycle. This polarization did not require
protein synthesis, suggesting direct phosphorylation of as-yet unidentified substrates. Actin
cytoskeleton depolarization and that of the secretory apparatus depend on activation of Cdc28p
by the mitotic Clb cyclins in budded G2 cells.50
Inactivation of Cdc28 in mitosis triggers redistribution of cortical patches to the neck for cytokinesis.123 Furthermore, when bud formation was
inhibited, a significant delay in nuclear division
ensued, suggesting a G1/S checkpoint that
monitors morphogenesis.124
424
Regulation by Rho and signalling Several types of
results suggest that the actin cytoskeleton is regulated by hormone and growth factor signals which
are transmitted via small GTP-binding or G
proteins in cellular signalling pathways to the
cytoskeleton as well as to the transcription apparatus. The prototype mammalian signalling protein
Ras has homologues called Rho (for Ras homologue). In S. cerevisiae CDC42 with RHO1-4
gene products constitute this family of G protein
homologues which regulate the actin cytoskeleton
(reviewed by Ridley177). Cdc42 was initially
defined as an essential protein necessary for
bud emergence.5 An assay in permeabilized cells
using fluorescently labelled actin monomers in the
assembly of actin structures has been used to show
that lack of nucleation activity in a cdc42 mutant
strain could be complemented in vitro by a constitutively active Cdc42 protein.127 Bem3p is the
GTP activating protein and Cdc24p the nucleotide
exchange factor which regulates the level of active
Cdc42p necessary for the organization of actin
filaments. In addition, Cdc42p and Bem1p (for
bud emergence) are key proteins in the cell-cycledependent reorganization of actin structures operating in bud emergence and in shmoo formation
following pheromone stimulation of mating.
Mutants of bem1 are mating deficient because they
are incapable of polarizing their actin cytoskeleton. Ste20p has been directly implicated with
actin since protein interaction assays revealed
Bem1p with actin as well as Ste5p and Ste20p, the
receptor of pheromone signals, at a branchpoint
for MAP kinase signalling pathways and the
cytoskeleton.122 Another Ste20-like kinase, Cla4p,
interacts with Cdc42p and is required for normal
cell growth and cytokinesis.51 Bud growth requires
the presence of an intact actin cytoskeleton and
depends on the activity of other Rho proteins in
addition to Cdc42p. Rho1p is concentrated in
areas of cell growth in the same regions as the actin
cortical cytoskeleton.138,139,238 Loss-of-function
rho3 and rho4 mutants show delocalized actin and
chitin.138,139 Additionally, the Slt2/Mpk1 map
kinase of the cell integrity pathway seems to be
implicated because kinase mutants show an alteration of actin polarization and an accumulation
of vesicles.140
How do these proteins interact? CAP (not to be
confused with Cap1p and Cap2p) is a component
of the adenyl cyclase complex whose N-terminal
domain is required for Ras responsiveness and
whose C-terminal part binds actin and is associ-
?. ?????? ??? ?. ????????
ated with morphological (budding) and nutritional
defects. Profilin overexpression counters these
morphological and nutritional defects by binding
not only to actin but perhaps also to polyphosphoinositides (PIP2).222 Binding studies of wildtype and mutant profilins with PIP2 are consistent
with profilin being a negative regulator of the
phosphoinositide signalling pathway, although
this has not been demonstrated formally.77,78,86
This could be an important regulation point for
Rho-mediated effects on the cytoskeleton. Profilin
and the SH3-domain-containing protein, Rvs167p,
showed a similar pattern of differential actinbinding interactions in a two-hybrid assay, hence
the suggestion that SH3 domains may be actinbinding motifs with similar consequences to
profilin-actin binding.10 The localization of CAP
to actin-containing cortical structures in vivo is
dependent on its SH3 domain (which binds to
proline-rich regions). The proline-rich region of
CAP is recognized by SH3 domains of several
other proteins including Abp1p.
Many other fascinating results are emerging
which establish cell signalling pathways in yeast
and possible connections with the actin cytoskeleton but an inclusive discussion is beyond the
scope of this review.
Interactions between microtubules and
microfilaments
The regulation of microtubule functions in cell
division depends on cell cycle regulation orchestrated by cyclin-dependent kinases. In addition,
the trigger for polarization of actin filament structures at START is directly dependent on G1
cyclin/Cdc28p complexes and depolarization corresponds to a G2 cyclin/Cdc28p control.123,124
Furthermore, not only does the timing of major
events seem to have a common control, a number
of other molecules indicate important overlaps or
liaisons between the, once believed to be, separate
filament systems. For example, TCP1, BIN2, BIN3
and ANC2 genes code for chaperonin-type
proteins which are necessary for both actin and
tubulin functions.38,220 Folding of vertebrate
Arp1p and у-tubulin was previously shown to be
chaperone-mediated142 but the effect of the yeast
chaperonin-type proteins on other ARPs has not
been reported. Perhaps one of the best examples of
a link between the microfilament and microtubule
systems is the ARP Arp1p itself. While clearly a
close structural analogue of actin, both genetic and
??????????? ??? ????????????? ????????????
biochemical studies have shown Arp1p to be
an important component of the heteroligomer
regulating the dynein microtubular motor complex.194 Indeed, actin itself has been shown to
be necessary for the proper orientation of
microtubules and nuclear migration.167
The cytoskeleton is also an illustration that one
should be careful not to assume that the same
structure should mean the same function. Most
yeast myosins, albeit structurally resembling vertebrate myosins, may not be motors. The two known
tropomyosins seem to have different functions in
addition to partially overlapping functions. Arp1p
and Arp2p, though clearly structurally close to
actin, are apparently implicated in different cellular functions. Even more striking are the more
distant ARPs; Arp4p is an example for which a
suggestion of cellular function is provided by the
localization in the nucleus,103,226 where actin has
never been definitively demonstrated.
Many intriguing questions remain, including
what distinguishes cable filaments from patch filaments, how and when do each of these microfilament structures interact with each other and
with microtubules, and whether the cytoskeleton
plays a role in the switch76 to filamentous growth.
Knowledge of the complete genome sequence will
greatly facilitate future analyses of cytoskeletal
components and provide the framework necessary
to understand the cytoskeletal functions and
interactions necessary to sustain eukaryotic life.
ACKNOWLEDGEMENTS
We thank R. Arkowitz and R. Martin for critically
reading the manuscript. M. Aigle, S. Brown, J.
Cooper, D. Drubin, A. M. A. Hoyt, T. Huffaker,
J. Kilmartin, D. Pellman, M. Snyder and M.
Winey are gratefully acknowledged for communicating data.
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ied as a
protein with essentially the same pattern of differential interactions. After recalling that profilin and
SH3 domains have similar crystal structures, and
that both bind polyproline, the suggestion was
ts
Lethal
?
?
?
?
Lethal
ts
Lethal
?
?
?
ts, rvs161Ф act1-1
lethal
ts, rvs167Ф act1-1
lethal
ANC2
ANC3
ANC4
AIP1
AIP3/BUD6
ARP2/ACT2
END3
PAN1
RAH1
RAH2
RAH3
RVS161/END6
RVS167
Gene disruption
Reduced viability upon starvation,
disorganized actin in salt, defective
endocytosis
Reduced viability upon starvation,
disorganized actin in salt, defective
endocytosis
*ts, delocalized patches
*ts and cs for growth
*ts for growth
?
Karyokinesis, cytokinesis defects
*Osmosensitive, mitochondrial
clumping, disorganized actin,
random budding, endocytosis
defect, vesicles accumulation
?Defective endocytosis,
disorganized actin
*Delocalized large patches,
abnormal buds, cytokinesis defect,
random budding
*ts and cs for growth, randomly
oriented cables, delocalized
patches, osmosensitive
*ts for growth, defective
morphogenesis, disorganized actin
Phenotype of ?null or
*conditional mutant
Actin-associated proteins of Saccharomyces cerevisiae.
ANC1/TFG3
Gene
name
Table 8.
Plasma membrane Two-hybrid reaction with actin,
GPA domain, SH3 domain
Cortical patches
221, 229
221, 229
10
10
150, V.
Moreau et al.,
submitted
38, 221, 229
221, 227, 229
Reference
48, 154, 200,
M. Aigle,
unpublished
10, 15, 48, 154
40
40
40
185, 186, 214,
240
EF hand Ca2+ domain, PIP2 motif 17, 175
Actin non-complementing, MF
and MT chaperonin, TCP-1
related
Actin non-complementing
Actin non-complementing
Two-hybrid reaction with actin
Two-hybrid reaction with actin
46% identity to actin
Actin non-complementing
Protein function or
sequence homology
EF hand Ca2+ domain, 2#EH
domain, SH3 domain, similarity
Sla1p, PIP2 motif
?
Suppresses osmosensitivity, not ts
act1-1
?
Suppresses osmosensitivity, not ts
act1-1
?
Suppresses osmosensitivity, not ts
act1-1, may be ABP in osmotic
stress recovery
Plasma membrane GPA domain, similarity to
amphiphysin
?
?
?
?
?
Cortical patches
?
Nucleus
Cellular
localization
420
?. ?????? ??? ?. ????????
*Osmosensitive, delocalized
patches, randomly oriented cables,
inositol auxotrophy
*Osmosensitive, delocalized
patches, random cables
*Osmosensitive, delocalized
patches, randomly oriented cables,
large-budded cells (G2/M delay),
chromosome loss
*Osmosensitive, delocalized
patches, random cables
*Osmosensitive, delocalized
patches, random cables
*Osmosensitive, delocalized
patches, random cables
?Large, delocalized patches
ts, sla1Ф abp1Ф
lethal
ts, sla2Ф abp1Ф
?Endocytosis deficient,
lethal sla2Ф sac6Ф disorganized actin
lethal
slc1Ф cap2Ф lethal *SD ts at 39)C, cell wall fragility,
osmosensitive, disorganized actin,
pseudomycelial growth
slc2Ф cap2Ф lethal *SD ts at 39)C, cell wall fragility,
osmosensitive, disorganized actin,
pseudomycelial growth
ts
Aberrant morphology, aberrant
chitin deposition, disorganized
actin, defective endocytosis
c old-sensitive
?
?
Nucleus
Golgi and ER
Proline-rich regions
?
?
C-terminus similarity to talin
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses act1-4 ts, but not
osmosensitivity
3#SH3 domain
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses ts, but not
osmosensitivity of act1-1
Suppresses ts, but not
osmosensitivity of act1-1
SD, semi-dominant; ts, temperature-sensitive; MF, microfilament; MT, microtubule; ER, endoplasmic reticulum.
VRP1/END5
SLC2
SLC1
SLA2/END4/
MOP2
SLA1
SAC7
SAC5
SAC4
SAC3
SAC2
SAC1
55, 154
108
108
91, 154, 157,
175
91
62
160
160
14, 160
160
42, 160, 232
??????????? ??? ????????????? ????????????
421
?. ?????? ??? ?. ????????
422
Table 9.
Proposed names of S. cerevisiae actin-related proteins (ARPs) according to actin relatedness.
ORF no.
YHR129c
YDL029w
YJR065c
YJL081c
YNL059c
YLR085c
YP9367.14
YOR3348c
YM9973.07
YD9727.02
Chr no.
% Identity
% Similarity
Proposed
gene name
Published
gene name
VIII
IV
X
X
XIV
XII
XVI
XV
XIII
IV
45З1&1З9
45З7&1З5
38З5&1З0
30З4&1З4
25З7&0З9
24З1&1З2
21З9&0З8
20З8&0З8
17З0&0З7
17З3&0З7
68З5&1З4
69З3&1З2
60З3&0З9
52З5&1З4
51З2&1З1
45З8&0З7
43З6&0З8
43З5&0З8
40З2&0З8
37З6&0З9
ARP1
ARP2
ARP3
ARP4
ARP5
ARP6
ARP7
ARP8
ARP9
ARP10
ACT3/ACT5
ACT2
ACT4
ACT3
?
?
?
?
?
?
made that SH3 domains may be actin-binding
motifs with somehow similar consequences to
profilin-actin binding.10
Very recently, an essential gene, PAN1, originally thought to code for poly(A) ribonuclease,185,186 has instead been shown to be a factor
necessary for normal organization of the actin
cytoskeleton.214 A pan1-4 mutant was unable to
reorganize the actin cytoskeleton during the cell
cycle, showed random budding patterns and
abnormal bud growth, and was defective in cytokinesis. In addition to containing an EF-hand
Ca2+ -binding domain and an SH3-binding
domain, Pan1p localizes to cortical actin patches.
It thus appears to be a new ABP.214
Actin-related proteins
Since the serendipitous finding of the first protein similar to actin, yet clearly removed from the
family of classical actins,197,198 many actin-related
proteins (ARPs) have been found in yeast, flies,
frogs and mammals. The finding that an Hsp70
protein with only an 11 amino acid conserved
homology in the ATPase binding pocket, as well as
sugar kinases and bacterial cell cycle proteins had
similar crystal structures to actin around what has
been called the ?actin fold? constitutes the creation
of a superfamily of actins with a wide variety of
functions.106 Actin-related proteins are part of the
superfamily of actins and have been reviewed
recently.68,153 Three classes of ARPs most closely
related to actin are widely present across species
and a nomenclature has been suggested.195 Which
members belong to Arp1p, Arp2p or Arp3p classes
is evident by virtue of their higher intra-class
homology. They show, respectively, 50?55%, 45?
Reference
41, 151
150, 198
97
87, 103, 226
50% and 35?40% identity with actin. The unique
Arp1p (actin-RVP121 or centractin41 homologue in
S. cerevisiae, chromosome VIII), named both
ACT5151 and ACT3,41 is less homologous to other
members of the Arp1p group (see Table 9). Functions for the closely related ARPs have been
suggested in various organisms and are beginning
to emerge in S. cerevisiae.
Arp1p is the best characterized of the ARPs. As
mentioned in the section on microtubule motors,
in mammalian cells Arp1p has been shown to
be part of a multi-protein dynactin complex
which activates dynein to move membrane vesicles
along microtubules.121 Part of this complex is a
37 nm mini-filament structure observed by electron
microscopy, which is estimated to contain one
molecule of actin for nine molecules of Arp1p and
is thought to be involved in the association of
cargo with the complex.194 In S. cerevisiae, loss
of Arp1p function results in mis-orientation of
the mitotic spindle and defective nuclear
migration.41,151 These phenotypes are identical to
those caused by deletion of the DHC1 gene coding
for the dynein heavy chain; thus a similar role is
proposed. What is more, an arp1Фdhc1Ф strain
does not appear to have any additional defects,
suggesting that the two proteins act in the same
process.151 No direct interaction with the actin
cytoskeleton has been established.
Arp2p was first reported197 as an essential protein whose deletion gave rise to spores which
produced uniformly arrested cells with a single
large bud. This suggested a possible role in cytokinesis.198 Since then, biochemical studies in
Acanthamoeba from the Pollard group isolated
Arp2p, in complex with Arp3p and five other
??????????? ??? ????????????? ????????????
proteins, on a profilin?agarose column.134 Threedimensional modelling suggested that Arp2p had
a profilin-binding site, while antibody staining
revealed the protein in the cortex just under the
plasma membrane.110 In S. cerevisiae, the Arp2
protein (as revealed by an anti-peptide antibody)
localized essentially in actin cortical patches at
sites of membrane growth.150 Analysis of one
strain with a temperature-sensitive mutation, act2H330L, in a loop region non-homologous to actin,
revealed multiple phenotypes resembling those
found for act1 mutants. Random budding, osmosensitivity, delocalized actin after prolonged incubation at the non-permissive temperature, and lack
of Lucifer Yellow uptake at all temperatures were
observed.150 It has also been found by analysis of a
membrane transporter, uracil permease, that the
internalization step of endocytosis is severely
blocked in the temperature-sensitive mutant act2H330L, while secretion of invertase and carboxypeptidase Y appeared normal (V. Moreau, J. M.
Galan, G. Devilliers, R. Tsapsis-Haguenaur and
B. Winsor, submitted). Arp2p may have additional
functions, as an ARP2 temperature-sensitive
mutant (act2-1) was isolated in a screen for
deficient nuclear import. Nuclear pores appeared
to be affected at early times. By indirect immunofluorescence, an HA-tagged Arp2p is associated
with the nucleus (C. Yan and T. Mele?se, in press).
The differences between these localization results
have not yet been resolved, but multiple roles and
interactions seem to be part of the explanation.
The function of Arp3p is unknown. The protein
has been found as part of the same seven-protein
complex as Arp2p, isolated on a profilin-agarose
from Acanthamoeba, and localized to the cortex.110,134 In contrast to these results, Murgia et al.
found the Dictyostelium Arp3 protein in mitochondria.155 In S. cerevisiae, its function is essential and
null mutant cells stop growth after gemination
without a uniform arrest phenotype97 in several
different strains (A. Madania and B. Winsor,
unpublished). Its localization in yeast should be
informative.
Distantly related ARPs ARPs are a good
example of a family of proteins where genomic
sequencing has greatly increased the number of
family members. Inspection of the S. cerevisiae
complete genomic sequence revealed the presence
of at least ten actin-related sequences, five new
genes for distantly related ARPs in addition to
Arp1 (ACT5/ACT3) on chromosome VIII, Arp2
423
(ACT2) on chromosome IV, Arp3 (ACT4) on
chromosome X, and the more distant ARPs on
chromosome X87 (described below, also called
ACT3) and on chromosome XIV,18 show substantial blocks of conserved residues when the primary
sequences are aligned with a group of 29 disparate
and representative actins (O. Poch and B. Winsor,
in press). However, the existing nomenclature is
somewhat confusing. We therefore analysed and
ordered the amino acid sequences of the new ARPs
in order to put forth a nomenclature for the newly
found S. cerevisiae open reading frames. The distantly related ARPs were named according to their
relatedness to representative actins (Table 9).
Functional studies on only one of the more
distantly related ARP genes, ARP4 (ACT3) in the
proposed nomenclature, have been reported. The
gene is essential,87 and the protein has been localized in the nucleus (without any apparent connection to actin filaments).226 In a transcription assay,
genetically identical mutant cells gave rise to positive or negative sectoring phenotypes for a reporter
gene. These epigenetic effects of the mutant protein
apparently result from effects on chromatin structure.103 The question remains, for many of the
ARPs, whether they are directly implicated in the
actin cytoskeleton or whether they carry out other
diverse functions.
Regulation of the actin cytoskeleton
Cell-cycle dependence The question of what initiates polarization of the actin cytoskeleton to
a specialized pre-bud site has been addressed
in elegant S. cerevisiae studies by Lew and
Reed.123,124 Various cyclin/Cdc28 kinase complexes control cell cycle progression including the
commitment point called START. Using both
kinase and cyclin mutants affected in G1 or G2,
polarization of the actin cortical cytoskeleton in
unbudded G1 cells was shown to depend on activation of Cdc28p by the G1 Cln cyclins at START
of the cell cycle. This polarization did not require
protein synthesis, suggesting direct phosphorylation of as-yet unidentified substrates. Actin
cytoskeleton depolarization and that of the secretory apparatus depend on activation of Cdc28p
by the mitotic Clb cyclins in budded G2 cells.50
Inactivation of Cdc28 in mitosis triggers redistribution of cortical patches to the neck for cytokinesis.123 Furthermore, when bud formation was
inhibited, a significant delay in nuclear division
ensued, suggesting a G1/S checkpoint that
monitors morphogenesis.124
424
Regulation by Rho and signalling Several types of
results suggest that the actin cytoskeleton is regulated by hormone and growth factor signals which
are transmitted via small GTP-binding or G
proteins in cellular signalling pathways to the
cytoskeleton as well as to the transcription apparatus. The prototype mammalian signalling protein
Ras has homologues called Rho (for Ras homologue). In S. cerevisiae CDC42 with RHO1-4
gene products constitute this family of G protein
homologues which regulate the actin cytoskeleton
(reviewed by Ridley177). Cdc42 was initially
defined as an essential protein necessary for
bud emergence.5 An assay in permeabilized cells
using fluorescently labelled actin monomers in the
assembly of actin structures has been used to show
that lack of nucleation activity in a cdc42 mutant
strain could be complemented in vitro by a constitutively active Cdc42 protein.127 Bem3p is the
GTP activating protein and Cdc24p the nucleotide
exchange factor which regulates the level of active
Cdc42p necessary for the organization of actin
filaments. In addition, Cdc42p and Bem1p (for
bud emergence) are key proteins in the cell-cycledependent reorganization of actin structures operating in bud emergence and in shmoo formation
following pheromone stimulation of mating.
Mutants of bem1 are mating deficient because they
are incapable of polarizing their actin cytoskeleton. Ste20p has been directly implicated with
actin since protein interaction assays revealed
Bem1p with actin as well as Ste5p and Ste20p, the
receptor of pheromone signals, at a branchpoint
for MAP kinase signalling pathways and the
cytoskeleton.122 Another Ste20-like kinase, Cla4p,
interacts with Cdc42p and is required for normal
cell growth and cytokinesis.51 Bud growth requires
the presence of an intact actin cytoskeleton and
depends on the activity of other Rho proteins in
addition to Cdc42p. Rho1p is concentrated in
areas of cell growth in the same regions as the actin
cortical cytoskeleton.138,139,238 Loss-of-function
rho3 and rho4 mutants show delocalized actin and
chitin.138,139 Additionally, the Slt2/Mpk1 map
kinase of the cell integrity pathway seems to be
implicated because kinase mutants show an alteration of actin polarization and an accumulation
of vesicles.140
How do these proteins interact? CAP (not to be
confused with Cap1p and Cap2p) is a component
of the adenyl cyclase complex whose N-terminal
domain is required for Ras responsiveness and
whose C-terminal part binds actin and is associ-
?. ?????? ??? ?. ????????
ated with morphological (budding) and nutritional
defects. Profilin overexpression counters these
morphological and nutritional defects by binding
not only to actin but perhaps also to polyphosphoinositides (PIP2).222 Binding studies of wildtype and mutant profilins with PIP2 are consistent
with profilin being a negative regulator of the
phosphoinositide signalling pathway, although
this has not been demonstrated formally.77,78,86
This could be an important regulation point for
Rho-mediated effects on the cytoskeleton. Profilin
and the SH3-domain-containing protein, Rvs167p,
showed a similar pattern of differential actinbinding interactions in a two-hybrid assay, hence
the suggestion that SH3 domains may be actinbinding motifs with similar consequences to
profilin-actin binding.10 The localization of CAP
to actin-containing cortical structures in vivo is
dependent on its SH3 domain (which binds to
proline-rich regions). The proline-rich region of
CAP is recognized by SH3 domains of several
other proteins including Abp1p.
Many other fascinating results are emerging
which establish cell signalling pathways in yeast
and possible connections with the actin cytoskeleton but an inclusive discussion is beyond the
scope of this review.
Interactions between microtubules and
microfilaments
The regulation of microtubule functions in cell
division depends on cell cycle regulation orchestrated by cyclin-dependent kinases. In addition,
the trigger for polarization of actin filament structures at START is directly dependent on G1
cyclin/Cdc28p complexes and depolarization corresponds to a G2 cyclin/Cdc28p control.123,124
Furthermore, not only does the timing of major
events seem to have a common control, a number
of other molecules indicate important overlaps or
liaisons between the, once believed to be, separate
filament systems. For example, TCP1, BIN2, BIN3
and ANC2 genes code for chaperonin-type
proteins which are necessary for both actin and
tubulin functions.38,220 Folding of vertebrate
Arp1p and у-tubulin was previously shown to be
chaperone-mediated142 but the effect of the yeast
chaperonin-type proteins on other ARPs has not
been reported. Perhaps one of the best examples of
a link between the microfilament and microtubule
systems is the ARP Arp1p itself. While clearly a
close structural analogue of actin, both genetic and
??????????? ??? ????????????? ????????????
biochemical studies have shown Arp1p to be
an important component of the heteroligomer
regulating the dynein microtubular motor complex.194 Indeed, actin itself has been shown to
be necessary for the proper orientation of
microtubules and nuclear migration.167
The cytoskeleton is also an illustration that one
should be careful not to assume that the same
structure should mean the same function. Most
yeast myosins, albeit structurally resembling vertebrate myosins, may not be motors. The two known
tropomyosins seem to have different functions in
addition to partially overlapping functions. Arp1p
and Arp2p, though clearly structurally close to
actin, are apparently implicated in different cellular functions. Even more striking are the more
distant ARPs; Arp4p is an example for which a
suggestion of cellular function is provided by the
localization in the nucleus,103,226 where actin has
never been definitively demonstrated.
Many intriguing questions remain, including
what distinguishes cable filaments from patch filaments, how and when do each of these microfilament structures interact with each other and
with microtubules, and whether the cytoskeleton
plays a role in the switch76 to filamentous growth.
Knowledge of the complete genome sequence will
greatly facilitate future analyses of cytoskeletal
components and provide the framework necessary
to understand the cytoskeletal functions and
interactions necessary to sustain eukaryotic life.
ACKNOWLEDGEMENTS
We thank R. Arkowitz and R. Martin for critically
reading the manuscript. M. Aigle, S. Brown, J.
Cooper, D. Drubin, A. M. A. Hoyt, T. Huffaker,
J. Kilmartin, D. Pellman, M. Snyder and M.
Winey are gratefully acknowledged for communicating data.
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