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Lessons from yeast: the spindle pole body
and the centrosome
John V. Kilmartin
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK
rstb.royalsocietypublishing.org
Review
Cite this article: Kilmartin JV. 2014 Lessons
from yeast: the spindle pole body and the
centrosome. Phil. Trans. R. Soc. B 369:
20130456.
http://dx.doi.org/10.1098/rstb.2013.0456
One contribution of 18 to a Theme Issue
‘The centrosome renaissance’.
Subject Areas:
cellular biology
Keywords:
spindle, pole, bodies, centrosomes
Author for correspondence:
John V. Kilmartin
e-mail: [email protected]
The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly
established. This involved a large variety of techniques which are described
here, and the potential use of some of these in the centrosome field is highlighted. In particular, very useful structural information on the SPB was
obtained from a reconstituted complex, the g-tubulin complex, and also
from a sub-particle, SPB cores, prepared by extraction of an enriched SPB
preparation. The labelling of SPB proteins with GFP at the N or C termini,
using GFP tags inserted into the genome, gave informative electron
microscopy localization and fluorescence resonance energy transfer data.
Examples are given of more precise functional data obtained by removing
domains from one SPB protein, Spc110p, without affecting its essential function. Finally, a structural model for SPB duplication is described and the
differences between SPB and centrosome duplication discussed.
1. Introduction
The functional equivalent of the centrosome in budding yeast is called the spindle pole body (SPB). It lies embedded in the nuclear membrane, which remains
intact during mitosis. The SPB is a cylindrical multi-layered organelle, with the
two most visible layers being the outer and central plaques (figure 1a). Attached
to one side of the SPB is a specialized part of the nuclear membrane called
the half-bridge. Here, the lipid bilayers are darkly stained on thin sections,
and a thin cytoplasmic half-bridge outer layer lies just above the outer nuclear
membrane. This part of the SPB has an important role in SPB duplication.
Almost all of the components of the SPB have been identified and their
locations are shown in figure 1b. This review will describe the approaches to identifying SPB components, excluding transiently associated components such as
those involved in mitotic exit and checkpoints. It will describe how the functions
of some of these SPB components were established, and what lessons can be
learnt that be helpful in establishing the functions of centrosome components.
2. Enrichment and extraction to prepare sub-particles
The first SPB components were identified from a monoclonal antibody screen of
a 600-fold enriched preparation [1]. The monoclonals were later used to screen
expression banks to identify the corresponding genes [2,3]. Subsequently,
further SPB genes were identified by mass spectrometry screening of a more
enriched SPB preparation [4]. This approach of enrichment [5,6] followed by
mass spectrometry [7,8] has also been successful with the centrosome. There
are additional benefits of enrichment. First, the enriched preparation can be
very useful in characterizing antibodies against low abundance components
of both SPBs [1] and centrosomes [9,10], which were not detectable in western
blots of whole cell lysates. Second, enriched preparations are very useful in
high-resolution cryo-electron microscopy (cryoEM) structural work [11–14],
where a high density of SPBs or centrosomes is required. Lastly, the enriched
preparation can be extracted to prepare sub-particles: for instance, treatment
of the enriched SPBs with heparin [1] gave sub-particles, SPB cores, morphologically similar to central plaques, which were later made essentially pure,
and contained Spc110p, Cnm67p, Spc42p, Spc29p, calmodulin (Cmd1p) and
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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(a)
spindle
CP
CP
OP
OP
Tub4p, Spc97 + 98p
Spc72p
Nud1p
Cnm67p
Spc42p
Bbp1p, Nbp1p
Ndc1p, Mps2p
Spc29p
Cmd1p
Spc110p
Tub4p, Spc97 + 98p
halfSPB bridge (HB)
cytoplasmic
microtubules
outer plaque (OP)
half-bridge outer layer
Cdc31p, Sfi1p
Kar1p, Mps3p
nuclear
membrane
nucleus (NM)
central plaque (CP)
inner plaque
spindle microtubules
Figure 1. (a) Thin section of a yeast spindle showing two SPBs. Also shown are
the central plaque (CP), outer plaque (OP), half-bridge (HB) and nuclear membrane (NM). Scale bar, 0.1 mm. (b) Diagram of the SPB showing the localization
of components, excluding those involved in mitotic exit and checkpoints.
some Nud1p [15]. Examination of the SPB cores by cryoEM
methods [11] showed that a particular layer in the SPB core
containing Spc42p was crystalline; in addition a purified
preparation of Spc42p sheets prepared from overexpressing
cells was also crystalline [3,11], suggesting that SPB assembly
started with a crystallization of Spc42p. Some preliminary
extractions have been described on centrosomes to check
their effects on parthenogenesis [16], and it seems likely
that some of these, together with future sub-particles, could
give useful structural data, given the current advances in
cryoEM single particle analysis and tomography [17,18].
3. Genetics and the analysis of phenotypes
Yeast has very powerful genetics and this has contributed to
the identification and functional analysis of SPB components.
One of the original Hartwell cell cycle genes, CDC31 [19],
was the yeast centrin [20] involved in SPB duplication [21]
and localized to the half-bridge [22]. The Hartwell cell cycle
genes were identified from screening random ts mutants
for their budding phenotype. When immunofluorescence
methods were developed for yeast [23,24], the same bank
was screened with anti-tubulin fluorescence, and MPS1 and
MPS2 were identified [25]. MPS1 encodes a conserved
kinase involved in SPB duplication [26], while MPS2 encodes
a membrane protein involved in SPB insertion into the
nuclear membrane [25,27]. Suppressor analysis of ts mutants
of components discovered by other methods has also been
very fruitful; thus, SPC98 [28], encoding the 90 kDa component of the SPB [1], was identified from a suppressor
analysis of tub4-1, a ts mutant in Tub4p, the yeast g-tubulin
4. Isolation of complexes
A very productive way of identifying and establishing the
function of SPB components was to tag them and use the
tag in a pulldown to identify binding partners. This isolation
can be the basis for a reconstitution giving larger quantities
and allowing a structural and in vitro functional approach.
All of this is illustrated most clearly in the case of the yeast
g-tubulin complex. This is composed of three proteins:
Tub4p, the g-tubulin homologue in Saccharomyces cerevisiae
[29,37,38], together with Spc98p and Spc97p [28,30]. The
g-tubulin complex was purified in a pulldown after attaching
protein A to either Spc98p or Spc97 [39]. This complex was
later reconstituted in vitro [40], and cryoEM reconstruction
showed that the complex had 13-fold symmetry and probably
acted as a template for microtubule initiation [41].
Some centrosome complexes have been isolated, such as
the g-tubulin ring complex [42] and the CP110 complex
[43], although at present for the latter only the identity of
the other components has been reported.
5. Two-hybrid analysis and fluorescence
resonance energy transfer
Two-hybrid analysis was very useful in establishing the pattern of interactions between the domains of different proteins,
for example the N and C terminal domains of the coiled-coil
proteins within the SPB core [15,44]. Only one SPB component was found from a two-hybrid genomic screen,
Spc72p [45]; this protein was also found from a mass
spectrometry screen [4].
Fluorescence resonance energy transfer (FRET) has also
been used to examine the interactions between the N and C
termini of core SPB components [46] after labelling with
CFP or YFP. This method has the additional advantage that
it is possible to estimate the distance between the domains.
Phil. Trans. R. Soc. B 369: 20130456
(b)
2
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NM
HB
homologue [29]. A further suppressor analysis using spc982 identified SPC97 [30]. These three proteins constitute the
yeast g-tubulin complex (see §4).
Analysis of ts phenotypes could also be helpful if the phenotype was clear; thus mps2-1 showed a very clear defect in
the insertion of the SPB into the nuclear membrane [25].
In this work and subsequently it was essential to examine
the cells by serial sectioning to obtain a clear view of the
phenotype.
Another very useful genetic approach in yeast was the
tagging of most of the genes with GFP [31]. Although this
screen did not identify any new SPB components, it did
give reassurance that probably all the components had been
identified, except possibly for very low abundance SPB proteins encoded by the 25% of the genome where the tagging
procedure was unsuccessful.
In the case of the centrosome, an RNAi screen for mitotic
phenotypes in Caenorhabditis elegans was very productive
in identifying several genes involved in the initial stages of
centriole assembly [32]. In mammalian cells, the increasing
ease with which genomes can be edited [33– 36] suggests
that both a deletion analysis and tagging with GFP are
possible and would be useful in defining centrosomal
components.
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6. Location of components
SPBs and centrosomes are very large organelles whose
construction relies on many coiled-coil proteins, and since
these are very often filamentous, they could be the equivalent
of connectors in the construction of these organelles. A useful
way of clearly establishing this is to change the length of the
coiled coil, which may lead to a predictable alteration in the
morphology of the organelle. If this change has little effect
on the function, a clearer conclusion can be drawn. This
approach is in contrast to the normal one of removal or disabling of the protein, which can lead to a local or complete
collapse of the organelle structure. This shows the important
structural role of the protein but does not lead to a detailed
definition of its role.
There was an early illustration of this approach for
the SPB in the case of Spc110p [2], a protein with probable
globular N- and C-terminal domains and a long central
coiled coil. ImmunoEM with monoclonal antibodies, most
of which recognized epitopes in the coiled coil [2], located
this part of the protein to the region between the central
D216-710
Figure 2. Removal of the Spc110p coiled coil (black boxes in the diagram)
causes the ends of the spindle microtubules to move closer to the central
plaque of isolated SPBs: the arrow shows the narrowing gap. Note that
the position of the ends of the microtubules initiated by the SPB is clearly
defined by their sealed ends [58]. Scale bar, 0.1 mm.
plaque and the inner plaque [1] where the spindle microtubules are initiated, suggesting the coiled coil might bridge
this gap. Partial deletion of the coiled coil decreased the
gap, while an almost complete deletion removed the gap.
So the spindle microtubules appear to grow directly from
the central plaque (figure 2), suggesting that the coiled coil
of Spc110p was responsible for the spacing of this gap.
Another illustration of this approach was in establishing the
essential function of calmodulin in yeast mitosis [59]. Calmodulin has several essential functions in yeast [60] including
one in mitosis which was defined by particular ts alleles
including cmd1-1 [61]. Because of the role of calmodulin in
calcium signalling, there was considerable interest in its role
in mitosis. Mutations in the essential calcium-binding amino
acids reduced the calcium binding to negligibly low amounts
yet this calmodulin was still functional [62], eliminating any
role for calcium. Calmodulin’s exact role in mitosis was investigated further by identifying a binding partner, Spc110p, and
further identifying the binding site of Spc110p which was
close to the C terminus [59,63]. Suppressors of cmd1-1 included
truncated versions of Spc110p, which removed the calmodulinbinding site and were functional since they fully complemented
the ts mutation [59]. This suggested that the essential function
of calmodulin in mitosis is to stabilize an a-helix in the calmodulin-binding site of Spc110p, and that removal of this unstable
helix produces a functional protein which no longer requires
calmodulin binding.
Both of these experiments illustrate that clear conclusions
can be obtained if domains are removed from a protein
without affecting its main function.
8. Conservation
Although the SPB and centrosome are functionally equivalent,
most of the internal components, in particular some of those in
the SPB core, are highly divergent. Thus, Spc42p and Spc29p
homologues can only be found in the Saccharomyces clade
[64] and have not been identified in other fungi with SPBs.
SPB components which have a more direct involvement with
microtubules are much more conserved.
9. Spindle pole body duplication
One of the most interesting problems in the case of both the
SPB and the centriole is their duplication at the start of
the cell cycle to produce a single copy. Centriole duplication
3
Phil. Trans. R. Soc. B 369: 20130456
7. Structural role of components
D489-710
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From the earliest days in the characterization of SPB components, a considerable effort was put into their localization
within the organelle by immunoEM [1,22]. Accurate localization has always been very helpful in establishing the
function of the SPB components; for instance, in the case of
Spc110p, suggesting particular experiments to show that it
acted as a spacer protein (see §7). One problem with immunoEM is often antibody accessibility, but this can be helped
by the use of the small secondary probe Fab-Nanogold followed by silver intensification [47]. However, in some
instances, for example the localization of Cnm67p, no signal
was obtained with an anti-Cnm67p, presumably because
the antigen is buried deep within the SPB. This problem
was overcome by labelling Cnm67p with GFP at the C terminus [15], and this fusion was shown to be functional, as were
all the SPB proteins tested (except Cdc31p and Tub4p), since
they complemented the deletions. The success with using
GFP-labelled protein in immunoEM is probably for two
reasons: first, the compact GFP is probably on the exterior
and so more accessible in a densely packed structure; and
second, the anti-GFP antibodies always have a very high
titre [48] which facilitates the immunoEM. An additional
advantage of GFP labelling is that the GFP can be placed
on either the N or the C terminus; thus, in the case of the
filamentous centrin-binding protein Sfi1p, it was possible to
determine the distinct locations of both the N and C termini
[49,50]. Placement of GFP at the C terminus was not always
successful, thus Spc29p-GFP was functional but gave no
immunoEM signal; however, when GFP was at the N terminus, staining was seen both at the SPB and the satellite [15].
Given the increasing ease with which homologous recombination can be applied to the genomes of vertebrate and
mammalian cells [33–36], it should be possible to tag
genes with GFP or other direct labels [51,52]. CryoEM of
centrosomes isolated from cell lines with directly labelled
centrosomal components should give high-resolution localization of these components. These approaches will complement
the new super-resolution light microscopy techniques [53–57],
as they can be carried out on unfixed material.
wt
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N C N
N C
after
anaphase
satellite
assembly
cell cycle
start
N C N
Spc110p
assembly of Spc42p and probably Spc29p
enlarges the satellite to form the duplication
plaque: the membranous part of the bridge
elongates under the duplication plaque and
the distal end fuses; the outer plaque starts
to assemble
insertion: we propose that the membranous
part of the bridge retracts across the nuclear
face of the duplication plaque exposing it to
nucleoplasm; nuclear SPB components
including Spc110p assemble
N C
N C N
side-by-side SPBs
connected by the bridge
C N
dissociation of the C termini of Sfi1p,
fission of the bridge to leave a half-bridge
with each SPB and formation of the
mitotic spindle
Figure 3. Role of Sfi1p in SPB duplication.
is currently a very active area. Its control by the kinase Plk4
seems clear [65], although what is happening structurally
is less clear, particularly in what exactly connects the procentriole at a set distance to the mother. SPB duplication has
been examined by EM and immunoEM. The process,
shown schematically in figure 3, starts with a doubling in
length of the half-bridge and the formation of a small structure called the satellite at the distal cytoplasmic end of the
bridge [66]. ImmunoEM showed the satellite contains SPB
core components [15] which then assemble into a larger structure called the duplication plaque which contains the same
SPB core components. In some unknown way, this plaque
is inserted into the nuclear membrane and the rest of the
SPB then assembles. The transition from satellite to duplication plaque and insertion and spindle assembly is very
fast, and rare examples of these stages could only be found
from observations at the right time point in synchronized
cells [15]. Based on these results, a structural model was proposed for SPB duplication [15] and later modified after the
centrin-binding protein Sfi1p was characterized [50]. Yeast
centrin (Cdc31p) has an essential function in SPB duplication
[21] and localizes to the half-bridge [22]. Sfi1p was found
from a centrin pulldown in yeast [49]; it, like centrin, localizes
to the half-bridge and has an essential function during SPB
duplication [49]. Sfi1p has about 20 continuous centrinbinding repeats which form a long a-helical filament [50],
long enough to span the half-bridge where the centrin is
localized [22]. This was confirmed by immunoEM using
GFP placed at either the N or C terminus [49,50], which
showed the N terminus of Sfi1p close to the SPB while the
C terminus was at the distal end of the half-bridge. This
suggested that the doubling in length of the half-bridge
during SPB duplication was due to an end-to-end dimerization of Sfi1p at its C terminus (figure 3) and led to a
structural model for SPB duplication [50]. This model proposes that since the N terminus of Sfi1p is close to the SPB,
it can bind directly or indirectly to the core SPB components.
The end-to-end dimerization of Sfi1p during the initial stages
of SPB duplication, when the half-bridge doubles in length,
would then give a fresh N terminus of Sfi1p at the distal
end of the bridge to initiate the assembly of the daughter
SPB. Provided the critical concentration for the assembly of
core SPB components is high, then they would only assemble
at the mother SPB or at the new Sfi1p N terminus, and in this
way only a single copy of the daughter SPB would be produced. Once the daughter SPB is assembled the bridge is
split so each SPB now has a half-bridge and a spindle is
now formed. It is interesting that while ts mutants in the centrin-binding domains of Sfi1p described above show a block
in SPB duplication, ts mutants in the C-terminal domain
arrest with paired SPBs [67,68], suggesting that the fission
of the bridge is blocked. After cell division, the single SPBs
with half-bridges can then go through another cycle of
elongation and daughter SPB assembly.
Phil. Trans. R. Soc. B 369: 20130456
elongation of the half-bridge
by dimerization of Sfi1p at
the C terminus
Cnm67p, Nud1p
Spc42p, Spc29p(?)
N C N
duplication
plaque
4
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Cnm67p, Nud1p
Spc42p, Spc29p
satellite
N C N
Sfi1p
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bridge. In centrioles the connection is different, from the proximal side of the mother to the proximal end of the daughter.
However, despite these differences, there is a fundamental similarity in which a single point of assembly from a single
organelle, the mother, ensures a single copy of the daughter.
Acknowledgements. Thanks are due to Sean Munro, Matthew Freeman
and Hugh Pelham for support, and to M. S. Robinson for discussion.
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Sfi1p is conserved and when expressed localized to the centrosome in HeLa cells [49], but it does not seem likely that
vertebrate Sfi1p is involved in centriole duplication because it
is possible to delete all the centrin genes in chicken DT40 cells
without affecting duplication [69]. Also there are clear structural
differences in the nature of the connection between mother and
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