Commentary
3485
Microtubule dynamics in the budding yeast mating
pathway
Jeffrey N. Molk and Kerry Bloom*
Department of Biology, University of North Carolina, 622 Fordham Hall, Chapel Hill, NC 27599, USA
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 26 July 2006
Journal of Cell Science 119, 3485-3490 Published by The Company of Biologists 2006
doi:10.1242/jcs.03193
Summary
In order for haploid gametes to fuse during fertilization,
microtubules (MTs) must generate forces that are sufficient
to move the nuclei together. Nuclear movements during
fertilization rely on microtubule-associated proteins
(MAPs), many of which have been characterized
extensively during mitosis. A useful model system to study
MT-dependent forces before nuclear fusion, or karyogamy,
is the mating pathway of budding yeast. Dynamic MTs are
guided to the mating projection (shmoo tip) when plusend-binding proteins interact with polarized actin
microfilaments. If two shmoo tips are in proximity they
may fuse, dissolving the MT-cortical interactions.
Introduction
Eukaryotic fertilization requires microtubule (MT)-dependent
nuclear movements for gamete fusion. MTs are polarized
polymers nucleated at centrosomes or spindle pole bodies
(SPBs) that are composed primarily of ␣- and ␤-tubulin
dimers. MTs grow and shorten through rapid tubulin addition
and subtraction at their plus ends, a process known as dynamic
instability (Inoue and Salmon, 1995; Kirschner and Mitchison,
1986). Dynamic instability is not inhibited when MT plus ends
interact with attachment sites or the plus ends of oppositely
oriented MTs. Consequently, MTs can facilitate the movement
of subcellular structures to which they attach. MT plus ends
are linked to cortical sites or oppositely oriented MTs by plusend-binding proteins, a subset of which are plus-end-tracking
proteins (+TIPs) that preferentially accumulate on growing
MTs (Akhmanova and Hoogenraad, 2005; Schuyler and
Pellman, 2001). When a MT encounters an attachment site, the
plus-end-binding proteins act as an adaptor to tether the
dynamic plus end to the attachment site. After the MTs attach,
growth and shrinkage continues, and because MTs are
anchored at microtubule-organizing centers, the attached
structures can oscillate, a behavior seen during chromosome
directional instability in mitosis (Skibbens et al., 1993).
Alternatively, directed movement may dominate as a
consequence of persistent MT polymerization or
depolymerization, such as during anaphase chromosome
movement. A model system that allows us to examine MT
attachment, MT-dependent oscillations, and MT-MT
interactions is the mating pathway of Saccharomyces
cerevisiae. In this pathway, nuclei are oriented towards the site
of cell fusion by MTs and attach to the cell cortex. After the
two cells fuse, the nuclei are then drawn together by MT-MT
Subsequently, oppositely oriented MT plus ends interact
and draw the nuclei together. The plus-end-binding
proteins in the yeast mating pathway are conserved in
metazoan cells and may play a role in higher eukaryotic
fertilizaton. Thus, understanding the mechanism of plus
end orientation and karyogamy in budding yeast will reveal
mechanisms of MT-dependent force generation conserved
throughout evolution.
Key words: Kar3p, Bik1p, Kar9p, Nuclear congression,
Microtubules
interactions. Thus, information about MT dynamics revealed
by studies of the mating pathway is relevant to processes
ranging from chromosome segregation to metazoan
fertilization that are critical for cellular survival.
MT dynamics during S. cerevisiae mating
When budding yeast cells of opposite mating type are mixed,
a signal transduction cascade is initiated to induce the mating
pathway (Fig. 1A) (Bardwell, 2005). The mating response
includes the polarization of the unbudded cell and formation
of a mating projection known as the shmoo (MacKay and
Manney, 1974; Tkacz and MacKay, 1979). Cells of opposite
mating types fuse at the shmoo tips (Rose, 1996).
MT dynamics in the mating pathway were first examined in
live cells by imaging of dynein or tubulin fused to GFP
(Maddox et al., 1999). This revealed that MTs nucleated from
the SPB are guided to the shmoo tip by the plus end, and this
orients the nucleus (Maddox et al., 1999). The plus ends
become oriented towards and attach persistently to the shmoo
tip while growing and shrinking (Hasek et al., 1987; Maddox
et al., 1999). The coordinated growth of MTs pushes the
nucleus away from the shmoo tip, whereas shortening of MTs
at the plus end draws the nucleus towards the shmoo tip,
generating nuclear oscillations over time. Unlike higher
eukaryotes, in which tubulin dimers are lost at the minus end,
in budding yeast MT dynamics are regulated by proteins
exclusively at the plus end (Maddox et al., 1999; Maddox et
al., 2000; Tanaka et al., 2005).
After cell fusion, MTs that were originally attached to the
shmoo tip interact with MTs from the other cell to form a
bridge between the two nuclei (Hasek et al., 1987; Read et al.,
1992). The cross-linked MTs then coordinately depolymerize,
3486
Journal of Cell Science 119 (17)
Journal of Cell Science
drawing the nuclei together for karyogamy (Maddox et al.,
1999; Molk et al., 2006; Rose, 1996). Inhibitor studies and
genetic analysis of tubulin mutants have demonstrated that
MTs are required for karyogamy (Delgado and Conde, 1984;
Huffaker et al., 1988). [Class I karyogamy mutants exhibit
defective nuclear congression whereas class II karyogamy
mutants do not undergo nuclear fusion (Kurihara et al., 1994).]
Genetic analyses have similarly demonstrated that plus-endbinding proteins are required for nuclear congression and for
karyogamy (Berlin et al., 1990; Meluh and Rose, 1990;
Schwartz et al., 1997) (Fig. 1B).
MT dynamics in the mating pathway are thus crucial for
orientation of the nucleus to the shmoo tip and nuclear
congression. They are also required at mitosis in the first
zygotic division. Here, we review what is known about these
processes and highlight some outstanding questions.
Formation of the MT-shmoo tip attachment
There are three steps in the formation of the MT-shmoo tip
attachment (Fig. 2 and Table 1): (1) polarization of the actin
cytoskeleton and formation of the shmoo tip; (2) nuclear
orientation – guidance of MT plus ends toward the shmoo tip
along actin cables; (3) attachment of MT plus ends to the
shmoo tip cell cortex.
Polarization
Cell polarization is initiated when pheromones bind to
cell surface receptors that are coupled to a heterotrimeric
A
Unbudded
Shmoo
Fused
G-protein signaling cascade (reviewed by Bardwell, 2005).
Disassociation of the G␣ subunit, Gpa1p, from the G␤␥
subunits activates Cdc42p, a key component of the cell
polarization pathway, and induces the formation of the
polarisome, a protein complex that includes Pea2p, Spa2p,
Bud6p, and Bni1p (Bardwell, 2005; Etienne-Manneville, 2004;
Pruyne and Bretscher, 2000). Gpa1p is required for localization
of Bni1p to the future shmoo site, and this event is coupled
to Cdc42p activation to polarize the cell (Matheos et al.,
2004). As the cell polarizes, new cell-surface materials are
synthesized to form the shmoo (Tkacz and MacKay, 1979).
After shmoos form, cells polarize towards and mate with the
partner expressing the highest pheromone concentration – this
process is termed courtship (Jackson and Hartwell, 1990). If
cells are treated with synthetic mating pheromone, a default
mating pathway is initiated and the shmoo forms next to the
bud scar from previous divisions (Dorer et al., 1995). In both
cases, the major cytoskeletal component required for cell
polarization is actin (Hasek et al., 1987; Read et al., 1992).
Orientation
After the cell polarizes, MT plus ends are transported along
actin cables to the shmoo tip; because the minus ends are
attached to the SPB, the result is orientation of the nucleus to
the shmoo tip (Fig. 2B). Nuclear orientation requires the +TIP
Bim1p and Kar9p. Bim1p, the budding yeast EB1 (end binding
protein) ortholog, binds to MTs and preferentially localizes to
growing plus ends (Maddox et al., 2003; Tirnauer et al., 1999).
Preanaphase
Anaphase
GFP-Tub1p
B
MT
Plus ends
Shmoo tip
SPB
MT
Plus ends
SPB
Bik1-3xGFP
Fig. 1. Microtubule (MT) and plus-end-tracking protein
(+TIP) distribution in the budding yeast mating pathway.
(A) MT morphologies through the first zygotic division.
Upper panel shows fluorescent images of GFP fused to
tubulin (GFP-Tub1p) in the mating pathway. Lower panel
are corresponding DIC images. (B) +TIP localization in
pheromone-treated cells. Schematic of mating cell is
shown on the left. Formation of the shmoo after cell
polarization allows MT plus ends to interact with the
shmoo tip. Bik1p-3xGFP localizes to MT plus ends in the
shmoo tip, free MT plus ends, and to the SPB (right,
bright-field image is displayed in lower right corner). Bars,
2 ␮m.
MT dynamics during S. cerevisiae mating
Bim1p interacts with Kar9p to link MTs to the actin network.
Kar9p is transported to MT plus ends and serves as an adaptor
protein between Bim1p and the actin-associated type V myosin
motor Myo2p (Hwang et al., 2003; Korinek et al., 2000; Lee
et al., 2000; Maekawa et al., 2003; Yin et al., 2000). Once
Kar9p links the plus end to the actin network, Myo2p moves
along polarized actin pulling the nucleus to the shmoo tip.
When both mating partners have defects in nuclear orientation,
such as kar9⌬ cells, karyogamy can still occur when the plus
A
Kar9p
Bim1p
Kip2p
Bik1p
Journal of Cell Science
Kar3p
Cik1p
B
Myo2p
Actin
Tpm1p
C
Unknown
protein
complex
Fig. 2. Schematic of +TIP function during nuclear orientation to the
shmoo tip. (A) In unbudded cells, Kar9p (red) and Bik1p (purple) are
transported along the MT (green) by the motor protein Kip2p
(brown) to the plus end where Bim1p (blue) binds. Kar3p (cyan) and
Cik1p (yellow) form heterodimers that localize near the plus ends.
(B) After cell polarization, the MT plus end interacts with actin
cables (red) via Myo2p (gray). Myo2p links to Bim1p through Kar9p
and moves the nucleus towards the shmoo tip. (C) Once at the shmoo
tip, the plus end interacts persistently with the shmoo tip. This
interaction could be mediated by proteins at the shmoo tip cortex that
act as attachment factors.
3487
ends of MTs stochastically interact (Molk et al., 2006). When
karyogamy mutants, such as kar9⌬, are mated to a wild-type
cell, no defects in nuclear congression occur (Berlin et al.,
1990; Kurihara et al., 1994). In this unilateral condition, it is
likely that the wild-type protein diffuses quickly into the other
cell body after fusion and binds MT plus ends, driving nuclear
congression. Therefore, nuclear orientation is not necessary for
karyogamy, but proteins such as Kar9p and Myo2p that guide
plus ends to the shmoo tip are required for high-fidelity nuclear
congression. Because the plus end is the site of MT-MT crosslinking for nuclear congression, guidance of plus ends to the
shmoo tip significantly increases the probability that MTs from
the different nuclei will interact.
Attachment
After MTs have been transported to the shmoo tip along
microfilaments, their plus ends interact with the cell cortex
(Maddox et al., 1999; Maddox et al., 2003). Plus-end-binding
proteins keep polymerizing and depolymerizing plus ends at
the shmoo tip. Thus far, only Bim1p has been proposed to link
polymerizing plus ends to the shmoo tip (Maddox et al., 2003).
Depolymerizing plus ends attach to the shmoo tip via Kar3p
and Bik1p. Kar3p is a kinesin 14 motor protein that has minusend-directed motility (Endow et al., 1994; Maddox et al., 2003;
Meluh and Rose, 1990). Kar3p forms a heterodimer with the
light chain Cik1p, and this heterodimer is targeted to the plus
end, where Kar3p promotes depolymerization (Barrett et al.,
2000; Maddox et al., 2003; Sproul et al., 2005). In kar3⌬ cells,
MTs lose their persistent attachment to the shmoo tip when
switching to depolymerization and shorten back to the SPB
(Maddox et al., 2003). Therefore, Kar3p is proposed to anchor
depolymerizing plus ends to the shmoo tip, preventing their
detachment and shortening.
Bik1p, the CLIP-170 ortholog in budding yeast, helps
maintain depolymerizing MTs at the shmoo tip and is required
for nuclear congression (Berlin et al., 1990; Lin et al., 2001;
Molk et al., 2006). CLIP-170 is a MT-binding protein that
was originally characterized as a linker between MTs and
membranes in metazoan cells (Vaughan, 2005). In budding
yeast, Bik1p may directly interact with the plasma membrane,
anchoring MTs to the shmoo tip. In pheromone-treated kar3⌬
cells, Bik1p localizes to the plus end, which suggests Bik1p
localization does not depend on Kar3p (Molk et al., 2006).
However, in kar3⌬ cells MTs detach from the shmoo tip when
they switch to shortening (Maddox et al., 2003), which
demonstrates that Bik1p is necessary but not sufficient for MT
interactions with the shmoo tip.
In addition to plus-end-binding proteins, other proteins
involved in cell polarization could play a role in MT–shmootip attachments. Kip2p is a plus-end-directed kinesin-like
protein that transports both Kar9p and Bik1p along MTs in
mitotic cells but kip2⌬ cells do not exhibit karyogamy defects
(Carvalho et al., 2004; Maekawa et al., 2003; Miller et al.,
1998). It is unknown whether, after nuclear orientation, actin
cables are required to maintain MT attachments. In myo2-17,
myo2-18 and myo2–20 mutants that lack functional Myo2p
myosin motors, MTs appear detached from the shmoo tip but
the SPB remains near the base of the mating projection (Hwang
et al., 2003). This phenotype is reminiscent of kar3⌬ cells, and
Myo2p may play a direct role in attachment of MTs to the
shmoo tip. Furthermore, proteins that establish cell polarity
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Table 1. Summary of proteins involved in budding yeast karyogamy
Protein
Metazoan ortholog
Nuclear orientation
Shmoo tip attachment
Frequency of nuclear
congression or karyogamy*
Kip2p
Kar9p
Myo2p
Bik1p
Bim1p
Kar3p
Kinesin-7
APC†
Type V Myosin
CLIP-170
EB1
Kinesin-14
+
–
–
+
–
+
Detach on MT shortening
NA
?
Detach on MT shortening
NA
Detach on MT shortening
100%
50%
?
20%
~1%
<1%
*References for phenotypes: Miller et al., 1998, Meluh and Rose, 1990; Molk et al., 2006, Schwartz et al., 1997.
†
Weak homology to adenomatous polyposis coli protein.
NA, not applicable; ?, unknown.
Journal of Cell Science
and are found at the plasma membrane, such as Gpa1p or
polarisome components, may link the MT to the shmoo tip.
Thus, it is likely that additional proteins are required to couple
MTs to the shmoo tip.
Models for MT attachment to the shmoo tip
How do MTs attach to the cell cortex at the shmoo tip? The
leading hypothesis is the cortical anchorage model in which
polymerizing MTs have Bim1p on their plus ends and are kept
at the shmoo tip by Kar9p that is linked to the actin network
or polarity proteins at the cell cortex (Fig. 3A) (Maddox et
al., 2003; Miller et al., 1999). When MTs switch to
depolymerization, the plus ends are held at the shmoo tip by
Bik1p and Kar3p. Kar3p is anchored to the cell cortex by an
unknown attachment factor. Candidates for this include a
component of the heterotrimeric G-protein complex or the
polarisome.
An alternative, but not mutually exclusive, model is the plus
end cycling hypothesis (Fig. 3B). In this model, MT plus ends
are not anchored to the cell cortex; instead they are transported
by Myo2p towards the shmoo tip and are maintained at the
ends of actin cables. In this hypothesis, Myo2p does not
disassociate immediately from the end of the actin cable. By
remaining bound, the Myo2p-Kar9p-Bim1p complex holds the
MT at the shmoo tip while it grows or shortens. Kar9p could
reinforce this linkage by interacting with actin or polarisome
components (Miller et al., 1999). This would result in
‘attachment’ where the affinity of a MT for the shmoo tip is
defined by the ability of Myo2p and/or Kar9p to persistently
Fig. 3. Models for MT interaction with the shmoo tip. (A) As
hypothesized by Maddox et al. (Maddox et al., 2003), an unknown
binding factor may interact with Kar3p to keep this motor near the
cortex. Bim1p may link the plus end to the shmoo tip through Kar9p.
During polymerization, Bim1p and Kar9p maintain the interactions
between the plus end and the shmoo tip. A switch to
depolymerization will cause Bim1p to leave the plus end while
Kar3p maintains the attachment of the MT to the tip. (B) Plus end
cycling hypothesis. In this model, the MT plus end can detach from
the shmoo tip at some frequency governed by the affinity of +TIPs
for either the cortex or the ends of actin cables. After detachment, the
MT shrinks back to the SPB while a newly nucleated MT grows and
interacts with the actin network. The newly nucleated MT is
transported by Myo2p along the actin network towards the shmoo
tip, generating movement of the nucleus towards the mating
projection. Once at the shmoo tip, the MT attaches and begins to
polymerize, moving the nucleus away from the shmoo tip. Therefore,
the combination of MT transport and MT dynamics at the shmoo tip
generates nuclear oscillations.
associate with actin. Since Bim1p preferentially associates
with growing MTs in the shmoo tip, Bim1p may maintain the
attachment of polymerizing MT plus ends by recruiting Kar9p
to strengthen the MT-actin interactions. As MTs shorten,
Kar3p and Bik1p would keep plus ends at the shmoo tip, but
at some frequency, MTs could be released (Molk et al., 2006).
A released plus end could then be replaced by newly nucleated
MTs guided by Myo2p into the shmoo tip. The movement of
A
Polymerization
Kar3p
Unknown Kar3p
binding factor
Bim1p
Kar9p
Depolymerization
B
Growth
Attachment
Detachment
Transport
MT dynamics during S. cerevisiae mating
Journal of Cell Science
the new plus end into the shmoo tip would pull the nucleus
toward the mating projection. The cycle of MT attachment,
growth, detachment, and replacement would define a single
nuclear oscillation that is repeated until cell fusion.
One way to distinguish between the two models would be
an analysis of shmoo tip attachment in tropomyosin mutants in
which actin cables are defective. The cortical anchorage model
predicts that actin cables are not required once the MT interacts
with the shmoo tip. The plus end cycling model requires actin
cables for MT–shmoo-tip attachment.
MT dynamics during nuclear congression
After cell fusion, MTs rapidly associate during nuclear
congression (Maddox et al., 1999). Nuclear congression
requires Bik1p and Kar3p (Fig. 4A) (Berlin et al., 1990; Meluh
and Rose, 1990). In early models for nuclear congression based
on genetic analysis, Kar3p was proposed to cross-link and slide
oppositely oriented MTs, which would result in extensive MT
overlap (Fig. 4B) (Rose, 1996). This model predicts that plus
ends would be located near the SPBs. However, live cell
fluorescence microscopy has demonstrated that MTs do not
slide past one another over long distances and that crosslinking occurs only near the plus ends (Molk et al., 2006).
Therefore, we favor a new model for nuclear congression that
focuses on plus end interactions (Fig. 4B). In this model, the
opposing plus ends at the shmoo tip are juxtaposed and crosslinking occurs with a minimum of MT-MT sliding. The crosslinking is initiated by the Kar3p-Cik1p heterodimer and Bik1p
could also play a role. Once plus ends interact, we propose that
the MTs switch to persistent shortening to drive both nuclei
together.
3489
What other proteins are required for nuclear congression?
Bim1p is necessary for efficient karyogamy (Schwartz et al.,
1997) and may play a role in MT-MT interactions during
nuclear congression. It is puzzling that bim1⌬ cells have a
severe karyogamy defect but Bim1p is proposed to function on
polymerizing MTs. Bim1p could be required for MT growth
after the cell wall breaks down to link the plus ends. After the
polymerizing plus ends are linked by Bim1p, Kar3p may
maintain the linkage after the switch to shortening occurs.
Other plus-end-binding proteins that regulate MT dynamics
could also be required for nuclear congression. Additionally,
we do not know how the cell regulates the switch from the
dynamic instability that occurs before cell fusion to persistent
depolymerization after plus ends interact. Since Kar3p has
been shown to depolymerize MT plus ends in vitro (Sproul et
al., 2005), local regulation of Kar3p, either through proteinprotein interactions or post-translational modification, could
trigger the switch.
Mitosis during the first zygotic division
Little is known about mitosis during the first zygotic division.
Thus far, it appears spindle elongation during the first zygotic
division is similar to vegetative divisions (Maddox et al., 1999).
A failure to perform karyogamy produces a single cell that has
two haploid nuclei, each of which generates its own mitotic
spindle. In experiments using kar1 mutants with two spindles,
the response to DNA damage was determined to inhibit
anaphase onset locally within a single nucleus (Demeter et al.,
2000). A similar analysis of other karyogamy mutants may
uncover characteristics about how cell division occurs in the
presence of multiple spindles.
A
Sliding cross-bridge model
B
Wild type
Nuclear orientation
defect
Plus-end model
MT interaction
defect
Fig. 4. Karyogamy in budding yeast.
(A) Models for nuclear congression in
budding yeast. (Left) Sliding cross-bridge
model for nuclear congression. In this
model, Kar3p-Cik1p slides MTs past one
another and then depolymerization occurs
at the SPBs. (Right) Plus end model for
nuclear congression. MTs are linked at the
plus end by Kar3p-Cik1p and then
depolymerize, drawing both nuclei
together for karyogamy. (B) MT dynamics
in the wild type and karyogamy mutants.
(Left) Wild-type MT dynamics during
nuclear congression. (Middle) MT
dynamics when nuclear orientation is
defective. MTs grow and shrink the
cytoplasm and rely on stochastic
interactions to cross-link and perform
nuclear congression. (Right) MT
dynamics when cross-linking is defective.
MTs undergo dynamic instability in the
cytoplasm but never interact. A failure to
undergo karyogamy does not block the
zygotic bud from forming, resulting in the
assembly of two mitotic spindles within
the same cell.
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Conclusions and future perspectives
MT plus ends and plus-end-binding proteins are required for
efficient mating in budding yeast. Plus ends are crucial for
attachment to the shmoo tip and MT-MT interactions during
nuclear congression. Plus-end-binding proteins required for
MT dynamics in mating include Bim1p, Bik1p and Kar3p.
Bik1p and Kar3p function at the kinetochore in budding yeast
(Lin et al., 2001; Middleton and Carbon, 1994; Tanaka et al.,
2005), which suggests MT linkages could be similar at the
shmoo tip and centromere. Thus, the shmoo tip attachment site
could also serve as a model for kinetochore-MT attachments.
Future studies of MT-binding proteins may reveal further
karyogamy factors that are necessary for nuclear congression.
There are some differences between yeast karyogamy, in which
dynein is not required, and metazoan fertilization, which does
require cytoplasmic dynein (Molk et al., 2006; Payne et al.,
2003). However, plus-end-binding proteins may well have
conserved functions in yeast and metazoans that facilitate
nuclear movements. Therefore, understanding how MT
dynamics affect shmoo tip attachment and nuclear congression
could reveal underlying mechanisms that apply to a variety of
problems in cell biology.
We thank David Bouck, Richard Cheney, Julian Haase, Ted
Salmon, David Stone and members of the Bloom and Salmon
laboratories for helpful discussions and critical reading of the
manuscript. We would like to acknowledge Paul Maddox for initiating
the live cell studies of karyogamy and his insight into the mechanisms
that govern shmoo tip attachment. This work was supported by the
National Institutes of Health grant GM-32238 (to K.B.).
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