Mechanisms of cytokinesis in budding yeast
Cytokinesis is essential for cell proliferation in all domains of life. Because the core components and mechanisms of cytokinesis are conserved from fungi to humans, the budding yeast Saccharomyces cerevisiae has served as an attractive model for studying this fundamental process. Cytokinesis in budding yeast is driven by two interdependent cellular events: actomyosin ring (AMR) constriction and the formation of a chitinous cell wall structure called the primary septum (PS), the functional equivalent of extracellular matrix remodeling during animal cytokinesis. AMR constriction is thought to drive efficient plasma membrane ingression as well as to guide PS formation, whereas PS formation is thought to stabilize the AMR during its constriction. Following the completion of the PS formation, two secondary septa (SS), consisting of glucans and mannoproteins, are synthesized at both sides of the PS. Degradation of the PS and a part of the SS by a chitinase and glucanases then enables cell separation. In this review, we discuss the mechanics of cytokinesis in budding yeast, highlighting its common and unique features as well as the emerging questions. © 2012 Wiley Periodicals, Inc.
Mechanisms of Cytokinesis in Budding Yeast
and Erfei Bi
Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania
Institute of Chemistry and Biochemistry, Laboratory of Membrane Biochemistry and Molecular Cell Biology, Freie Universit
Takustrabe 6, Berlin, Germany
Received 11 May 2012; Accepted 15 June 2012
Monitoring Editor: William Bement
Cytokinesis is essential for cell proliferation in all
domains of life. Because the core components and
mechanisms of cytokinesis are conserved from fungi to
humans, the budding yeast Saccharomyces cerevisiae has
served as an attractive model for studying this funda-
mental process. Cytokinesis in budding yeast is driven
by two interdependent cellular events: actomyosin ring
(AMR) constriction and the formation of a chitinous
cell wall structure called the primary septum (PS), the
functional equivalent of extracellular matrix remodeling
during animal cytokinesis. AMR constriction is thought
to drive efﬁcient plasma membrane ingression as well as
to guide PS formation, whereas PS formation is thought
to stabilize the AMR during its constriction. Following
the completion of the PS formation, two secondary
septa (SS), consisting of glucans and mannoproteins,
are synthesized at both sides of the PS. Degradation of
the PS and a part of the SS by a chitinase and gluca-
nases then enables cell separation. In this review, we dis-
cuss the mechanics of cytokinesis in budding yeast,
highlighting its common and unique features as well as
the emerging questions.
2012 Wiley Periodicals, Inc
Key Words: actomyosin ring constriction, targeted mem-
brane deposition, septum formation, cell separation, septins
ytokinesis, the process of partitioning cellular constit-
uents from one cell into two cells, is fundamental to
all cellular life. When coupled with cell polarization, cyto-
kinesis leads to the generation of diverse cell types. Thus,
cytokinesis is important not only for cell proliferation but
also for cell differentiation. Inhibition of cytokinesis can
be detrimental or beneﬁcial. On one hand, failure in cyto-
kinesis causes polyploidy that may render cells tumori-
genic, a century-old hypothesis [Boveri, 1914; Boveri,
2008] that has been supported by recent experimental evi-
dence [Fujiwara et al., 2005]. On the other hand, regu-
lated inhibition of cytokinesis leads to the formation of
cells with polyploidy that may be important for their nor-
mal functions [Davoli and de Lange, 2011]. For example,
megakaryocytes, which produce platelets by shedding their
cytoplasmic processes, can attain up to a 64N nuclear
content [Tomer et al., 1988; Lordier et al., 2008]. Adult
hepatocytes are often binucleated [Gentric et al., 2012].
In addition, aneuploidy caused by cytokinesis failure is
thought to confer an evolutionary advantage by readily
increasing gene dosage [Rancati et al., 2008; Gordon
et al., 2012]. Thus, detailed mechanistic studies of cytoki-
nesis are important for understanding normal cellular
function as well as human disease.
The budding yeast Saccharomyces cerevisiae divides
asymmetrically, with each division cycle producing two
cells of distinct sizes and fates. The newly born daughter
cell or the bud is approximately two-thirds of its mother
in size. A transcriptional repressor, Ash1, is localized
exclusively in the daughter cell to silence its ability in
mating-type switching [Bobola et al., 1996; Jansen et al.,
1996], whereas aging factors are restricted to the mother
cell [Shcheprova et al., 2008; Khmelinskii et al., 2011;
Zhou et al., 2011]. Immediately following cytokinesis
(complete membrane closure between two forming cells),
a transcriptional program called the RAM pathway (
Ace2 activity and Cellular Morphogenesis) is
activated exclusively in the daughter cell to ensure proper
cell separation [Bidlingmaier et al., 2001; Colman-Lerner
et al., 2001; Nelson et al., 2003] (see more discussion
later). In this review, we focus on the mechanisms of cyto-
kinesis not of cell polarization.
Cytokinesis in budding yeast and animal cells can be
viewed as an integrative process of several spatiotemporally
coordinated events: (1) division site speciﬁcation; (2) acto-
myosin ring (AMR) assembly, contraction, and disassembly;
(3) targeted membrane deposition and septum formation
*Address correspondence to: Erfei Bi, Department of Cell and
Developmental Biology, University of Pennsylvania Perelman
School of Medicine, Philadelphia, Pennsylvania 19104-6058.
Published online 31 July 2012 in Wiley Online Library
Cytoskeleton, October 2012 69:710–726 (doi: 10.1002/cm.21046)
2012 Wiley Periodicals, Inc.
[functional equivalent of localized extracellular matrix
(ECM) remodeling in animal cells]; and (4) cell cycle con-
trol of the division machinery. The mechanisms for specify-
ing the division site in different organisms vary widely. For
example, the division site in the budding yeast S. cerevisiae,
the ﬁssion yeast Schizosaccharomyces pombe, and animal cells
is determined by the bud-site-selection program, the nuclear
position, and the spindle position, respectively [Rappaport,
1961; Balasubramanian et al., 2004]. In contrast, the core
components and mechanisms involved in other aspects of
cytokinesis are largely conserved [Balasubramanian et al.,
2004; Pollard, 2010]. Because of this conservation, the bud-
ding yeast S. cerevisiae has served as an attractive model for
elucidating the general mechanisms of cytokinesis.
In budding yeast, efﬁcient cytokinesis depends on the
interplay between the AMR and the primary septum (PS),
a chitinous and electron-lucent structure that is formed
only during cytokinesis (Fig. 1A). AMR constriction is
followed closely by the centripetal growth of the PS (Fig.
1A). Upon the completion of the PS (Fig. 1B), two sec-
ondary septa (SS), which are electron-dense structures
with chemical composition similar to the general cell wall,
are laid down on both sides of the PS (Fig. 1C). Diges-
tion of the PS and a part of the SS by RAM pathway-
controlled hydrolytic enzymes results in cell separation
[Bidlingmaier et al., 2001; Colman-Lerner et al., 2001;
Nelson et al., 2003]. Strikingly, disruption of the AMR,
for example, by deleting MYO1, which encodes the sole
myosin-II gene in budding yeast, causes the formation of
misoriented PS (Fig. 1D) [Rodriguez and Paterson, 1990;
Fang et al., 2010], suggesting that the AMR may guide
PS formation [Fang et al., 2010]. Reciprocally, blocking
PS formation by deleting CHS2, which encodes the chitin
synthase II in budding yeast, results in asymmetric AMR
constriction, suggesting that the PS may stabilize the
AMR during its constriction [Bi, 2001; Schmidt et al.,
2002; VerPlank and Li, 2005]. Thus, the AMR and the
PS appear to be functionally interdependent during
The ‘‘cytokinesis machine’’ in budding yeast, including
the components of the AMR and those involved in PS for-
mation and cell separation, is assembled at the division site
(or the mother-bud neck) sequentially during the cell cycle
(Figs. 2 and 3). Here, we discuss the structures and func-
tions of the major components involved in cytokinesis.
Septins and Their Essential Role in
Septin Structures In Vitro
Septins are GTP-binding and ﬁlament-forming proteins
that are conserved from yeast to humans with a notable
absence in higher plants [Pan et al., 2007; Nishihama
et al., 2011]. There are a total of seven septin genes in
budding yeast, ﬁve of which (CDC3, CDC10, CDC11,
CDC12, and SHS1) are expressed vegetatively and the rest
(SPR3 and SPR28) expressed exclusively during sporula-
tion [Longtine et al., 1996; McMurray and Thorner,
2009; Oh and Bi, 2011]. The ﬁve mitotic septins are
Fig. 1. Mechanics of cytokinesi s in budding yeast. (A–C) Cytokinesis in wild-type (WT) cells. AMR, actomyosin ring; PS, pri-
mary septum; PM, plasma membrane; CW, cell wall; BS, bud scar; and SS, secondary septa. (D) Cytokinesis in myo1D cells. Note
that the PS is misoriented in myo1D cells.
CYTOSKELETON Cytokinesis in Budding Yeast 711 n
thought to form distinct octameric complexes (Shs1/Cdc11-
Cdc3, Cdc10, and Cdc12 as the core and Cdc11 replacea-
ble with Shs1 [Bertin et al., 2008; Garcia et al., 2011].
Like septin complexes in C. elegans and humans [J ohn
et al., 2007; Sirajuddin et al., 2007], the Cdc11- or Shs1-
containing complexes are nonpolar ‘‘rods,’’ with the possible
exception of a heterooctamer (Cdc11-Cdc12-Cdc3-Cdc10-
Cdc10-Cdc3-Cdc12-Shs1) that may form in vivo [Bertin
et al., 2008; Garcia et al., 2011]. The Cdc11-containing
complexes polymerize end-to-end into linear ﬁlaments that
are often in pairs [F razier et al., 1998; Bertin et al., 2008;
Garcia et al., 2011]. Strikingly, the Shs1-containing com-
plexes assemble into small rings consisting of staggered
short ﬁlaments [Garcia et al., 2011]. The ring size increases
with the addition of increasing concentrations of the
Cdc11-containing complexes. Ho w these in vitro assembled
septin structures are precisely correlated to the in vivo sep-
tin rings or hourglasses (see below) are not entirely clear.
Septin Structures and Dynamics In Vivo
Upon the launch of a new cell cycle in late G1, all ﬁve
mitotic septins are recruited simultaneously to the pre-
sumptive bud site and assembled into a cortical ring (Fig. 3),
a process that is controlled by the small GTPase Cdc42
[Iwase et al., 2006]. Shortly after bud emergence, the sep-
tin ring is somehow expanded into an hourglass spanning
the mother-bud neck (Fig. 3). At the onset of cytokinesis,
the hourglass is triggered by the mitotic exit network
(MEN) to split into two cortical rings that sandwich the
AMR (Fig. 3) [Cid et al., 2001; Lippincott et al., 2001].
Fig. 2. Localization of core cytokinesis proteins during the cell cycle. The generic names of the proteins or their key domains/
motifs are indicated in red in the parentheses. For the cell diagrams, blue, nucleus; orange, septin hourglass or rings; green, Myo1;
and red, actin ring.
n 712 Wloka and Bi CYTOSKELETON
The MEN consists of a small GTPase (Tem1), a kinase
cascade (Cdc15 and Dbf2/Dbf20-Mob1 kinases), and a
phosphatase (Cdc14) [Balasubramanian et al., 2004; Steg-
meier and Amon, 2004]. Activation of the kinase cascade
by Tem1 results in the release of the phosphatase Cdc14
from the nucleolus into the nucleus and the cytoplasm.
The released Cdc14, in turn, promotes mitotic exit by
inactivating CDK1 activity and promotes cytokinesis by a
mechanism that is not fully understood (see more discus-
sion later). The precise architecture of the septin rings and
hourglasses remains unclear, but undoubtedly, the hour-
glass contains septin ﬁlaments both perpendicular and par-
allel to the mother-daughter axis [Byers and Goetsch,
1976; DeMay et al., 2011; Bertin et al., 2012]. Fluores-
cence recovery after photobleaching analysis indicates that
the nascent septin ring is dynamic, the hourglass is stable,
and the double rings are dynamic again [Caviston et al.,
2003; Dobbelaere et al., 2003]. In addition, the septin
hourglass splitting is accompanied by a 90
change in sep-
tin ﬁlament orientation [Vrabioiu and Mitchison, 2006;
DeMay et al., 2011]. Thus, septins undergo cell cycle-trig-
gered organizational changes but the underlying mecha-
nisms remain largely unknown. Interestingly, in cdc10D
cells, a septin ring/hourglass is assembled at the division
site [Frazier et al., 1998; Versele et al., 2004; Wloka et al.,
2011] but it disappears precisely at the onset of cytokine-
sis [Wloka et al., 2011], suggesting that Cdc10 is required
for the assembly and/or maintenance of the septin double
ring during cytokinesis [Wloka et al., 2011]. Bud4, an
anillin-related protein involved in bud-site selection [Sand-
ers and Herskowitz, 1996], is selectively required for the
maintenance of the septin ring at the mother side during
cytokinesis [Wloka et al., 2011]. Similarly, anillin in ani-
mal cells [Field et al., 2005; Maddox et al., 2005; Gold-
bach et al., 2010] and anillin-like proteins such as Mid2
in ﬁssion yeast [Berlin et al., 2003; Tasto et al., 2003] are
also involved in septin localization and/or organization.
Septin Functions in Cytokinesis
Septins are essential for cytokinesis in budding yeast, as
temperature-sensitive mutants of CDC3, CDC10, CDC11,
and CDC12 fail to form colonies and arrest as nondivided
cells containing multiple nuclei at the nonpermissive tem-
perature [Hartwell, 1971]. Similarly, septins are required
for cytokinesis in some animal cells [Neufeld and Rubin,
1994; Fares et al., 1995; Kinoshita et al., 1997; Estey
et al., 2010]. In contrast, septins are not required for cell
viability but are required for efﬁcient cell separation in
the ﬁssion yeast S. pombe [Martin-Cuadrado et al., 2005;
Wu et al., 2010].
Septins play two distinct roles in cytokinesis in budding
yeast. Before the onset of cytokinesis, the septin hourglass
is required for AMR assembly, as Myo1 fails to localize to
the division site in septin mutants [Bi et al., 1998; Lip-
pincott and Li, 1998a] and Myo1 is required for actin
ring assembly [Bi et al., 1998]. After the onset of cytoki-
nesis, the septin double ring is thought to function as a
Fig. 3. Assembly and function of the division machinery
during the cell cycle. PM, plasma membrane; MEN, mitotic
exit network; RAM, regulation of Ace2 activity and cellular
morphogenesis. Red arrows, MEN-controlled septin hourglass
splitting and RAM-controlled cell separation. Red lines, actin
cables involved in exocytosis; red circles, actin patches involved
in endocytosis. The upper vertical green bar at the right indi-
cates Bni5-mediated Myo1 targeting from late G1 to the onset
of telophase. T he lower vertical green bar indicates Iqg1-medi-
ated Myo1 targeting from the onset of anaphase to the end of
cytokinesis. Solid arrow indicates demonstrated biochemical
interaction. Dashed arrow indicates localization dependency
without demonstrated interact ion.
CYTOSKELETON Cytokinesis in Budding Yeast 713 n
diffusion barrier that cages diffusible factors at the division
site to promote the completion of cytokinesis [Dobbelaere
and Barral, 2004]. However, in cdc10D and bud4D cells
where the septin double ring is defective, AMR assembly
and constriction occurs efﬁciently [Wloka et al., 2011].
Strikingly, membrane trafﬁcking components such as
Myo2 (myosin-V) and Exo84 (an exocyst subunit) are
delivered to the division site and constrict with the AMR
in the mutant cells. Importantly, these cells are able to
complete cytokinesis and cell separation. Together, these
observations suggest that the septin diffusion barrier is dis-
pensable for cytokinesis [Wloka et al., 2011] and support
the hypothesis that the septin hourglass is essential for
AMR assembly, which, in turn, guides targeted membrane
deposition and PS formation during cytokinesis independ-
ently of the septin double ring. However, septins are col-
lectively essential for cell viability and cytokinesis in all
genetic backgrounds. In contrast, myo1D cells, which lack
the AMR, are viable in most genetic backgrounds, despite
their severe defects in cytokinesis [Watts et al., 1987;
Rodriguez and Paterson, 1990; Bi et al., 1998], suggesting
that septins probably play an additional role in cytokinesis
aside from its role in AMR assembly. Indeed, when
MYO1 is deleted, the secretory cargo Chs2 is caged
between the septin double ring [Wloka et al., 2011].
When the double ring is conditionally inactivated in a
myo1D background, Chs2 drifts away from the division
site [Wloka et al., 2011]. These data suggest that the sep-
tin double ring and the AMR share a role in restricting
the factors required for PS formation during cytokinesis,
with the latter playing the dominant role. This conclusion
is supported by the observation that myo1D and cdc10D
are synthetically lethal [Wloka et al., 2011]. It is also pos-
sible that the double septin ring plays additional roles in
SS formation and/or cell separation.
AMR Assembly, Constriction, and
At least seven classes of proteins, including the septins,
myosin-II heavy chain, the essential light chain (ELC) of
myosin-II, IQGAP, Rho, formins, and tropomyosins, are
required for AMR assembly in budding yeast (Fig. 2).
Other proteins may also play a nonessential or ﬁne-tuning
role in AMR assembly that requires more sensitive assays
In order to understand the mechanism of AMR assem-
bly, it is essential to understand how each of the major
ring components is targeted to the division site and inter-
acts with each other. Myo1, the sole myosin-II heavy
chain in budding yeast, forms a two-headed structure with
a rod tail, similar to all other ‘‘conventional’’ myosin-IIs in
animal cells [Fang et al., 2010]. Myo1 targets to the divi-
sion site through a biphasic mechanism (Fig. 3) [Fang
et al., 2010]. In the ﬁrst phase, Myo1 targeting is medi-
ated by the septin-binding protein Bni5 [Lee et al., 2002],
which covers the time from late G1 to the onset of telo-
phase [Fang et al., 2010]. The second phase of targeting
is mediated by its ELC Mlc1 and Iqg1, the sole and
essential IQGAP in budding yeast [Epp and Chant, 1997;
Lippincott and Li, 1998a], which covers the time from
the onset of anaphase to the end of cytokinesis [Fang
et al., 2010]. These targeting mechanisms overlap from
the onset of anaphase to the onset of telophase. Bni5
interacts directly with the septins [Lee et al., 2002] and
also with a speciﬁc targeting domain (amino acids 991-
1180) of Myo1. Thus, the biochemical mechanism for the
ﬁrst-phase targeting is relatively clear. In contrast, it
remains unknown how Mlc1 and/or Iqg1 interact with
the septins and also with a distinct targeting domain
(amino acids 1224-1307) of Myo1 to enable the second-
phase targeting [Fang et al., 2010]. The Bni5-mediated
Myo1 recruitment might facilitate motor-dependent retro-
grade ﬂow of actin cables nucleated by the formin Bni1 at
the bud cortex [Fang et al., 2010], and this targeting
mechanism may be species-speciﬁc. The second phase of
targeting is clearly coupled with AMR assembly [Fang
et al., 2010], as Mlc1, Iqg1, and Myo1 are all essential
for actin ring formation [Epp and Chant, 1997; Bi et al.,
1998; Lippincott and Li, 1998a; Boyne et al., 2000; Shan-
non and Li, 2000]. In addition, the second targeting
mechanism involves conserved proteins and is likely con-
served throughout evolution [Fang et al., 2010]. Indeed, a
similar mechanism appears to dictate myosin-II targeting
in the ﬁssion yeast S. pombe [Laporte et al., 2011; Padma-
nabhan et al., 2011].
Mlc2 is the sole regulatory light chain (RLC) for Myo1
[Luo et al., 2004]. It displays an identical localization pat-
tern to that of Myo1 throughout the cell cycle (Fig. 2),
and its localization to the division site depends on its
binding to Myo1 [Luo et al., 2004]. Cells deleted for
MLC2 do not exhibit defects in actin ring formation but
display a mild defect in Myo1 disassembly during and/or
toward the end of cytokinesis [Luo et al., 2004]. As
described above, Mlc1 is the ELC for Myo1 but its bind-
ing to the IQ motif of Myo1 is not required for AMR as-
sembly [Luo et al., 2004]. Mlc1 is also a light chain for
Myo2 [Stevens and Davis, 1998], a myosin-V in budding
yeast that plays an essential role in vesicle and organelle
transport [Bretscher, 2003], but cells expressing Myo2
that lacks the Mlc1-binding site are still able to assemble
an actin ring [Wloka and Bi, unpublished data]. Thus,
the Mlc1–Myo2 interaction is not required for AMR as-
sembly. Mlc1 is also a light chain for Iqg1 and is required
for Iqg1 localization to the division site [Boyne et al.,
2000; Shannon and Li, 2000]. Iqg1 is required for Myo1
targeting during cytokinesis (see above). It is also required
for actin ring assembly, presumably by binding to actin
n 714 Wloka and Bi CYTOSKELETON
ﬁlaments at the division site through its calponin homol-
ogy domain (CHD) [Epp and Chant, 1997; Boyne et al.,
2000; Shannon and Li, 2000]. Thus, Mlc1 contributes to
Myo1 targeting and AMR assembly through an Iqg1-
mediated interaction with the tail of Myo1. Mlc1 localizes
to the division site in large-budded cells before septin
hourglass splitting (Fig. 2). This localization depends on
the septins but is largely independent of its interactions
with Myo1, Myo2, and Iqg1 [Luo et al., 2004]. It
remains unknown how Mlc1 interacts with the septins
before cytokinesis and how it is maintained at the division
site during cytokinesis.
Rho1, formins, and tropomyosins are also required for
actin ring assembly (Fig. 2). Bni1 and Bnr1 are two for-
mins in budding yeast that share an essential role in
nucleating linear actin ﬁlaments for actin cable and actin
ring assembly to mediate polarized bud growth and cyto-
kinesis, respectively [Moseley and Goode, 2006; Park and
Bi, 2007]. Bnr1 localizes to the division site in a septin-
dependent manner from late G1 to the onset of telophase,
which coincides with spindle disassembly, whereas Bni1
localizes to the division site from the onset of telophase to
the end of cytokinesis [Pruyne et al., 2004; Buttery et al.,
2007]. Both formins contribute to actin ring assembly
with Bni1 playing a predominant role [Vallen et al.,
2000; Tolliday et al., 2002]. Bnr1 targets to the septin
hourglass via distinct domains during different phases of
the cell cycle [Gao et al., 2010] but details surrounding
its interactions with the septins remain to be determined.
The mitotic exit phosphatase Cdc14 is not only required
for the timed disappearance of Bnr1 from the bud neck
but also required for the timed appearance of Bni1 at the
division site during cytokinesis [Bloom et al., 2011]. Con-
sistently, Bni1 fails to localize in the MEN mutants such
as cdc15-2 cells [Bloom et al., 2011]. However, another
study indicates that Bni1 is able to localize to the division
site in the cdc15-2 cells [Yoshida et al., 2006]. It is unclear
what accounts for the discrepancy between the studies.
Rho1 is thought to promote actin ring assembly by acti-
vating the formin Bni1 [Tolliday et al., 2002]. The local-
ization and activation of Rho1 at the division site is, in
turn, controlled by its guanine-nucleotide-exchange factor
(GEF), which is regulated by the Polo kinase Cdc5 [Yosh-
ida et al., 2006, 2009]. This cascade of activation-coupled
protein recruitment to the division site deﬁnes a mecha-
nism for the cell cycle control of actin ring assembly.
Tpm1 (the major isoform) and Tpm2 (the minor iso-
form), two functionally redundant tropomyosins in bud-
ding yeast [Drees et al., 1995], are also required for actin
ring assembly, presumably by stabilizing the formin-
nucleated actin ﬁlaments [Tolliday et al., 2002].
Together, these observations suggest a simple mod el
for AMR assembly. The formin-nucleated actin ﬁla-
ments are captured by the CHD of Iqg1 and organized
into a ring s tructure using Myo1 ﬁlaments as the tem-
plate. Currently, it is not known whether Myo1 forms
bipolar ﬁlaments during cytokinesis. However, Myo1 is
shown to be dynamic during G2/M and become immo-
bile during cytokinesis, suggesting that Myo1 undergoes
a cell cycle-regulated organizational change at the divi-
sion site [Dobbelaere and Barral, 2004]. Strikingly, a
C-terminal fragment of Myo1, which deﬁnes a ‘‘puta-
tive assembly domain’’ [Fang et al., 2010], is required
for establishing the immobile state of Myo 1 during
cytokinesis [Wloka and Bi, unpublished data]. Thus,
Myo1 is likely to form higher-order structures during
The MEN is required for AMR constriction but not its
assembly [Vallen et al., 2000; Lippincott et al., 2001;
Luca et al., 2001]. There are three possible ways by which
the MEN could control AMR constriction. First, the
MEN could control AMR constriction by controlling sep-
tin hourglass splitting [Cid et al., 2001; Lippincott et al.,
2001]. However, in the MEN mutants forced to exit mi-
tosis by overexpression of the CDK1 inhibitor Sic1, the
septin hourglass is split into two cortical rings but AMR
constriction still cannot occur [Meitinger et al., 2010;
Meitinger et al., 2011], suggesting that the MEN controls
AMR constriction independently of septin hourglass split-
ting. Second, the MEN could control AMR constriction
by controlling PS formation. The MEN activity is
required for the proper localization of the proteins
involved in PS formation such as Chs2, Inn1, Hof1, and
Cyk3 (see more discussion on these proteins later) at the
division site [Nishihama et al., 2009; Meitinger et al.,
2010]. Consistently, MEN mutants are defective in PS
formation [Meitinger et al., 2010]. However, blocking PS
formation by deleting CHS2 or INN1 causes asymmetric
AMR constriction that lasts only 3 to 4 min [VerPlank
and Li, 2005; Nishihama et al., 2009]. In contrast, the
AMR displays little or no constriction in the mutants
with or without forced mitotic exit by Sic1 overexpres-
sion. Thus, the MEN is unlikely to control AMR con-
striction via PS formation. Finally, the MEN could
control AMR constriction by directly regulating AMR
components. Iqg1 is a potential target. The AMR is
assembled normally but fails to constrict in cells carrying
an iqg1 allele that encodes a truncated protein lacking the
GRD (GAP-related domain) [Shannon and Li, 1999]. In
addition, Tem1 in either GDP- or GTP-bound state inter-
acts directly with the GRD of Iqg1 [Shannon and Li,
1999]. How the Tem1–GRD interaction controls AMR
constriction remains to be investigated.
One of the fundamental differences between muscle con-
traction and AMR constriction is that the number of
CYTOSKELETON Cytokinesis in Budding Yeast 715 n
‘‘contractile units’’ remains the same during muscle con-
traction [Huxley and Hanson, 1954; Huxley, 1969],
whereas AMR constriction is coupled with disassembly or
the loss of contractile proteins [Schroeder, 1972; Bi,
2010]. Indeed, Myo1 is progressively lost during AMR
constriction [Tully et al., 2009; Wloka et al., 2011]. To
date, three different mechanisms are known to regulate
AMR disassembly. First, Mlc2, the RLC for Myo1,
appears to play a role in Myo1 disassembly, as Myo1-GFP
lingers at the division site for longer time in mlc2D cells
than in wild-type cells [Luo et al., 2004]. This conclusion
is supported by the observation that myosin-II puriﬁed
from RLC-null cells of Dictyostelium discoideum assembles
into ﬁlaments normally but shows a clear defect in disas-
sembly [Chen et al., 1994]. Interestingly, deletion of the
IQ2 motif, the Mlc2-binding site, in Myo1 does not
cause any obvious defect in Myo1 disassembly, suggesting
that the RLC-binding site may be inhibitory to myosin-II
disassembly in the absence of its RLC [Luo et al., 2004].
A similar autoinhibitory mechanism has been observed in
ﬁssion yeast [Naqvi et al., 2000] and Dictyostelium
[Uyeda and Spudich, 1993].
Second, the motor domain of Myo1 plays a role in
AMR disassembly during cytokinesis. Myo1 lacking the
entire head domain, which includes the motor domain
and the binding sites for ELC and RLC, is able to assem-
ble a ‘‘headless’’ AMR that constricts with a 20-30%
decreased rate in comparison to the AMR in wild-type
cells [Lord et al., 2005; Fang et al., 2010]. This ‘‘headless’’
Myo1 is clearly defective in disassembly, despite its ability
to carry out cytokinesis [Lord et al., 2005; Fang et al.,
2010]. The light chain-binding sites are not responsible
for the observed disassembly defect, as Myo1 lacking both
IQ1 and IQ2 motifs constricts normally [Luo et al.,
2004]. Together, these observations suggest that absence
of the motor domain may account for the obser ved disas-
sembly defect displayed by the headless Myo1. This con-
clusion is supported by the observation that inhibition of
the motor activity of a mammalian myosin-II by blebbis-
tatin causes defects in AMR disassembly [Straight et al.,
Finally, AMR disassembly is regulated by the anaphase
promoting complex/cyclosome (APC/C)-mediated degra-
dation of Iqg1 [Ko et al., 2007; Tully et al., 2009]. APC/
C is an E3 ligase that ubiquitinates Iqg1 and targets it for
degradation by the 26S proteasome [Ko et al., 2007; Tully
et al., 2009]. In the absence of Cdh1 (an activator of the
APC/C), Iqg1, Myo1, Mlc1, and Mlc2 all linger at the
division site and also colocalize in ‘‘ectopic patches’’ for
more than 10 min after AMR constriction [Tully et al.,
2009]. Furthermore, deletion of CDH1 exacerbates the
defect of mlc2D cells, suggesting that the RLC-mediated
regulation of Myo1 and the APC/C-mediated degradation
of Iqg1 deﬁne two distinct mechanisms for controlling
AMR disassembly [Tully et al., 2009].
Targeted Membrane Deposition,
Septum Formation, and Cell
Membrane Addition at the Division Site
The ultimate goal in cytokinesis is to achieve membrane
closure between the progenies of a dividing cell, regardless
of cell types. This process requires new membrane inser-
tion at the division site in animal, fungal, plant, and per-
haps bacterial cells [Hales et al., 1999; Strickland and
Burgess, 2004; Oliferenko et al., 2009]. In budding yeast,
immediately after the launch of a new cell cycle in late
G1, the growth machinery including the polarity regula-
tors such as Cdc42, the actin cytoskeleton, and the secre-
tory pathways are all directed toward the bud cortex to
drive bud emergence and bud enlargement while the
mother cell stays constant in size [Bi and Park, 2012]. At
the onset of telophase, the same growth machinery is
redirected toward the mother-bud neck to promote cyto-
kinesis (Fig. 3). For example, the formin Bni1 is translo-
cated from the bud cortex to the bud neck to nucleate the
assembly of actin cables and the actin ring (Fig. 3). The
actin cables guide Myo2 (myosin-V)-powered transport of
post-Golgi vesicles from both the mother and the daugh-
ter to the bud neck (Figs. 2 and 3). These vesicles carry
various cargoes including Chs2, the chitin synthase II that
is essential for PS formation [Sburlati and Cabib, 1986;
Shaw et al., 1991; VerPlank and Li, 2005], some of the
exocyst subunits including Exo84 that is required for vesi-
cle tethering at the plasma membrane (PM) [Guo et al.,
1999; Boyd et al., 2004], and v-SNARE [
Soluble N-ethylmaleimide-sensitive factor Attachment
Receptor] proteins that are required for subse-
quent vesicle fusion (Figs. 2 and 3). Once the vesicles are
delivered to the division site, they are likely ‘‘captured’’ by
the AMR perhaps through the ‘‘track switching’’ of Myo2
from the actin cables to the actin ring (Fig. 3) [Fang
et al., 2010]. This capture mechanism ensures that mem-
brane addition at the division site is coupled with AMR
constriction in time and space.
The dimension of the division site for different cell
types can vary tremendously. For example, the bud neck
of haploid yeast cells is about 1 lm in diameter [Bi et al.,
1998; Lippincott and Li, 1998a], whereas animal cells are
usually over 15-20 lm in diameter [Schroeder, 1972; Car-
valho et al., 2009]. Since animal and fungal cells need to
complete cytokinesis within a similar time (<20 min) and
their post-Golgi vesicles are similar in size (80-100 nm in
diameter) [Novick et al., 1980; Lehman et al., 1988;
Rambourg et al., 1989; Rossi and Brennwald, 2011], a
large difference in dimension at the division site means
that vastly different degrees of vesicle fusions are required
to seal the division site during cytokinesis [Fang et al.,
2010]. For example, assuming that there is no AMR
n 716 Wloka and Bi CYTOSKELETON
constriction and the vesicle size is 100 nm in diameter,
only 50 vesicles are required to seal the division site in
budding yeast. In contrast, for an animal cell of 20 lmin
diameter, 20,000 vesicles are required to achieve the same
feat. These differential demands for membrane insertion
at the division site probably explain, at least in part, the
differential requirements for AMR constriction in different
cell types. For example, budding yeast cells lacking the
entire AMR, such as the cells harboring myo1D, are viable
despite displaying defects in cytokinesis [Watts et al.,
1987; Rodriguez and Paterson, 1990; Bi et al., 1998]. In
this case, a small amount of vesicles delivered to the divi-
sion site is presumably sufﬁcient to drive cytokinesis,
albeit less efﬁciently than the cells with a functional
AMR. In contrast, AMR constriction is presumably
required to reduce the membrane demand during cytoki-
nesis in animal cells, which explains why inhibition of
AMR constriction inhibits cytokinesis in these cells
[Straight et al., 2003].
In addition to AMR constriction and targeted membrane
deposition, ECM remodeling is also a hallmark of fungal
and animal cytokinesis [Bi and Park, 2012]. Defects in
chondroitin synthesis cause embryonic lethality with cyto-
kinesis arrest in Caenorhabditis elegans and mice [Mizugu-
chi et al., 2003; Izumikawa et al., 2010]. In budding
yeast, targeted vesicle fusion not only increases the surface
area at the division site but also delivers enzymatic cargoes
such as Chs2 for PS formation [Bi and Park, 2012]. The
PS, a thin electron-lucent structure spanning the mother-
bud neck (Fig. 1), consists of chitin, a polymer of b-1,4-
linked N-acetylglucosamine that is synthesized by the chi-
tin synthase II (Chs2) [Sburlati and Cabib, 1986; Shaw
et al., 1991]. There are two other chitin synthases in bud-
ding yeast. The chitin synthase I (Chs1) is thought to
play a role in cell wall repair after cell separation [Cabib
et al., 1989; Cabib et al., 1992]. The chitin synthase III
(with Chs3 as its catalytic subunit) is responsible for syn-
thesizing chitin in the ‘‘bud scar’’ and lateral cell wall
[Bulawa, 1993; Orlean, 1997; Lesage and Bussey, 2006].
During early stage of budding, Chs3 localizes to the
mother side of the bud neck to catalyze the formation of
a ‘‘chitin ring’’ (Fig. 2) [Chuang and Schekman, 1996;
DeMarini et al., 1997], which plays a role in bud-neck in-
tegrity [Schmidt et al., 2003] and becomes the ‘‘bud scar’’
on the mother cell surface after cytokinesis and cell sepa-
ration [Bi and Park, 2012]. Chs3 also localizes to the divi-
sion site during cytokinesis (Fig. 2) [Chuang and
Schekman, 1996; DeMarini et al., 1997] but its function
remains unclear. Deletion of CHS1 and CHS3 together
does not cause any obvious defect in cytokinesis [Oh
et al., 2012]. In contrast, deletion of CHS2 abolishes PS
formation and causes severe defects in cytokinesis but not
cell lethality [Sburlati and Cabib, 1986; Shaw et al.,
1991]. The viability of the chs2D cells depends on the for-
mation of a ‘‘remedial septum’’ that requires Chs3 activity
during cytokinesis [Shaw et al., 1991; Cabib and Schmidt,
2003]. Thus, Chs2 plays a major role in cytokinesis by
catalyzing PS formation, whereas Chs3 plays a minor and
undeﬁned role in the normal division process but is
required for the remedial septum formation in the absence
PS formation is r egulated through Chs2 at multiple
levels by cell cycle signals. During mitosis, Chs2 is syn-
thesized and held at the ER through phosphoryl ation by
CDK1 [Chuang and Schekman, 1996; Zhang et al.,
2006; Teh et al., 2009]. At the o nset of cyto kinesis,
Chs2 is triggered to exit the ER by removing the CDK1-
mediated phosphorylation through the action of Cdc14
phosphatase, a component of the MEN [Chin et al.,
2011]. Chs2 then enters the secretory pathway and is
delivered to the bud neck by the exocytic machiner y
[Chuang and Schekman, 1996; VerPlank and Li, 2005].
Approximately h alfway through AMR constriction, Chs2
is phosphorylated by t he neck-localized Dbf2-Mob1 ki-
nase, another component of the MEN, causing Chs2 dis-
removal from the division site by the endocytic machin-
ery [Oh et al., 2012]. Together, these obser vations sug-
gest that PS formation must be controlled exquisitely
during the cell cycle, and that targeted membrane depo-
sition is coupled with localized cell wall remodeling dur-
ing cytokinesis. Chs2 function and/or PS formation is
also regulated by other cytokinesis proteins, which will
be discussed l ater in the context of AMR-PS coordina-
tion during cytokinesis.
Secondary Septum Formation
At the end of AMR constriction and PS formation, two
electron-dense SS are laid down on both sides of the PS
(Fig. 1). These septa are made of 1,3-b-
mannoproteins [Lesage and Bussey, 2006]. The glucans
are synthesized by two 1,3-b-
D-glucan synthases (GSs),
each of which consists of a putative catalytic subunit and
a regulatory subunit. The catalytic subunits for the two
GSs are Fks1/Gcs1 and Fks2/Gcs2, respectively [Douglas
et al., 1994; Inoue et al., 1995; Mazur and Baginsky,
1996], whereas the regulatory subunit for both GSs is
Rho1 [Mazur and Baginsky, 1996; Qadota et al., 1996].
Fks1 plays a major role in cell wall synthesis during vege-
tative growth, whereas Fks2 mainly functions during
nutritional starvation, mating, and sporulation [Levin,
2005; Park and Bi, 2007]. Cells can tolerate the deletion
of a single gene but not both, suggesting that Fks1 and
Fks2 share an essential role in cell wall synthesis and SS
formation [Inoue et al., 1995; Mazur and Baginsky,
CYTOSKELETON Cytokinesis in Budding Yeast 717 n
Rho1 may also contribute to SS formation by control-
ling Chs3 localization at the division site [Yoshida et al.,
2009]. In chs3D cells, the SS appear to be morphologically
distinct from those in wild-type cells [Shaw et al., 1991],
suggesting a role of Chs3 in SS formation. In rho1
mutants, Chs3 fails to translocate from internal stores
(chitosomes) to the PM [Valdivia and Schekman, 2003],
which presumably explains why Chs3 fails to localize to
the division site in these mutants [Yoshida et al., 2009].
The localization of Rho1 at the division site is regulated
by two sequentially acting mechanisms [Yoshida et al.,
2009]. During late mitosis, the Polo kinase Cdc5 controls
Rho1 localization and activation through Tus1, a GEF for
Rho1 [ Yoshida et al., 2006], and the activated Rho1 is
thought to activate the formin Bni1 to promote actin ring
assembly [Yoshida et al., 2006]. After AMR constriction
and PS formation, phospholipids of the PM control Rho1
localization and activation directly and also indirectly
through Rom2, another GEF for Rho1. During this pe-
riod, activated Rho1 is thought to promote SS formation
by activating GSs and also controlling Chs3 localization.
Cell Separation: the RAM Signaling Network
After the SS formation is completed, the PS and a part of
the SS is quickly degraded by the endochitinase Cts1
[Kuranda and Robbins, 1991] and several glucanases
including Dse4/Eng1 and Egt2 [Kovacech et al., 1996;
Baladron et al., 2002], resulting in cell separation [Yeong,
2005; Lesage and Bussey, 2006]. The daughter cell-speciﬁc
transcriptional factor Ace2 controls the expression of both
Cts1 and Eng1 [King and Butler, 1998; O’Conallain
et al., 1999; Colman-Lerner et al., 2001], whereas Ace2
and its paralog Swi5 collectively control the expression of
Egt2, with Swi5 being the principal regulator [Kovacech
et al., 1996].
The asymmetric localization and activation of Ace2
depends on the RAM pathway, a conserved signaling net-
work known to play a role in polarized cell growth and
cell separation [Colman-Lerner et al., 2001; Nelson et al.,
2003; Mazanka et al., 2008]. The RAM pathway consists
of the Cbk1 kinase and its regulatory or activating subunit
Mob2 (Cbk1-Mob2 complex), and a few upstream and
mutually interacting proteins including Kic1 (a PAK /
Ste20 family kinase), Sog2, Hym1, and perhaps Tao3
[Nelson et al., 2003]. All these upstream components are
required for Cbk1 activation as well as its daughter nu-
cleus-restricted localization [Nelson et al., 2003]. Cbk1
activation also depends on MEN [Brace et al., 2011].
Direct phosphorylation by Cbk1 activates Ace2, which
initiates and then maintains the asymmetric localization of
Ace2 in the daughter nucleus [Mazanka et al., 2008]. Af-
ter cell separation, Ace2 exits the daughter nucleus and is
kept in the cytoplasm in late G1 by the CDKs Pho85
and Cdc28 [Mazanka and Weiss, 2010].
Coordination of AMR Constriction
and PS Formation during
Efﬁcient cytokinesis requires spatiotemporal coordination
of AMR constriction and PS formation. As described ear-
lier, the AMR is not essential for cell viability but is
required for efﬁcient cytokinesis and cell separation [Watts
et al., 1987; Rodriguez and Paterson, 1990; Bi et al.,
1998]. Cells defective in AMR assembly such as myo1D
and bni1D cells form misoriented PS [Vallen et al., 2000;
Fang et al., 2010], suggesting that the AMR may guide
PS formation during cytokinesis. Surprisingly, the guiding
role can be largely accomplished by a ‘‘headless’’ AMR
assembled by the Myo1 tail, whereas the Myo1 head plays
a ﬁne-tuning role in this process [Lord et al., 2005; Fang
et al., 2010]. Thus, the AMR may function as a scaffold
guiding PS formation during cytokinesis in addition to its
traditional role in force generation [Fang et al., 2010].
Reciprocally, PS formation is required for proper AMR
constriction [Bi, 2001; Schmidt et al., 2002; VerPlank
and Li, 2005]. Cells deleted for CHS2 or INN1 display
more severe defects in cell growth (nearly inviable) and
cytokinesis than myo1D cells, failing to form the PS
[Sburlati and Cabib, 1986; Shaw et al., 1991; Nishihama
et al., 2009] and showing asymmetric AMR constriction
that may be caused by partial detachment of the ring
from the PM [VerPlank and Li, 2005; Nishihama et al.,
2009]. Thus, the PS is thought to stabilize the AMR dur-
ing its constriction [Bi, 2001; Schmidt et al., 2002; Ver-
Plank and Li, 2005]. Yeast cells have high turgor pressure
that pushes the PM outward [Levin, 2005; Lesage and
Bussey, 2006]. Thus, it is not surprising that AMR con-
striction and, more importantly, PS formation are
required to drive PM ingression, counteracting the turgor
pressure during cytokinesis.
Coordination of the AMR and PS formation occurs at
multiple levels. First, septins are required for AMR assem-
bly [Bi et al., 1998; Lippincott and Li, 1998a] as well as
PS formation [Roh et al., 2002], thus deﬁning a coordina-
tion mechanism. Second, Iqg1 is required for AMR as-
sembly and presumably PS formation, as deletion of
IQG1, but not MYO1, causes cell lethality in most genetic
backgrounds [Epp and Chant, 1997; Bi et al., 1998; Lip-
pincott and Li, 1998a]. In addition, increased dosage of
Cyk3, a cytokinesis protein involved in PS formation (see
below), suppresses the lethality of iqg1D cells without
restoring the AMR [Korinek et al., 2000]. Because Mlc1
is required for the localization of Iqg1 to the division site
[Boyne et al., 2000; Shannon and Li, 2000], and because
mlc1D cells are inviable in most genetic backgrounds [Ste-
vens and Davis, 1998; Boyne et al., 2000; Shannon and
Li, 2000], Mlc1 is presumably required for both AMR as-
sembly and PS formation. Thus, Mlc1 and Iqg1 deﬁne
another level of AMR-PS coordination that likely acts
n 718 Wloka and Bi CYTOSKELETON
Table I. Major Proteins Involved in Cytokinesis in Budding Yeast
Standard name Generic name and /or feature Functions in cytokinesis Selected references
Assembly of the actomyosin ring
Cdc3 Septins (GTP-binding and
A ‘‘scaffold’’ for AMR assembly
before cytokinesis and a
possible ‘‘diffusion barrier’’
[Hartwell, 1971; Longtine
et al., 1996; Mino
et al., 1998; Takizawa et al.,
2000; Dobbelaere and Barral,
2004; Wloka et al., 2011]
Bni5 Septin-interacting protein Required for Myo1 targeting to
the division site before
[Lee et al., 2002; Fang
et al., 2010]
Myo1 Sole myosin-II heavy chain Required for AMR assembly [Bi et al., 1998; Lippincott
and Li, 1998a]
Mlc1 Essential light chain (ELC) for
Myo1, and also a light chain
for Iqg1 and Myo2
Required for Myo1 targeting to
the division site during
cytokinesis, required for Iqg1
localization to the division
site, and thus required for
[Stevens and Davis, 1998;
Boyne et al., 2000; Shannon
and Li, 2000; Wagner et al.,
2002; Luo et al., 2004; Fang
et al., 2010]
Mlc2 Regulatory light chain
(RLC) for Myo1
Involved in AMR disassembly [Luo et al., 2004]
Iqg1 Sole and essential IQGAP Required for Myo1 targeting to
the division site during
cytokinesis, required for actin
ring assembly, and thus
required for AMR assembly.
Also required for
[Epp and Chant, 1997;
Shannon and Li, 1999;
Boyne et al., 2000; Ko et al.,
2007; Tully et al., 2009;
Fang et al., 2010]
Bni1 Formin Plays a major role in actin ring
[Vallen et al., 2000; Pruyne
et al., 2004; Buttery et al.,
Bnr1 Formin Plays a minor role in actin ring
Rho1 Rho GTPase Required for actin ring
assembly by activating
[Tolliday et al., 2002; Yoshida
et al., 2006; Yoshida et al.,
Act1 Actin Essential for actin ring
[Epp and Chant, 1997; Bi
et al., 1998; Lippincott
and Li, 1998a]
Tpm1 Tropomyosin Plays a major role in stabilizing
the actin ﬁlaments in the
[Drees et al., 1995]
Tpm2 Tropomyosin Plays a minor role in stabilizing
the actin ﬁlaments in the
[Drees et al., 1995]
Primary septum formation
Myo2 Myosin-V Powers vesicle movement along
actin cables during bud
growth and cytokinesis
[Pruyne et al., 1998; Bretscher,
2003; VerPlank and Li,
Exo84 Exocyst subunit Part of the exocyst complex
required for vesicle tethering
to the PM during bud
growth and cytokinesis
[Guo et al., 1999; Boyd et al.,
CYTOSKELETON Cytokinesis in Budding Yeast 719 n
Table I. (Continued)
Standard name Generic name and /or feature Functions in cytokinesis Selected references
Chs2 Chitin synthase II Predominantly synthesizes the
chitin of the PS
[Sburlati and Cabib, 1986;
Shaw et al., 1991]
Secondary septum formation
Fks1 (Gsc1) Catalytic subunit of
1,3-b–glucan synthase (GS)
Required for cell wall synthesis
during budding and SS
formation during cytokinesis
[Douglas et al., 1994; Mazur
et al., 1995]
Fks2 (Gsc2) Catalytic subunit of
1,3-b–glucan synthase (GS)
Required for cell wall synthesis
during budding and SS
formation during cytokinesis
Rho1 Rho GTPase and serves as
the regulatory subunit of
1,3-b-glucan synthases (GS s)
Required for the activation of
Fks1 and Fks2 for their role
in cell wall synthesis during
budding and SS formation
[Mazur and Baginsky, 1996;
Qadota et al., 1996; Yoshida
et al., 2009]
Chs3 Catalytic subunit of chitin
Required for the synthesis of
chitin in the ‘‘bud scar’’ and
the cell wall and may also
play a role in SS formation
[Shaw et al., 1991; Chuang
and Schekman, 1996;
DeMarini et al., 1997;
Yoshida et al., 2009]
Ace2 The downstream transcription
factor of the RAM pathway
Plays an essential role in cell
separation by controlling the
transcription of the chitinase
Cts1 and the glucanases
Eng1 and Egt2
[King and Butler, 1998;
O’Conallain et al., 1999;
Colman-Lerner et al., 2001;
Nelson et al., 2003]
Cts1 Endochitinase Digestions of chitin in the PS [Kuranda and Robbins, 1991;
King and Butler, 1998;
O’Conallain et al., 1999;
Colman-Lerner et al., 2001]
Eng1 (Dse4) Glucanase Digestion of a part of the
glucan in the SS
[Baladron et al., 2002]
Egt2 Glucanase? Digestion of a part of the
glucan in the SS
[Kovacech et al., 1996]
Chs1 Chitin synthase I Involved in septum repair at
low pH after cell separation
[Cabib et al., 1989; Cabib
et al., 1992]
Coordination of actomyosin ring and primary septum formation
Septins GTP-binding and
Required for AMR assembly
and PS formation
[Hartwell, 1971; Longtine
et al., 1996 (p 297); Mino
et al., 1998; Takizawa et al.,
2000; Roh et al., 2002;
Dobbelaere and Barral,
Mlc1 ELC Required for AMR assembly
and PS formation
[Stevens and Davis, 1998;
Boyne et al., 2000; Shannon
and Li, 2000; Wagner et al.,
2002; Luo et al., 2004; Fang
et al., 2010]
Iqg1 IQGAP Required for AMR assembly
and PS formation
[Epp and Chant, 1997;
Shannon and Li, 1999;
Boyne et al., 2000; Korinek
et al., 2000; Ko et al., 2007;
Tully et al., 2009; Fang
et al., 2010]
n 720 Wloka and Bi CYTOSKELETON
downstream of the septins, as septins are required for the
localization of Mlc1 and Iqg1 to the bud neck [Boyne
et al., 2000; Shannon and Li, 2000; Luo et al., 2004].
Finally, Inn1 interacts with Iqg1 on the AMR side and
with Hof1 and Cyk3 on the PS side (Fig. 2) [Sanchez-
Diaz et al., 2008; Jendretzki et al., 2009; Nishihama
et al., 2009; Meitinger et al., 2010]. Inn1 contains a C2-
like domain in its N-terminal region and multiple PXXP
motifs in its C-terminal region and plays an essential role
in PS formation [Nishihama et al., 2009]. Inn1 interacts
with the SH3 domains of Hof1 and Cyk3 via distinct
PXXP motifs [Nishihama et al., 2009]. Hof1 contains a
F-BAR domain in its N-terminal region and a SH3 do-
main in its C-terminal region. Deletion of HOF1 is not
lethal but often causes asymmetric AMR constriction and
PS formation [Nishihama et al., 2009; Meitinger et al.,
2010]. In addition, hof1D and myo1D are synthetically le-
thal [Vallen et al., 2000], suggesting that Hof1 is involved
in AMR-independent cytokinesis, presumably in PS for-
mation. Cyk3 contains a SH3 domain in its N-terminal
region and a transglutaminase-like domain near the mid-
point of the protein [Makarova et al., 1999; Korinek
et al., 2000; Nishihama et al., 2009]. Like HOF1, dele-
tion of CYK3 does not cause cell lethality. Strikingly,
increased dosage of Cyk3 causes an increase in Chs2-de-
pendent chitin synthesis at the division site [Oh et al.,
2012] as well as the formation of PS-like structures at ec-
topic positions near the bud neck [Meitinger et al., 2010].
In addition, increased dosage of Cyk3 also rescues the
growth and PS formation defects of inn1D cells [Nishi-
hama et al., 2009]. These observations suggest that Cyk3
is a potent activator of Chs2 in vivo [Oh et al., 2012].
Because cyk3D is synthetically lethal with either myo1D or
hof1D [Korinek et al., 2000], Cyk3 and Hof1 likely share
an essential role in promoting PS formation in AMR-in-
dependent cytokinesis. Inn1 localizes to the division site
after Iqg1 but before Cyk3, whereas Hof1 displays a com-
plex pattern of localization during the cell cycle [ Vallen
et al., 2000; Sanchez-Diaz et al., 2008; Jendretzki et al.,
2009; Nishihama et al., 2009; Meitinger et al., 2010]. To-
gether, these observations suggest that Iqg1-Inn1-Hof1/
Cyk3 deﬁnes another level of AMR-PS coordination that
presumably acts downstream of Iqg1.
Conclusions and Perspectives
Cytokinesis deﬁnes a classic problem in cell biology. It has
been studied for over a century. Like any other biological
process, progress in cytokinesis has been fueled by the de-
velopment of model organisms and technologies. Before
the early 1990s, myosin-II and actin were known to be
enriched at the cleavage furrow, leading to the idea of a
contractile AMR driving cytokinesis in animal cells
[Schroeder, 1972; Satterwhite and Pollard, 1992]. With
the development of genetic model systems (ﬁssion yeast,
budding yeast, Drosophila, Dictyostelium, etc.), genomic
approaches, mass spectrometry, and live-cell imaging,
more than 100 proteins are known to play a role in cyto-
kinesis (Fig. 2 and Table I) [Balasubramanian et al., 2004;
Echard et al., 2004; Skop et al., 2004; Pollard, 2010;
Goyal et al., 2011]. Functional information has been
gained on individuals or binary complexes of this growing
inventory of cytokinesis proteins. Parallel studies in multi-
ple systems using diverse approaches have led to a low re-
solution but important sketch of cytokinesis. It is a
complex process that integrates cell signaling, force genera-
tion, membrane trafﬁcking, and ECM remodeling, and all
these subcellular process must coordinate in time and
Table I. (Continued)
Standard name Generic name and /or feature Functions in cytokinesis Selected references
Inn1 C2 domain, 8 PXXP motifs Constricts together with the
AMR, may interact with
Iqg1, required for PS
formation, and interacts with
the SH3 domains of Hof1
and Cyk3 via distinct PXXP
[Sanchez-Diaz et al., 2008;
Jendretzki et al., 2009;
Nishihama et al., 2009]
Hof1 F-BAR domain, SH3 domain Constricts together with the
AMR, involved in PS
formation, and interacts
[Kamei et al., 1998; Lippincott
and Li, 1998b; Vallen et al.,
2000; Nishihama et al.,
2009; Meitinger et al., 2010]
Cyk3 SH3 domain and a transgluta-
Constricts together with the
AMR, likely activates Chs2
in PS formation, interacts
with Inn1, and functions as a
of iqg1D and inn1D cells
[Makarova et al., 1999;
Korinek et al., 2000;
Jendretzki et al., 2009;
Nishihama et al., 2009;
Oh et al., 2012]
CYTOSKELETON Cytokinesis in Budding Yeast 721 n
space to achieve cytokinesis with high efﬁciency and
Despite the impressive progress in the last 30 years,
major questions regarding the mechanisms of cytokinesis
remain unanswered. For example, it is not known how
myosin-II and actin ﬁlaments are organized into ‘‘contract-
ile units’’ that power the ingression of the PM. Are they
organized into sarcomere-like structures as in muscles or
something entirely different? What are the functions of
other ring components such as IQGAP and F-BAR pro-
teins? Do they facilitate AMR assembly and if so, how?
How is AMR assembly regulated by the cell cycle machin-
ery? Equally elusive is the mechanism underlying AMR
disassembly during its constriction. Are the ‘‘contractile
units’’ reduced in number or size or both during AMR
constriction? Do the RLC- and IQGAP-mediated disas-
sembly mechanisms apply to other fungi and animal cells?
Another major question is to deﬁne at the molecular level
how exactly Chs2 is activated at the division site for PS
formation or how localized ECM remodeling occurs in
animal cells, and how this process is linked to the AMR.
All these major questions must be addressed in order to
appreciate the elegance of the orchestra called cytokinesis.
The authors thank Dr. Younghoon Oh for critically read-
ing the manuscript and the members of the Bi lab for
stimulating discussions. This work was supported by a
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