Cytokinesis-Based Constraints on Polarized Cell Growth
in Fission Yeast
K. Adam Bohnert, Kathleen L. Gould*
Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of
The rod-shaped fission yeast Schizosaccharomyces pombe, which undergoes cycles of monopolar-to-bipolar tip growth, is an
attractive organism for studying cell-cycle regulation of polarity establishment. While previous research has described
factors mediating this process from interphase cell tips, we found that division site signaling also impacts the re-
establishment of bipolar cell growth in the ensuing cell cycle. Complete loss or targeted disruption of the non-essential
cytokinesis protein Fic1 at the division site, but not at interphase cell tips, resulted in many cells failing to grow at new ends
created by cell division. This appeared due to faulty disassembly and abnormal persistence of the cell division machinery at
new ends of fic1D cells. Moreover, additional mutants defective in the final stages of cytokinesis exhibited analogous
growth polarity defects, supporting that robust completion of cell division contributes to new end-growth competency. To
test this model, we genetically manipulated S. pombe cells to undergo new end take-off immediately after cell division.
Intriguingly, such cells elongated constitutively at new ends unless cytokinesis was perturbed. Thus, cell division imposes
constraints that partially override positive controls on growth. We posit that such constraints facilitate invasive fungal
growth, as cytokinesis mutants displaying bipolar growth defects formed numerous pseudohyphae. Collectively, these data
highlight a role for previous cell cycles in defining a cell’s capacity to polarize at specific sites, and they additionally provide
insight into how a unicellular yeast can transition into a quasi-multicellular state.
Citation: Bohnert KA, Gould KL (2012) Cytokinesis-Based Constraints on Polarized Cell Growth in Fission Yeast. PLoS Genet 8(10): e1003004. doi:10.1371/
Editor: David P. Toczyski, University of California San Francisco, United States of America
Received April 15, 2012; Accepted August 15, 2012; Published October 18, 2012
Copyright: ? 2012 Bohnert, Gould. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (http://www.nih.org) grant T32-CA119925 to KAB and by the Howard Hughes Medical
Institute (http://www.hhmi.org), of which KLG is an Investigator. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Many cells polarize in response to intrinsic and extrinsic signals.
As cell polarization is generally multifaceted, cells must integrate
both negative and positive cues for successful cellular morpho-
genesis. In various organisms, the cell cycle provides a platform on
which these cues are organized (for reviews, see [1,2]), thereby
ensuring distinct polarization events occur at the appropriate
location, time, and context.
The fission yeast Schizosaccharomyces pombe represents a geneti-
cally tractable organism for studying cell cycle regulation of
growth polarity (for reviews, see [3,4]). Wild-type S. pombe extend
solely at their two cell tips, lengthening their rod-shaped bodies
while retaining fairly constant widths. After cell division, S. pombe
grow only at old ends, so-called because they served as ends of the
dividing mother cell. Then, at a point in G2 known as new end
take off (NETO), new ends, which arise from cell division, also
initiate growth . NETO is not required for cell viability, and
myriad mutants defective in this process have been identified [3,4].
Yet, beyond requirements for S-phase completion and a minimal
interphase cell size , additional cell cycle controls on NETO
have not been identified.
As in other cell polarization events, cytoskeletal rearrangements
accompany growth transitions in S. pombe. Prior to NETO,
microtubule plus end-associated proteins Tea1 and Tea4 ride
growing microtubule ends to both cell tip cortices [6–9], where
they anchor based on their association with membrane proteins
[10,11]. Upon NETO, Tea4 recruits formin For3, which had
before only been tethered to old ends, into a complex with itself
and Tea1 at new ends . As over-expression of a Tea1-For3
fusion can drive NETO prematurely , this association likely
brings For3 into the proximity of formin activators at new ends,
stimulating For3 catalysis of F-actin cables that will deliver growth
cargo to this tip. Not surprisingly, loss of Tea1, Tea4, and/or For3
impairs fission yeast polarization and elongation [8,9,12,13]. Actin
patches, which guide endocytic vesicle internalization and
constitute a second F-actin structure, also re-polarize to both cell
tips upon NETO . Disruption of proteins comprising these
structures similarly jeopardizes growth polarity establishment [15–
17]. Thus, alteration in protein composition at cell tips is coupled
tightly to cytoskeletal rearrangements.
In addition to promoting cell tip growth, several tip-localized
cell polarity factors, including Tea1 and Tea4, direct the cell
division plane away from cell ends and towards the cell middle for
cytokinesis , the process by which daughter cells undergo
physical separation following nuclear division. However, whether
the process of cytokinesis reciprocally modulates cell polarity is
unclear. Some observations hint that the cell division machinery
may play a role in directing cell polarity. As was previously noted,
new ends formed by cell division initiate growth well after old
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ends. In mutants in which cells remain physically connected at
division sites for multiple cell divisions, internal cells can grow,
though this occurs sub-apically adjacent to septa [19,20].
Moreover, many polarity factors localize to the cell division site
[4,21–23]; nonetheless, only cell tip-localized populations of these
polarity proteins have been demonstrated to contribute to growth
polarity in S. pombe.
As in most eukaryotes, cytokinesis occurs in S. pombe through the
assembly and constriction of an actomyosin-based cytokinetic ring
(CR) . In addition to actin and myosin, several accessory
proteins regulate the dynamics and organization of this structure.
For one, Cdc15, which contains an N-terminal F-BAR domain
and a C-terminal SH3 domain characteristic of the pombe Cdc15
homology protein family , has been posited to link CR
proteins to the cortical membrane at the division site . Cdc15-
binding proteins at the CR include formin, myosin, and the C2
domain protein Fic1 [27,28]. Fic1 localizes to both interphase cell
tips and the cell division site , though its specific functions at
these sites have not been described. Fic1’s budding yeast ortholog,
Inn1, contributes to cytokinesis by linking the CR to the ingressing
membrane and by participating in septum formation [29,30].
Septa form in both budding and fission yeasts as cell wall is
deposited behind the constricting CR . A conserved signaling
network, known as the septation initiation network (SIN) in S.
pombe, triggers septum deposition during cytokinesis . Together
with the CR, septa provide mechanical force for membrane
closure at the cell division site . Subsequent septum
degradation allows for abscission [34,35]. Clearly, various
remodeling events must occur at the cell division site for
cytokinesis to complete efficiently. Whether such remodeling
events also influence daughter cell behavior has never been
While wild-type S. pombe classically grow in a single-celled form,
multiple fission yeasts, including S. pombe, possess the ability to
assume an invasive, hyphal-like state [20,36]. The ability of
pathogenic fungi to undergo such a morphogenetic switch
contributes significantly to fungal infections . Though non-
pathogenic, S. pombe, similar to the budding yeast Saccharomyces
cerevisiae , can transition into invasive growth as a foraging
response to low nutrients . Invasive S. pombe form structures
that technically qualify as pseudohyphae, for, unlike as in hyphal
growth, cytokinetic constriction occurs [39,40]. Pseudohyphae
likely maintain their hyphal-like structure due to cellular adherence
and preferential growth at old ends [39,40]. Intriguingly, it has been
postulated that single-celled fission yeast evolved from multicellular,
filamentous fungi,with transcriptional networksthat ensure efficient
cell separation playing predominant roles in the evolution of a
single-celled state . Though S. pombe pseudohyphae do not
commonly exhibit aborted cytokineses or multicellularity, it is an
attractive hypothesis that inefficient, but not entirely defective,
cytokinesis might somehow mark new ends to impair their growth
and promote the dimorphic switch in S. pombe.
In this manuscript, we define a novel cell cycle control on S.
pombe growth polarity, namely that the process of cytokinesis
imposes limitations on new end growth competency. Here, we
focus on Fic1, which we show to be involved in the re-
establishment of polarized cell growth at new ends following cell
division. Specifically, we demonstrate that Fic1 polarity function
requires its localization to the CR but not to interphase cell tips,
and that its protein-protein interactions at the CR, including that
with Cdc15, promote bipolar cell growth in the ensuing cell cycle.
We present evidence that loss of Fic1 impairs disassembly of the
cell division apparatus, with parts of this machinery persisting at
new ends following CR constriction. Additional mutants defective
in late cytokinesis also exhibit impaired new end growth.
Importantly, premature activation of NETO signaling does not
fully rescue bipolar growth in cells with late cytokinesis defects,
suggesting that cytokinesis-based constraints on S. pombe growth
polarity play a central role in defining new end growth
competency. We propose that such constraints can provide a
mechanistic understanding of how S. pombe and possibly other
fungi transition into invasive hyphal-like growth.
The S. pombe Cytokinesis Factor Fic1 Promotes the
Establishment of Bipolar Cell Growth
Recently, our laboratory identified Fic1, which was implicated
in cytokinesis based on its protein and genetic interactions and its
localization to the CR . In addition to defects in cytokinesis,
deletion of S. pombe fic1+, which is a non-essential gene, resulted in
an abnormally high percentage of cells that grew only from one
end (i.e., monopolar cells) (Figure 1A–1C). Tip growth was judged
using calcofluor staining, as birth scars formed at previous division
sites do not stain well with calcofluor and growth can be assessed
using the position of these scars relative to cell tips (Figure S1A)
. The growth defects observed upon fic1+disruption suggested
that Fic1 not only participates in cytokinesis but also in the
establishment of bipolar cell growth.
Although the upstream NETO factors Tea1 and Tea4 localized
normally to both cell tips in fic1D cells (Figure S1B–S1C), other
cell tip proteins implicated in growth polarity regulation exhibited
unusual localization patterns in this mutant. Unlike wild-type cells
with mostly bipolar actin patch distribution (Figure 1D–1E), a
variety of mutants defective in bipolar cell growth exhibit
monopolar actin patches [8,21–23]. As in such mutants, the actin
patch marker Crn1-GFP  accumulated preferentially at one
cell end in a high percentage of fic1D cells (Figure 1D–1E).
Signaling through Rho GTPases controls actin patch organization
in S. pombe [13,43], and the Rho1 activator Rgf1 , which was
GFP-tagged and imaged with the spindle pole body marker Sid4-
RFP , likewise predominated on one end of many fic1D cells
Many processes, including cell growth, are often regulated
differently in distinct cellular regions. In the rod-shaped
fission yeast Schizosaccharomyces pombe, new cell ends
created by cell division initiate growth long after old cell
ends inherited from mother cells. Though distributions of
cell tip factors contribute to this growth pattern, we have
found that the process of cytokinesis, which executes
physical separation of daughter cells at the end of the cell
cycle, also plays an important role in defining new end-
growth competency. Defects in completing cytokinesis
and remodeling the division site curb new end growth
even when protein complexes that drive tip elongation
constitutively associate with new cell ends. Moreover,
when parts of the cytokinetic machinery persist at the
division plane following constriction, S. pombe cells
become highly invasive. We believe that these findings
provide insight into growth transitions in pathogenic
fungi, as well as into the evolution of the single-celled
state from multicellular hyphal forms. Additionally, we
speculate that cytokinesis-based constraints on growth
polarity might be conserved in mammalian cells, which
have been reported to likewise polarize only distally to the
cleavage furrow at the conclusion of cell division.
Control of Growth Polarity by Cytokinesis
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(Figure 1F–1G). Not surprisingly, in both wild-type and fic1D cells,
Rgf1-GFP and Crn1-RFP concentrated at the same ends (Figure
S1D), which were confirmed by calcofluor staining to be the
growing ends of fic1D cells (Figure S1E). Consistent with Fic1
affecting both actin and Rho networks, deletion of fic1+was
synthetically sick with deletion of genes encoding factors involved
in F-actin nucleation (WASp Wsp1) and Rho GTPase regulation
(RhoGEF Rgf1 and RhoGAP Rga1) (Figure S1F). Thus, we
conclude that the absence of Fic1 upsets patterning of some but
not all polarity factors.
To discern whether new and/or old ends were defective in
resuming growth following cell division in fic1D cells, we
performed time-lapse DIC imaging to trace birth scars in live
cells. As expected, nearly all wild-type cells underwent NETO
prior to subsequent septation (Figure 2A and 2C). However,
following roughly two-thirds of fic1D cell divisions, either one or
both daughter cells failed to initiate new end growth prior to the
next septation (Figure 2B–2C). The most predominant growth
pattern in fic1D cells was that in which one daughter cell
underwent NETO while the other did not (Figure 2B–2C), with
nearly 70% of those daughter cells that did not exhibit NETO
being the younger daughter cell. Unlike tea1;D cells, in which one
daughter cell commonly fails at its new end and the other daughter
cell fails at its old end (Figure 2D) [8,22,23], fic1D cells were
Figure 1. Loss of the cytokinesis protein Fic1 causes defects in S. pombe growth polarity. (A) Live-cell images of calcofluor-stained wild-
type and fic1D cells. Birth scars remain unstained and appear as dark bands across cells. Arrowheads indicate monopolar cells, i.e. cells that have only
grown at one end, with birth scars abutting cell ends. (B) Quantification of (A), with three trials per genotype and n.300 for each trial. Data are
presented as mean 6 SEM for each category. (C) Quantification of septated cells in (A) and (B), with three trials per genotype and n.200 for each trial.
Data are presented as mean 6 SEM for each category. (D) Live-cell GFP images of crn1-GFP and fic1D crn1-GFP cells. (E) Quantification of (D), with
three trials per genotype and n.200 for each trial. Data are presented as mean 6 SEM for each category. (F) Live-cell GFP (in green) and RFP (in
magenta) merged images of rgf1-GFP sid4-RFP and fic1D rgf1-GFP sid4-RFP cells. (G) Quantification of (F), with three trials per genotype and n.200
for each trial. Data are presented as mean 6 SEM for each category (Bars=5 mm).
Control of Growth Polarity by Cytokinesis
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paper towel. These methods were established in previous studies
To assay whether specific mutants rescued invasiveness of an
asp1D strain on 0.3% agar , 1 ml containing 106cells was
spotted on 0.3% YE agar as well as onto 2% agar as a control.
Plates were incubated at 29uC for 12 days, at which point colony
growth and/or biofilm formation were visualized.
Schematic of phenotypes scored by calcofluor staining. Black
bands represent birth scars. (B) Live-cell bright field (BF) and GFP
images of tea1-GFP, tea4-GFP, fic1D tea1-GFP, and fic1D tea4-GFP
cells. (C) Quantification of (B), with three trials per genotype and
n.200 for each trial. Data are presented as mean 6 SEM for each
category. (D) Live-cell BF, GFP (in green), RFP (in magenta), and
GFP/RFP merged images of rgf1-GFP crn1-RFP and fic1D rgf1-
GFP crn1-GFP cells. (E) Live cell calcofluor (in magenta), GFP (in
green), and calcofluor/GFP merged images of a calcofluor-stained
fic1D rgf1-GFP cell. (F) Serial 10-fold dilutions of cells of the
indicated genotypes. Cells were spotted on YE agar and incubated
at 25uC, 29uC, 32uC, or 36uC. In the upper panel, all cells were
spotted on the same plate for each temperature, though some
intervening rows were removed in the figure presentation
Polarity and cytoskeletal defects of fic1D cells. (A)
analysis. Lysates from cells of the indicated genotypes were blotted
with an anti-GFP antibody, as well as with anti-Cdc2 as a loading
Fragments of Fic1 used for structure-function
cation of monopolar Crn1-GFP in cdc15DSH3 crn1-GFP and
imp2D crn1-GFP cells that were non-dividing or had only one
division site. Three trials were performed per genotype, with
n.100 for each trial. Data are presented as mean 6 SEM. (B)
Live-cell GFP images of cdc15DSH3 crn1-GFP and imp2D crn1-
GFP cells scored in (A). Cells with monopolar Crn1-GFP are
outlined with magenta dotted lines. (C) Schematic of Fic1
protein domain organization, with residues of interest marked,
PxxP motifs (*) numbered, the region responsible for Cdc15
binding  indicated, and the sequence spanning the terminal
two PxxPs given. (D) Yeast two-hybrid identification of the
Cdc15 binding site on Fic1. S. cerevisiae strain PJ69-4A was co-
transformed with bait and prey plasmids, which were either
empty or expressed mutants/regions of Fic1 or Cdc15. P253A
and P257A mutations were used to distinguish between PxxPs
10 and 11 as the motif responsible for Cdc15 binding. Two-
hybrid interaction was judged by growth of transformants
carrying both plasmids on selective media lacking histidine and
adenine (-His, -Ade). None of the prey plasmids transactivated.
(E) Live-cell bright field (BF), GFP (colored green), mCherry
(mCh) (colored magenta), and GFP/mCh merged images of a
fic1-GFP cdc15-mCherry interphase cell tip. (F) Live-cell BF and
GFP images of fic1-P257A-GFP cells. Arrowheads mark Fic1 in
CRs. (G) Yeast two-hybrid identification of Fic1 binding to
Cyk3’s SH3 domain. S. cerevisiae strain PJ69-4A was co-
transformed with bait and prey plasmids, which were either
empty or expressed Fic1 or Cyk3’s SH3 domain. Two-hybrid
interaction was judged by growth of transformants carrying both
plasmids on selective media lacking histidine and adenine (-His,
-Ade). None of the prey plasmids transactivated. (H) Live-cell
Analysis of Fic1-interacting proteins. (A) Quantifi-
BF and GFP images of fic1-P257A-GFP imp2D cyk3D cells.
Arrowheads mark CR localization (Bars=5 mm, except for S3E
where Bar=2 mm).
Serial 10-fold dilutions of cells of the indicated genotypes. Cells
were spotted on YE agar plates that were incubated at 25uC,
27uC, 29uC, or 32uC. Mutation of cdc16 causes SIN hyper-
activation, whereas mutants of spg1, cdc7, or sid2 exhibit loss of
SIN function. fic1D was previously shown to be synthetically
lethal with sid2-250 . All cells were spotted on the same
plate for each temperature, though some intervening rows were
removed in the figure presentation. (B) fic1D and spg1-106 were
mated, and tetrads were pulled on YE agar at 25uC. Genotypes
were assessed by replica plating to YE agar at 36uC and to
minimal medium lacking uracil. Images of colonies from a
tetratype are also given. (C) Fixed-cell GFP images of G2-
arrested cdc25-22 fic1D cells expressing acyl-GFP. Enlarged
images of cells’ division planes are also given. (D) Live-cell DIC,
GFP (colored green), mCherry (mCh) (colored magenta), and
GFP/mCh merged images of a fic1D rlc1-mCherry3 cell
expressing LifeAct-GFP. Images are single z-planes. The solid
white arrow in the GFP image indicates the division plane
(which entirely lacks GFP signal), and the dashed white arrow in
the GFP image indicates an abnormal actin mass flanking the
division plane. (E) Live-cell bright field (BF) and GFP movies of
eng1-GFP and fic1D eng1-GFP cells, with images acquired every
3 min. Representative images are shown for different times.
Yellow arrows denote Eng1-GFP at the division site. (F)
Quantification of times from ingression to Eng1-GFP disap-
pearance from the division plane in movies scored in (E), with
n.15 for each genotype. Data are presented in box-and-whisker
plots showing the median (line in the box), 25th–75thpercentiles
(box), and 5th–95thpercentiles (whiskers) for each genotype
(Bars=5 mm, except for enlarged regions in S4C where
Analysis of cytokinesis defects of fic1D cells. (A)
mutants. (A) Live-cell images of calcofluor-stained cells of the
indicated genotypes (scored in Figure 6C–6D). Arrowheads
indicate monopolar cells. For cells just completing division,
daughter cells were scored as monopolar as long as ingression of
the mother cell had progressed to such a degree that birth scars
could be easily identified at new ends. (B) Quantification of times
from septum splitting to initiation of growth at new ends in cells
of the indicated genotypes that undergo NETO prior to the next
septation in Figure 6A–6B. Data are presented as mean 6 SEM
for each genotype. (C) Live-cell DIC and GFP images of cps1-191
cells expressing acyl-GFP. Cells were shifted to 36uC for 3 h
before imaging. Arrows indicate membrane bridges linking
daughter cells. (D) Table of negative genetic interactions between
deletion of genes encoding ESCRT-related proteins (ESCRT-III
deubiquitinase Sst2) and deletion/loss-of-function alleles of genes
encoding cytokinesis factors (Imp2, myosin Myo2, b-glucan
synthase Cps1, SIN GTPase Spg1, SIN kinase Sid2, and formin
Cdc12) (Bars=5 mm).
Polarity and cytokinesis defects of late cytokinesis
images of calcofluor-stained cdc10-V50 and cdc10-V50 tea1-for3
cells arrested in G1. Arrowheads indicate monopolar cells. (B)
Quantification of (A), with three trials per genotype and n.300 for
Premature NETO in tea1-for3 cells. (A) Live-cell
Control of Growth Polarity by Cytokinesis
PLOS Genetics | www.plosgenetics.org 18October 2012 | Volume 8 | Issue 10 | e1003004
each trial. Data are presented as mean 6 SEM for each category
defects. (A) Images of pseudohyphae for strains of the indicated
genotypes in 2% agar. (B) Invasive growth assay for tea1;D agar.
Cells were spotted on rich medium and incubated for 20 days at
29uC (top panel). Colonies were then rinsed under a stream of
water and rubbed off (bottom panel). (C) Quantification of
pseudohyphae in (B), with n$3 for each genotype. Data are
presented as mean 6 SEM for each genotype. Data for wild-type
and fic1D strains are included for comparison. (D) Image of tea1;D
agar. (E) Image of tea1-for3 fic1D pseudohyphae in 2% agar
Pseudohyphae of mutants with growth polarity
S. pombe strains used in this study.
We thank Drs. Mohan Balasubramanian, Fred Chang, Paul Nurse, Shelley
Sazer, and Kaoru Takegawa for strains and reagents used in this study. We
also thank Fulvia Ferde and Maitreyi Das for valuable suggestions and
members of the Gould laboratory for helpful discussions and critical
reading of this manuscript.
Conceived and designed the experiments: KAB KLG. Performed the
experiments: KAB. Analyzed the data: KAB KLG. Wrote the paper: KAB
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