Meiotic actin rings are essential for proper sporulation in fission yeastHongyan Yan and Mohan K. Balasubramanian

Journal of Cell Science 125, 2544� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.113316

There was an error published in J. Cell Sci. 125, 1429-1439.

The doi number for this paper did not match the doi number of the version published as an Advance Online Publication.

The correct doi number should be 10.1242/jcs.jcs091561.

We apologise for this error.

2544 Erratum

Meiotic actin rings are essential for proper sporulationin fission yeast

Hongyan Yan1,2 and Mohan K. Balasubramanian1,2,3,*1Cell Division Laboratory, Temasek Life Sciences Laboratory, 2Department of Biological Sciences, 3Mechanobiology Institute, 1 Research Link,National University of Singapore, 117604, Singapore

*Author for correspondence (mohan@tll.org.sg)

Accepted 17 August 2011Journal of Cell Science 125, 1429–1439� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.091561

SummarySporulation is a unique form of cytokinesis that occurs following meiosis II in many yeasts, during which four daughter cells (spores) are

generated within a single mother cell. Here we characterize the role of F-actin in the process of sporulation in the fission yeastSchizosaccharomyces pombe. As shown previously, we find that F-actin assembles into four ring structures per ascus, referred to as themeiotic actin ring (MeiAR). The actin nucleators Arp2/3 and formin For3 assemble into ring structures that overlap with Meu14, a

protein known to assemble into the so-called leading edge, a ring structure that is known to guide forespore membrane assembly.Interestingly, F-actin makes rings that occupy a larger region behind the leading edge ring. Time-lapse microscopy showed that theMeiAR assembles near the spindle pole bodies and undergoes an expansion in diameter during the early stages of meiosis II, followed byclosure in later stages of meiosis II. MeiAR closure completes the process of forespore membrane assembly. Loss of the MeiAR leads to

excessive assembly of forespore membranes with a deformed appearance. The rate of closure of the MeiAR is dictated by the function ofthe septation initiation network (SIN). We conclude that the MeiAR ensures proper targeting of the membrane biogenesis machinery tothe leading edge, thereby ensuring the formation of spherical spores.

Key words: Fission yeast, Meiotic actin ring, SIN, Cytokinesis

IntroductionIn many organisms, cytokinesis is achieved through the use of an

actomyosin ring containing F-actin, type II myosin and several

other proteins (Balasubramanian et al., 2004; Fujiwara and

Pollard, 1976; Fujiwara et al., 1978; Glotzer, 2001; Glotzer,

2005; Schroeder, 1968; Schroeder, 1973). The contraction of

actomyosin rings is presumed to guide membrane assembly

during cytokinesis (Dobbelaere and Barral, 2004). In recent

years, the fission yeast Schizosaccharomyces pombe has been

used as a model organism for the study of cytokinesis, because of

the ease with which genetic and cell biological analysis are

applied in this organism (Gould and Simanis, 1997; McCollum

and Gould, 2001; Wu and Pollard, 2005; Wu et al., 2006). During

mitosis, fission yeast cells divide through the use of an

actomyosin-based contractile ring (Balasubramanian et al.,

1992; Balasubramanian et al., 1994; Balasubramanian et al.,

1998; Chang et al., 1997; Eng et al., 1998; Fankhauser

et al., 1995; Le Goff et al., 2000; Marks et al., 1986;

McCollum et al., 1995; Naqvi et al., 2000; Wong et al., 2000;

Wu et al., 2003). The actomyosin ring is assembled at the onset

of mitosis and constricts concomitant with the centripetal

addition of new membranes and the division septum at the end

of mitosis (Balasubramanian et al., 2004; Wu and Pollard, 2005;

Wu et al., 2006).

In fission yeast, contraction of the actomyosin ring, as well as

the formation of new membrane and the division septum, are

regulated by a signaling cascade termed the septation initiation

network (SIN) (McCollum and Gould, 2001; Simanis, 2003); the

functional equivalent in budding yeast is called the mitotic exit

network (MEN) (Balasubramanian et al., 2004; Bardin and

Amon, 2001). The SIN is a GTPase-regulated protein kinase

cascade consisting of three protein kinases (Cdc7, Sid1 and Sid2)

and one small GTPase, Spg1 (Fankhauser and Simanis, 1994;

Guertin et al., 2000; Hou et al., 2000; Schmidt et al., 1997;

Sparks et al., 1999). Mob1 and Sid2 form a complex and are

downstream of the SIN pathway (Balasubramanian et al., 2004;

Hou et al., 2000). Cdc16 and Byr4 function as part of a two-

component GTPase-activating protein (GAP) that inhibits Spg1

(Furge et al., 1998; Furge et al., 1999; Li et al., 2000). All of the

SIN components seem to be anchored to the spindle pole body

(SPB) through the SIN scaffold complex containing Sid4 and

Cdc11 (Krapp et al., 2001; Li et al., 2000; Tomlin et al., 2002).

Interestingly, during meiosis, a different type of cytokinesis

regulates the formation of gametes (spores) in S. pombe and

several other yeasts and fungi (Neiman, 2005; Shimoda, 2004).

An extensive body of genetic and genomic approaches has led to

the identification of the SPO (sporulation defective) and MEU

(meiotic expression upregulated) genes, products of which have

been shown to be important for forespore membrane assembly

and proper sporulation (Hirata and Shimoda, 1992; Yamamoto,

1996). Characterization of the MEU gene products have revealed

an important role for the leading edge proteins (LEPs), such as

Meu14, which assembles into a pair of rings per mitotic spindle

and guides forespore membrane assembly (Okuzaki et al., 2003).

In addition, analysis of many of the SPO and MEU gene products

has led to the identification of elements of the membrane

trafficking machinery, such as v-SNAREs and t-SNAREs

that specifically function during forespore membrane assembly

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(Edamatsu and Toyoshima, 2003; Gotte and von Mollard, 1998;Nakamura et al., 2001; Nakamura et al., 2005). In addition, theactin cytoskeleton and some of its regulators have also been

shown to assemble in the vicinity of the LEP ring, suggestinga role for F-actin in forespore membrane assembly and/orsporulation (Itadani et al., 2006; Toya et al., 2001).

Recent studies have shown that, as in mitosis, sporulation is

dependent on the SIN. SIN proteins localize to SPBs duringmeiosis and the pathway is activated during the second meioticdivision. In some SIN mutant cells, the forespore membrane(FSM) assembly is defective, resulting in unencapsulated nuclei

(Krapp et al., 2006; Ohtaka et al., 2008; Perez–Hidalgo et al., 2008;Yan et al., 2008). Although SIN has been analyzed, it is not fullyunderstood how F-actin participates in this process and whether

SIN regulates actin cytoskeletal function during meiosis.

In this study, we have characterized the dynamics and function ofthe actin cytoskeleton during meiosis and sporulation. Wecharacterize the meiotic actin ring (MeiAR), which contains F-

actin and its nucleators, and appears to participate in properassembly of the forespore membrane. We also report that the rate ofclosure of the meiotic actin ring is strongly influenced by the SIN.

ResultsF-actin assembles into ring structures during sporulation

Previous studies have shown that F-actin assembles into ring-like

structures during meiosis II in fission yeast (Itadani et al., 2006;Ohtaka et al., 2007; Petersen et al., 1998). Consistent with this,we found that F-actin assembles into rings in meiosis II

(Fig. 1A). Staining of fixed wild-type cells with FITC–phalloidin and DAPI revealed a striking array of F-actinrearrangements. In cells undergoing horsetail movement

(Fig. 1A, cell 1) of chromosomes, F-actin was detected inpatches throughout the forming ascus. Similarly, F-actin wasdetected in patches in cells that had completed meiosis I

(Fig. 1A, cells 2 and 3). Around the time of initiation ofchromosome segregation in meiosis II (Fig. 1A, cells 4 and 5), F-actin started to accumulate near the nuclei. In cells in whichchromosome segregation had initiated during meiosis II (Fig. 1A,

cells 6 and 7), F-actin organization was apparent as four ringstructures, which we term meiotic actin rings (MeiARs).Following full segregation of chromosomes (Fig. 1A, cells 8–

12) the diameter of the F-actin rings decreased. In cells that hadcompleted sporulation (Fig. 1A, cell 13), F-actin was detected inrandomly organized patches.

Because previous studies had not investigated the dynamics of

F-actin during sporulation, we used green fluorescent protein(GFP) fused to the calponin homology domain (CHD) of Rng2 todetect the dynamics of F-actin ring assembly (Karagiannis et al.,2005). The GFP–CHD fusion, which binds to all F-actin

structures, has been used previously to study the dynamicsof F-actin in the mitotic cell cycle (Karagiannis et al., 2005).We also performed live-cell imaging to analyze the spatial

relationship between F-actin and Meu14 (a marker of the leadingedge) during sporulation. Before meiosis II, GFP–CHD patcheswere randomly distributed in the cytoplasm (Fig. 1B, 20 minutes,

–3 minutes). At metaphase II, the number of GFP–CHD patchesin the cytoplasm gradually decreased, whereas bright GFP–CHDsignals started to accumulate at each of the four SPBs (Fig. 1B,

Fig. 1C, visualized with the nuclear envelope marker Cut11–mCherry). Subsequently, the four bright GFP–CHD dots eachformed a donut-like structure, indicative of the initiation of

assembly of the FSM (Fig. 1B). During this stage, the size of the

GFP–CHD rings began to expand to the maximum diameter(Fig. 1B) (0–20.5 minutes) and GFP–CHD overlapped with the

leading edge ring protein marker Meu14–mCherry (Fig. 1C, 0–20 minutes, bottom panel). However, after anaphase II (Fig. 1C),

the GFP–CHD rings started to shrink in diameter until theydisappeared completely, which indicates closure of the FSMs

(Fig. 1B, 20.5–58.5 minutes and 1C, 24–66 minutes). During thisshrinking stage, the fluorescence of the ring-like structure of

GFP–CHD was rather broad and followed the leading edge

Meu14 rings (Fig. 1C, right panel). Finally, the GFP–CHDdiffused into each newly formed spore (Fig. 1B, 61.5 minutes

and 1C, 72 minutes). The results are shown schematically(Fig. 1D). These studies established that F-actin assembled into

rings that follow the leading edge during FSM assembly.

Actin nucleators and actin interactors localize to the MeiAR

Assembly of actin filaments from their monomeric subunitsrequires the actin nucleators Arp2–Arp3 (Arp2/3) and formins

(Baum and Kunda, 2005; Campellone and Welch, 2010;Chesarone and Goode, 2009). The Arp2/3 complex is known to

nucleate and organize actin into a dense network of short, branchedactin filaments (Goley and Welch, 2006). By contrast, formins are

required for unbranched actin cable formation during interphase

and mitosis (Chang et al., 1997; Evangelista et al., 2003; Feierbachand Chang, 2001; Pruyne et al., 2002). The mYFP–Arp2/3

complex (Wu and Pollard, 2005) colocalized with Meu14–mCherry at the leading edge throughout the entire process of

FSM assembly (Fig. 2A). For3–GFP also colocalized andconstricted in concert with the Meu14–mCherry rings (Fig. 2A).

The type I myosin Myo1 is known to recruit the Arp2/3 complex,

and is involved in actin polymerization during mitosis (Itadaniet al., 2006; Lee et al., 2000). Consistently, Myo1 was also

detected at the leading edge (Fig. 2B). We noticed that asignificant fraction of F-actin in the MeiAR trailed the leading

edge (Fig. 2A–C). These experiments suggested that F-actinpolymerized at the leading edge potentially through the action of

the nucleators such as Arp2/3 complex and the formin For3.

We then tested whether other known actin cytoskeletal

regulators and cytokinetic proteins localized to the MeiAR. Ofthe proteins tested, we found that the fimbrin Fim1 (Nakano et al.,

2001; Wu et al., 2001) and Sla2 (an actin-patch protein related to

budding yeast End4) (Castagnetti et al., 2005; Ge et al., 2005;Gourlay et al., 2003; Holtzman et al., 1993) localized to the

MeiAR (Fig. 2D). Interestingly, the myosin regulatory lightchain Rlc1 was also detected at the MeiAR (Fig. 2E), although

other actomyosin ring components, such as Rng2–IQGAP, the F-BAR protein Cdc15, the UCS protein Rng3 and the formin Cdc12

were not readily detected at the MeiAR.

For3 and Arp2/3 are important for MeiAR assembly andfunction

We then set out to understand the role of the several of the

components of the MeiAR in forespore membrane assembly andsporulation. To this end, we first tested the role of For3 in

sporulation. Whereas wild-type cells assembled spherical sporesof roughly equal size (Fig. 3A), for3D cells generated spores with

an abnormal morphology and size (Fig. 3A). These effects weremore obvious when GFP–Psy1 was used as a marker for spore

membranes (Fig. 3A, bottom panels). In this analysis we found

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that whereas wild-type spores had smooth membranes underlying

the spherical spores, for3D spores were of abnormal morphology

and had small protrusions of membranous material (Fig. 3A,B).

Time-lapse microscopy showed that the protrusions of

membranous material arose around the end of forespore

membrane assembly (Fig. 3C).

To test whether the observed effects were related to MeiAR

assembly and function defects in for3D cells, we imaged the

Fig. 1. F-actin dynamics during meiosis in S. pombe. (A) F-actin localization during various stages of sexual differentiation in representative cells. F-actin was

stained with phalloidin (green) and DNA was stained with DAPI (red) in fixed wild-type cells (MBY1238) undergoing meiosis and sporulation. (B) Time-lapse

images showing the dynamics of GFP–CHD in cells undergoing FSM formation. Images were taken using a LSM510 Meta inverted confocal microscope.

(C) Time-lapse images showing the dynamics of GFP–CHD (green), Cut11–mCherry (red, marked the nuclear envelope and spindle pole body, SPB) and Meu14–

mCherry (red, leading-edge protein) in cells (MBY5998) undergoing FSM formation. Images (top panel) were captured every 6 minutes. The images in the

bottom panel were captured every 2 minutes. Arrows show the localization of Cut11–mCherry at the SPBs during meiosis II. The time, with 0 minute being the

time at which FSM formation begins, is indicated at the bottom of each image. Enlarged image (right panel) shows that GFP–CHD colocalized with Meu14–

mCherry at the leading edge of FSM, but GFP–CHD was rather broad and positioned slightly behind the Meu14 rings at late meiosis II. Images were taken using a

spinning-disk confocal microscope. Scale bars: 10 mm. (D) The schematic representation shows the dynamics of CHD (green), Cut11 (pink), Meu14 (red) and

nuclei (blue) in cells during the FSM formation.

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Fig. 2. Localization of actin nucleators and interactors to the leading edge during FSM formation in S. pombe. (A) Time-lapse images showing the

colocalization of For3–GFP (green) and Meu14–mCherry (red) (top panel, MBY6237), mYFP–Arp3 (green) and Meu14–mCherry (red) (middle panel,

MBY6228) and mYFP–Arp2 (green) and Meu14–mCherry (red) (bottom panel, MBY6206) in cells undergoing FSM formation. Enlarged images (right panels)

show that mYFP–Arp2, mYFP–Arp3 and For3–GFP colocalized with Meu14–mCherry at the leading edge of FSM at late meiosis II, respectively. (B) Time-lapse

images showing the colocalization of Myo1–GFP (green) with Meu14–mCherry (red) at the leading edge in cells (MBY6048) undergoing FSM assembly.

Enlarged image (right panel) shows that Myo1–GFP colocalized with Meu14–mCherry at the leading edge of FSM at late meiosis II. (C) Time-lapse images

showing the colocalization of GFP–CHD (green) with Arp3–mCherry (red) at the leading edge of FSM in cells (MBY6241) undergoing FSM assembly. Enlarged

image (right panel) shows that GFP–CHD colocalized with Arp3p–mCherry at the leading edge of FSM, but GFP–CHD signal was broader and positioned slightly

behind the Arp3–mCherry rings at late meiosis II. The time, with 0 minute being the time at which FSM formation begins, is indicated at the bottom of each

image. (D) Time-lapse images showing the colocalization of Fim1–GFP (green) with Meu14–mCherry (red) (top panel, MBY7589), Sla2–GFP (green) and

Meu14–mCherry (red) (bottom panel, MBY7591) at the leading edge in cells undergoing FSM assembly. Enlarged images (right panel) show that Fim1–GFP,

Sla2–GFP colocalized with Meu14–mCherry at the leading edge of FSM at late meiosis II. Images were taken using an inverted confocal microscope except that

For3–GFP image in A (bottom panel), Fim1–GFP and Sla2–GFP images in D were taken using a spinning-disk confocal microscope. (E) Rlc1–3GFP colocalizes

with Meu14–mCherry to the leading edge in the meiotic cells (MBY7609). Arrows show the meiotic cells. Images were taken using an Olympus microscope.

Scale bars: 10 mm.

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behaviour of F-actin in wild-type and for3D cells undergoing

meiosis and sporulation. As described earlier (Fig. 1B) GFP–

CHD assembled into MeiARs that underwent normal assembly

and constriction (Fig. 3D). F-actin cables were also detected in

wild-type cells (Fig. 3D, arrows). By contrast, MeiARs were

significantly smaller in diameter in for3D cells (Fig. 3D,E) and

Fig. 3. For3 is required for proper sporulation. (A) Abnormally shaped spores and small protrusions of membranous material were observed in for3D spores.

Wild-type (MBY3664) and for3D cells (MBY7583) expressing GFP–Psy1 were induced to undergo meiosis at 31 C̊ for 2 days. Arrows show the protrusions of

GFP–Psy1 in asci. (B) Quantification of GFP–Psy1 morphology in wild-type (MBY3664) and for3D asci (MBY7583). Cells were induced to undergo meiosis on

YPD plates at 31 C̊ for 24 hours. n$500 for each genotype. (C) Time-lapse microscopy of GFP–Psy1 in for3D (MBY7583) shows that the protrusions of

membranous material arise around the end of FSM assembly. Images were taken at 31 C̊. The GFP–Psy1 formed cup-shaped and round FSMs (0–18 minutes) but

failed to terminate the assembly of FSMs (24–96 minutes), resulting in the protrusion of membranous material around the end of the FSM assembly

(102 minutes). Arrows show the protrusions of GFP–Psy1. Images were taken using a spinning-disk confocal microscope. (D) Time-lapse images showing the

dynamics of GFP–CHD in wild-type (MBY5998) and for3D cells (MBY6030) undergoing meiosis and sporulation. Arrows show F-actin cables detected during

FSM formation in the wild-type cell. By contrast, actin cables were undetectable in for3D cells. Images were taken using a spinning-disk confocal microscope.

(E) Comparison of the maximal diameter of GFP–CHD rings between wild-type (MBY5998) and for3D (MBY6030) cells. The maximum diameter of GFP–CHD

rings was measured by MetaMorph software. n$60 for each genotype. Means and s.d. are represented. (F) Graph shows the duration of the two phases of GFP–

CHD assembly and disassembly depicted in the upper panel of F in wild-type (MBY5998) and for3D (MBY6030) cells. The MeiAR formation process was

divided into two phases as follows: Phase I, from the initiation of MeiAR formation to the time at which the diameter of the MeiAR is maximal; Phase II, the time

at which the large MeiAR begins to reduce in size until MeiAR forms a dot (#1 mm). The depictions indicate the dynamics of GFP–CHD rings (green) and

Cut11–mCherry (black) during FSM formation. n$30 for each genotype. Means and s.d. are represented. Scale bars: 10 mm.

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actin cables were undetectable (Fig. 3D). In addition, the timetaken for the assembly and disassembly of MeiARs was

significantly increased in for3D cells (Fig. 3F, assembly isindicated as Phase I and disassembly is indicated as Phase II).These experiments established that For3 function was important,

although not essential, for aspects of MeiAR assembly, functionand proper sporulation.

Having established that For3 was important for MeiAR

function, we investigated whether the Arp2/3 complex wasimportant for MeiAR function and/or sporulation. To address thisquestion we tested the ability of arp3-c1 cells to sporulate at

the restrictive temperature of 19 C̊ (McCollum et al., 1996).Although arp3-c1 cells were capable of performing meiosis I andII, as evidenced from the presence of tetranucleated asci, sporeswere not detected in these asci (Fig. 4A). Unlike in wild-type

cells (Fig. 1A), F-actin was not observed in MeiARs, establishingthat Arp2/3 complex was also important for MeiAR assemblyand/or its maintenance. Collectively, these experiments establish

that actin is polymerized at the leading edge of the MeiAR andthat the nucleators and F-actin at the MeiAR participate in propersporulation.

Because the regulatory light chain for myosin II, Rlc1, wasdetected at the MeiAR, we addressed whether myosin II mightpower constriction of the MeiAR. Rlc1 is known to interact with

two myosin II heavy chains, Myo2 and Myp2 (Le Goff et al.,2000; Motegi et al., 2004; Naqvi et al., 2000). Meiotic cells of thetemperature sensitive myo2-E1 mutant and the myp2D null

mutant were able to assemble spores of normal appearance(Fig. 4B,C,D). Owing to the synthetic lethality of the myo2-E1myp2D double mutants, we have been unable to investigatepotential sporulation defects in these double mutants. Our studies

therefore established that Myo2 and Myp2 were individuallydispensable for sporulation.

F-actin is required for coordination of FSM assembly withprogression of the leading edge

To further investigate the role of F-actin in sporulation, wetreated cells with Latrunculin A (Lat A) to prevent actinpolymerization (Ayscough et al., 1997) and carried out time–

lapse analysis during sporulation. We compared themorphologies of the FSM and the nucleus in DMSO and Lat Atreated cells by live examination of the dynamics of GFP–Psy1,Meu14–mCherry and mCherry–Atb2. Psy1 is a fission yeast

homolog of mammalian syntaxin 1A, which is a t–SNARE(soluble NSF attachment protein receptor) protein involved inmembrane assembly (Maeda et al., 2009; Nakamura et al., 2001;

Nakamura et al., 2008; Shimoda, 2004). Psy1 disappears from theplasma membrane upon exit from meiosis I and reappears in thenascent FSMs (Maeda et al., 2009; Nakamura et al., 2001;

Nakamura et al., 2008). Thus, monitoring the movement of GFP–Psy1 allowed us to visualize the process of FSM formation.Observation of the mCherry–Atb2 allowed us to monitor the

progression through the meiotic cell cycle.

First, we established that Lat A treatment led to a completedispersal of actin nucleators For3 and Arp2/3 from the leading

edge as well as the loss of all detectable F-actin and the MeiAR(supplementary material Fig. S1). Time-lapse movies showedthat, in comparison to DMSO-treated cells, the localization of

GFP–Psy1 was significantly different in cells treated with Lat A.DMSO-treated cells developed FSMs marked by Psy1 andformed four nucleated forepores (Fig. 5A). In cells undergoing

meiosis II, treatment with Lat A led to excessive proliferation

of FSMs whose geometry was that of abnormal spheres in

which several bubble-like outgrowths were noticed (Fig. 5B).

Interestingly, these bubble-like structures were visualized just at

the point of the closure of FSM (Fig. 5B, arrows). The bubble

structures extended to form long tubes and stopped growing at

around the time spore walls would have been deposited. Normal

meiotic spindles were detected in Lat-A-treated cells, ruling out

the possibility that improper FSM growth was a consequence

of abnormal mitosis (Fig. 5B). To understand the detailed

morphology of the bubble structures, we examined them by

electron microscopy (EM). EM observation in wild-type cells

revealed four normal spores that encapsulate nuclei, rough

endoplasmic reticulum, Golgi complexes and mature spore walls

Fig. 4. Arp2/3 complex, but not type II myosin, is important for MeiAR

function during sporulation. (A) Sporulation is defective in the arp3-c1

cells (MBY7615) at the restrictive temperature of 19 C̊. Fresh arp3-c1

homothallic h90 cells were induced to form zygotes on YPD solid plates at

24 C̊ overnight and shifted to 19 C̊ for 2 days. Cells were fixed with

paraformaldehyde and stained with Alexa Fluor 488 phalloidin (green) and

DAPI (red). Arrows indicate the tetranucleated asci. Spores were not detected

in these asci. F-actin was observed in randomly organized clusters.

(B) Normal spores are formed in myo2-E1 mutant cells (MBY7620). Fresh

myo2-E1 homothallic h90 cells were induced to undergo sporulation on YPD

solid plate at 34 C̊ for 2 days. Asci with four normal spores are shown.

(C) Quantification of spore morphology in wild-type (MBY3664) and myo2-

E1 asci (MBY7620). Cells were induced to undergo meiosis on YPD plates at

34 C̊ for 2 days. n$500 for each genotype. (D) Normal spores are formed in

myp2D null mutant cells (MBY7758). Cells were induced to undergo meiosis

on YPD plates at 30 C̊ for 2 days. Scale bars: 10 mm.

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(Fig. 5C, i and iv). By contrast, in Lat-A-treated cells, many of

the bubbled spores containing haploid nuclei retained a canal-like

passage of cytoplasm between the mother and daughter spores

(Fig. 5D, ii) and displayed ruffled, seemingly fragile spore walls

(Fig. 5D, iii). Moreover, the fragile spore walls did not stain with

Anillin Blue, implying defects in spore wall formation and/or

maturation (data not shown). All these experiments established

that a key role of MeiAR during meiosis II is to coordinate the

FSM assembly with the progression of the leading edge.

Loss of SIN affects MeiAR dynamics

During cytokinesis coupled with progression of mitosis in

vegetative cells, the SIN is required for actomysin ring

maintenance, new membrane formation and cell wall

assembly (Balasubramanian et al., 2004; Krapp et al., 2004;

Krapp and Simanis, 2008; McCollum and Gould, 2001). Given

that the maintenance and constriction of the actomyosin ring in

mitosis depended on SIN, we assessed whether the rate of

progression or constriction of the MeiAR depended on the SIN.

Fig. 5. Actin is required for proper spore morphology in S. pombe. (A) Time-lapse images showing the dynamics of GFP–Psy1 (green), Meu14–mCherry (red)

and mCherry–Atb2 (red) during meiosis I, meiosis II and sporulation in cells (MBY6197) treated with DMSO. GFP–Psy1 signals were first observed at the plasma

membrane of the mother cell before the completion of meiosis I (0–61 minutes); the signals then preferentially diffused into the ascus before meiosis II

(79–90 minutes); subsequently, the GFP–Psy1 signals accumulated to form the cup–shaped FSMs during meiosis II (103–160 minutes). A closed compartment of the

FSM containing a single nucleus was eventually formed (229 minutes). (B) F-actin is required for the termination of FSM assembly and spore morphology. Time-

lapse images showing the dynamics of GFP–Psy1 (green), Meu14–mCherry (red) and mCherry–Atb2 (red) in cells (MBY6197) treated with 100 mM Lat A before the

initiation of meiosis II (top panel) and after the initiation of meiosis II (bottom panel). Images in top panel were taken at the initiation of meiosis II (0 minute).

The GFP–Psy1 formed cup-shaped and round FSMs (0–78 minutes) but failed in terminating the assembly of FSMs (105–276 minutes), resulting in the tubule-like

FSMs (105 minutes, white arrows). Images in bottom panel were taken after anaphase II. The GFP–Psy1 formed tubule-like FSMs (48–99 minutes, white arrows).

Images were taken using a spinning-disk confocal microscope. Scale bars: 10 mm in A,B. (C) EM images of FSMs (i–iii) and spores (iv–vi) in wild-type cells. (D) EM

images of bubbled FSMs and tubule shaped immature spore wall in wild-type cells treated with 100 mM Lat A. ii and iii are enlarged views of the indicated regions

show abnormal tubule-shaped FSMs and spore wall. N, nuclei. White arrow, bubbled FSM. Black arrow, tubule-like immature spore wall.

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To address this question, we made use of a strain in which the

gene encoding Sid4 was placed downstream of the promoter of

the rad21 gene, which is turned off during meiosis. In previous

work, we have shown that such cells assemble four abnormal

spores, two of which are larger and the other two smaller than

spores in wild-type cells (supplementary material Fig. S2) (Yan

et al., 2008). In fixed Prad21-sid4 cells, two of the four MeiARs

were larger than the other two in later stages of meiosis II

(Fig. 6A), consistent with a role for SIN in MeiAR progression

and constriction.

Fig. 6. SIN is required for MeiAR constriction. (A) F-actin localization during anaphase II in representative Prad21-sid4 cells (MBY3507). F-actin was stained

with phalloidin (green) and DNA was stained with DAPI (red) in fixed Prad21-sid4 cells. Two types of MeiARs with different diameters are shown: smaller, normal

constriction MeiARs (arrows) and broad, slow constriction MeiARs (asterisks). (B) Time-lapse images show the dynamics of GFP–CHD at the leading edge in

wild-type cells (top panel, MBY6896) and Prad21-sid4 cells (bottom panel, MBY6002). Top panel: each of the four MeiARs present in an ascus constricted with

similar kinetics in wild-type cells (arrows). Bottom panel: two of the MeiARs constricted with kinetics similar to wild-type cells (arrows), whereas the other two

were significantly slower (asterisks). The time, at which GFP–CHD ring (MeiAR) had the maximal diameter is indicated as 0 minute. (C) Graph shows the

quantification of the diameters of the GFP–CHD rings as a function of time in wild-type (MBY5998), Prad21-sid4 (normal MeiAR constriction, abnormal MeiAR

constriction) (MBY6002) and sid2-250 slk1D (MBY6346) cells from maximal diameters to the closure of the GFP–CHD rings. The diameter of GFP–CHD rings

was measured using MetaMorph software (n$30 for each genotype). Means and s.d. are represented. The depictions indicate the dynamics of GFP–CHD rings

(green) and Cut11–mCherry (black) during FSM formation in wild-type, Prad21-sid4 and sid2-250 slk1D cells. (D) Time-lapse images show the dynamics of Arp3–

mCherry and For3–GFP at the leading edge in Prad21-sid4 cells. Two types of MeiAR constriction are shown: two of the MeiARs in each genotype cell constricted

with kinetics similar to wild-type cells (arrows), whereas the other two were significantly slower (asterisks). Images were taken by a spinning disk confocal except

images in A were taken by an inverted confocal microscope. Scale bars, 10 mm.

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To understand the dynamics of the MeiAR in Prad21-sid4 cells,we carried out time-lapse imaging experiments. In wild-type

cells, each of the four MeiARs present in an ascus constrictedwith similar kinetics such that the closure of the MeiAR, fromapproximately a diameter of 3.1 mm to a diameter of under 1 mm

took roughly 21 minutes (Fig. 6B,C). By contrast, in Prad21-sid4

cells, two of the MeiARs constricted with kinetics similar towild-type cells, whereas the other two were significantly slower.The diameter of the two slower MeiARs only decreased to

approximately 1.6 mm in an even longer time frame (Fig. 6C).

In addition to the SIN scaffold protein mutants, we tested

whether downstream elements of the SIN were involved inregulating MeiAR dynamics. To this end, actin dynamics wastested in double mutants defective in the two SIN kinases Sid2and Slk1 that function at the end of the SIN cascade during

meiosis (Ohtaka et al., 2008; Perez-Hidalgo et al., 2008; Yanet al., 2008). A similar slow-down of MeiAR constriction wasalso detected in sid2-250 slk1D cells (Fig. 6C; supplementary

material Fig. S3). Collectively, the analysis of Prad21-sid4 andsid2-250 slk1D cells established that SIN function was importantfor proper closure of the MeiAR. Similar defects in MeiAR

closure rates were observed in Prad21-sid4 cells when Arp3 orFor3 were used as markers of the MeiAR (Fig. 6D).

DiscussionIn this study we have characterized the MeiAR, a ring structurecomposed of F-actin, its nucleators and binding proteins.

Previous studies have identified the presence of a ring-likestructure containing F-actin that assembles during sporulation,although the dynamics of this structure have not been studied in

detail (Itadani et al., 2006; Petersen et al., 1998). We haveconfirmed and extended these studies, through the dynamicanalysis of additional actin cytoskeletal proteins and the role of

the SIN.

We have shown that the MeiAR consists of among others, F-actin, its nucleators For3 and Arp2/3 and actin interactors Myo1,

Sla2, Fim1. In addition, previous work has shown that thecalmodulin-like protein Cam2 and Myo1 localize to the MeiAR(Itadani et al., 2006; Toya et al., 2001). Interestingly, although

For3 and Arp2/3 colocalize with the leading-edge protein Meu14,F-actin itself trails this structure. The presence of the nucleatingproteins at the leading edge suggests that the MeiAR might becomposed of de novo generated actin filaments rather than those

redistributed from other regions of the cell. The fact that For3 andArp2/3 localize to the ring suggests that linear actin cables andbranched actin filaments are nucleated in the vicinity of the

leading edge, and the assembled F-actin trails the leading edge.Detailed imaging using electron microscopy using myosin headS1 decoration will be necessary to establish the organization of

actin filaments in the MeiAR (Kamasaki et al., 2007). Becauseproteins such as Myo1 and Sla2 localize to the MeiAR, it is likelythat the MeiAR is also a site for endocytic events (Ge et al., 2005;

Toya et al., 2001). Myo1 has been shown to participate in actinpolymerization, through Arp2/3 and it is therefore also possiblethat Myo1 facilitates MeiAR assembly. The sporulation-defectivephenotype of myo1 mutants is consistent with both possible

molecular roles of Myo1.

We have shown that loss of function of For3 leads to defects in

the assembly, maintenance and constriction of the MeiAR, whichin turn generates spores of abnormal morphology. Because for3Dcells are also defective in actin cable (non-MeiAR) assembly, it is

possible that the loss of actin cables generates the sporulation

defective phenotype. We believe this is unlikely because many

properties of the MeiAR (such as time taken for assembly and

disassembly and the diameter of the ring) are disturbed in for3Dcells. Although we have been unable to carry out time-lapse

imaging experiments on arp3-c1 cells, as a result of technical

limitations, we have shown that loss of Arp2/3 complex function

leads to destabilization of the MeiAR and leads to sporulation

defects. Collectively, these experiments establish that linear actin

cables and branched actin filaments, generated at the MeiAR, are

important for proper sporulation.

We have shown that the regulatory light chain of myosin II,

Rlc1p, localizes to the MeiAR. Rlc1 is known to bind Myo2 and

Myp2, the two proteins related to myosin II heavy chains in

fission yeast (Le Goff et al., 2000; Motegi et al., 2000; Naqvi

et al., 2000). Cells defective individually for Myo2 and Myp2

function are able to generate normal spores, suggesting that

actomyosin contractility, as observed in mitotic cells, might not

operate during cytokinesis. However, we are unable to confirm

this effect because of the synthetic lethality observed in myo2-E1

myp2D double mutants. It is also possible that Rlc1p might

partner other myosins (rather than myosin II) to facilitate

sporulation.

What then is the function of the MeiAR? We have shown that

the loss of formin For3 leads to abnormally shaped spores, which

results from improper forespore membrane assembly. We have

also shown that arp3-c1 cells are defective in MeiAR assembly

and sporulation. Compelling evidence for the role of F-actin and

the MeiAR in sporulation was obtained by analyzing cells treated

with the actin polymerization inhibitor Lat A. In Lat-A-treated

cells, we noticed that FSM assembly was not arrested by

the absence of F-actin. Rather, we found that the integrity of

the leading edge ring (visualized by using Meu14) was

compromised, leading to excessive proliferation of disorganized

forespore membranes containing Psy1–syntaxin in cells devoid of

detectable F-actin. Thus the primary function of the MeiAR

might be to reinforce the organization and stability of the leading

edge ring.

Studies of the contractile actomyosin ring in mitotic cells have

shown that the stability of these rings and their constriction are

dependent on the SIN (Balasubramanian et al., 2004; Hachet and

Simanis, 2008; Mishra et al., 2004; Wu et al., 2006). Curiously,

we have found that the rate of migration of the leading edge was

significantly reduced in meiotic cells defective in SIN, leading to

abnormal FSM assembly and sporulation. These observations

reveal that despite the differential nature of cytokinesis in mitotic

and meiotic cells, SIN might play an important role in

coordinating membrane assembly events with actin cytoskeletal

dynamics. Future studies should address whether SIN kinases

phosphorylate related substrates in mitosis and meiosis to

coordinate membrane assembly with actin dynamics.

Materials and MethodsYeast strains, media and culture conditions

Fission yeast cells were grown on YES medium or minimal medium withappropriate supplements (Moreno et al., 1991). Yeast strains were constructedby either random spore germination method or by tetrad analysis. Growthtemperatures were 24 C̊ (permissive) for temperature-sensitive strains, and 30 C̊for all other strains. S. pombe cells were sporulated at 30 C̊, except fortemperature-sensitive strains which were sporulated at 24 C̊, 19 C̊ or 34 C̊ indifferent experiments. Solid yeast extract peptonedextrose (YPD) medium orEMM5S medium (for nmt1–gfp–chd) were used for inducing meiosis. Strains usedin this study are listed in supplementary material Table S1.

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F-actin and DAPI staining

Fission yeast cells, grown and induced to enter meiosis on solid YPD medium,were fixed with 4% paraformaldehyde at the growth temperature for 15 minutes.Fixed cells were then rinsed three times with phosphate-buffered saline (PBS). Tostain DNA and F-actin, fixed cells were permeabilized with 16 PBS, pH 7.4,solution containing 1% Triton X-100 for 5 minutes, followed by three washes with16 PBS. 1 mg/ml 49,6-diamidino-2-phenylindole (DAPI) was added to the cellsuspension to visualize the nuclei. Alexa-Fluor-488-conjugated phalloidin at afinal concentration of 100 ng/ml was used to stain F-actin.

Microscopy and data analysis

For time-lapse microscopy, cells were induced to undergo meiosis on solid YPDmedium or EMM5S (for strains containing nmt1–gfp–chd) for 16 hours at 28 C̊ or24 hours at the permissive temperature for temperature-sensitive strains. Cellswere spotted on a glass slide containing a 2% agar pad. For Latrunculin A (Lat A)treatment experiments, cells were treated with 100 mM Lat A or equal volume ofDMSO at room temperature for 20 minutes, and placed on EMM5S + 2% agarpads (containing 100 mM Lat A or DMSO). Time-lapse analysis was conducted atroom temperature (22–24 C̊).

We used a 1006/1.4 NA objective lens (Nikon, Melville, NY) on a spinningdisk confocal microscope (UltraView ERS, PerkinElmer Life And AnalyticalSciences, Waltham, MA) with 488 nm and 561 nm lasers and a cooled charge-coupled device camera (ORCA-AG, Hamamatsu, Bridgewater, NJ). Maximumintensity projections of color images were created using MetaMorph software.Images in figures are maximum intensity projections of Z-sections spaced at0.5 mm. For confocal microscopy (Zeiss LSM510; Thornwood, NY), images weretaken with a 1006/1.4 NA PlanApo objective lens and analyzed with LSM 5browser software. Z–stack images were taken at 0.5 mm intervals andreconstructed in three dimensions using the projection module. Somemicroscopic images were captured using Olympus IX71 fluorescencemicroscope equipped with a Photometrics Cool SNAP ES camera (Tucson, AZ).

Construction of arp3–mCherry, cut11–mCherry and meu14–mCherry

To generate fusion of arp3, cut11 and meu14 with mCherry at the chromosomelocus, DNA fragments containing the C–terminus of arp3, cut11, meu14 were firstamplified by PCR and were cloned into pJK210-mCherry (pCDL1207), yieldingpJK210-arp3-mCherry (pCDL1524), pJK210-cut11-mCherry (pCDL1526) andpJK210-meu14-mCherry (pCDL1527), respectively. These plasmids werelinearized and were transformed into the wild-type strain (MBY1238).Transformation was carried by the lithium acetate method (Okazaki et al.,1990). Integration was verified by colony PCR. Primers and plasmids are listed insupplementary material Table S2.

Electron microscopy

Cells were incubated on YPD plates at 28 C̊ for 20 hours. Cells were collected in200 ml YPD liquid medium and treated with 100 mM Lat A at room temperature.Cells were collected and fixed with 3% glutaraldehyde in potassium phosphatebuffer (pH 7.0) at different time points (30 minutes, 45 minutes and 60 minutesLat A treatment). After washing several times with 16 PBS, cells were post-fixedin 2% OsO4 for 2 hours at room temperature. The cells were then soaked in 0.5%aqueous solution of uranyl acetate for 2 hours and mounted on a copper grid toform a thin layer. The cells were then dehydrated by passing through gradedethanol and acetone and subsequently embedded in Spurr’s resin. Sections werestained with uranyl acetate and viewed and photographed using a JEOL 200CXelectron microscope (Peabody, MA) at 100 kV.

AcknowledgementsWe would like to thank the Yeast Genetic Resource Center (YGRC,Japan) and Tom Pollard (Yale University) for generously providingseveral yeast strains used in this study. We would like to thank allmembers of the Cell Division Laboratory for discussion and XieTang, Mithilesh Mishra, Meredith Calvert, Srinivasan Ramanujamand Kianhong Ng for critical reading of the manuscript. Wegratefully acknowledge X. Z. Ouyang for EM support.

FundingThis work was supported by research funds from the Temasek LifeSciences Laboratory and the Singapore Millennium Foundation.H.Y. was funded by a scholarship from the Singapore MillenniumFoundation.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.091561/-/DC1

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