Microtubule‐organizing centers of Aspergillus nidulans are … Zhang et al.pdf · required for sMTOC activity. This is the first evidence for a septum-specific protein, Spa10, as

Microtubule-organizing centers of Aspergillus nidulans areanchored at septa by a disordered protein

Ying Zhang,1† Xiaolei Gao,1† Raphael Manck,1

Marjorie Schmid,1 Aysha H. Osmani,2

Stephen A. Osmani,2 Norio Takeshita1,3 and

Reinhard Fischer 1*1Department of Microbiology, Institute for Applied

Biosciences, Karlsruhe Institute of Technology

(KIT) – South Campus, Fritz-Haber-Weg 4, Karlsruhe

D-76131, Germany.2Department of Molecular Genetics, Ohio State

University, 105 Biological Sciences Building, 484 W

12th Ave, Columbus, OH 43210, USA.3School of Life and Environmental Sciences,

University of Tsukuba, Ten-Nou-Dai, Tsukuba

305-8572, Japan.

Summary

Microtubule-organizing centers (MTOCs) are large,

multi-subunit protein complexes. Schizosaccharomy-

ces pombe harbors MTOCs at spindle pole bodies,

transient MTOCs in the division plane (eMTOCs) and

nuclear-envelope associated MTOCs in interphase

cells (iMTOCs). In the filamentous fungus Aspergillus

nidulans SPBs and septum-associated MTOCs were

described. Although comparable to S. pombe

eMTOCs, A. nidulans sMTOCS are permanent

septum-associated structures. The composition of

sMTOCs is poorly understood and how they are tar-

geted to septa was unknown. Here, we show that in

A. nidulans several SPB outer plaque proteins also

locate to sMTOCs while other SPB proteins do not,

including SfiA, a protein required for SPB duplication

in Saccharomyces cerevisiae and S. pombe and

PcpA, the anchor for g-TuSCs at the SPB inner pla-

que. The A. nidulans disordered protein Spa18Mto2

and the centrosomin-domain containing protein

ApsBMto1 were required for recruiting the g-TuRC

component GcpC to sMTOCs and for seeding MT for-

mation from septa. Testing different septum-

associated proteins for a role in sMTOC function,

Spa10 was identified. It forms a septal pore disc

structure, recruits Spa18 and ApsB to septa and is

required for sMTOC activity. This is the first evidence

for a septum-specific protein, Spa10, as anchor for a

specific class of MTOCs.

Introduction

Microtubule polymerization is a key feature of eukaryotic

cells. Because microtubules cannot be formed de novo,

eukaryotic cells employ large, multi-subunit protein com-

plexes, so-called microtubule-organizing centers

(MTOCs), to seed their formation (Kollman et al., 2011).

In higher eukaryotes one prominent MTOC is the cen-

trosome, consisting of two central centrioles surrounded

by pericentriolar material (PCM), with g-tubulin as a cru-

cial component for MT formation (L€uders, 2012). The

first evidence for MT polymerizing activity apart from

centrosomes came from plants, which lack obvious cen-

trosomes but contain extended MT arrays (L€uders and

Stearns, 2007). These MTs are polymerized from the

nuclear envelope and the cortex. As in the PCM, g-

tubulin is essential for MT polymerization in planta as

well. g-tubulin was also found along the lattice of MTs

giving rise to branched MTs, which was also described

recently in Xenopus cells (Petry et al., 2013; Hamada,

2014). There is also good evidence that secondary

MTOCs exist at other places in cells including the ER,

the cytoplasm and the Golgi apparatus (Efimov et al.,

2007; Sanchez-Huertas et al., 2016). Our knowledge of

the composition and functions of alternative MTOCs is

however rather scarce.

In fungi the functional equivalents of the centrosomes

are the spindle-pole bodies (SPBs) (Jaspersen and

Winey, 2004). SPBs are also associated with the

nucleus, as are the centrosomes of higher eukaryotes,

but unlike centrosomes they are embedded in the

nuclear envelope. They are composed of about 20 dif-

ferent proteins (Kilmartin, 2014). Whereas in Saccharo-

myces cerevisiae they represent stable structures in the

envelope and are duplicated within its membrane, in

Schizosaccharomyces pombe SPBs are only embedded

into the nuclear envelope prior to mitosis (Ding et al.,

Accepted 3 August, 2017. *For correspondence. E-mail [email protected]; Tel. (149) 721 6084 4630; Fax (149) 7216084 4509. †These authors contributed equally to this work.

VC 2017 John Wiley & Sons Ltd

Molecular Microbiology (2017) 106(2), 285–303 � doi:10.1111/mmi.13763First published online 30 August 2017

http://orcid.org/0000-0002-6704-2569

1997). In S. cerevisiae the SPB complex is comprised

of an outer, a central and an inner plaque. The plaques

are visible in the electron microscope as disc-like struc-

tures. The inner plaque seeds polymerization of microtu-

bules of the mitotic spindles and astral microtubules are

seeded from the outer plaque. During interphase the

outer plaque remains active and generates short cyto-

plasmic microtubules. At the onset of mitosis, these

microtubules are used to pull the nucleus towards the

bud neck (Rose and Fink, 1987; Pereira et al., 2000).

Both plaques share the essential components for MT

polymerization with g-tubulin being the defining protein.

g-tubulin, together with several core proteins called

GCPs (gamma complex proteins in human), assemble

to form a g-tubulin complex. In S. cerevisiae only two

proteins Spc97 (GCP2) and Spc98 (GCP3) assemble

with g-tubulin and form the g-tubulin small complex (g-

TuSC). In addition, the outer and inner plaques contain

several specific proteins. For instance Spc110 (Pcp1 in

S. pombe, PcpA in Aspergillus nidulans) is restricted to

the inner plaque and is responsible for recruitment of

the g-tubulin complexes (Supporting Information Fig.

S1A) (Flory et al., 2002; Chen et al., 2012). The duplica-

tion of the SPB prior to mitosis requires a number of

proteins extending from the central plaque along the

nuclear envelope. This structure is called the half bridge

and one of its characteristic proteins is Sfi1 (Avena

et al., 2014; Elserafy et al., 2014; Burns et al., 2015;

R€uthnick and Schiebel, 2016).

In S. pombe a few cytoplasmic MTs span the entire

cell during interphase. In addition to the SPBs, two other

types of MT organizers have been described (Piel and

Tran, 2009). In interphase several MTOCs are attached

to the nuclear envelope and generate the MT array

(Zimmerman et al., 2004). They are called iMTOCs.

During cytokinesis a MTOC appears temporarily at the

constricting ring in the equatorial plane, the eMTOC

(Heitz et al., 2001). The transient iMTOCs and the

eMTOC contain g-tubulin as do SPBs, and the g-tubulin

complex resembles the human protein complex with

GCP4–6 and Mzt1 as additional proteins (Horio et al.,

1991; Becker and Cassimeris, 2005; Dhani et al., 2013;

Masuda et al., 2013). The larger g-tubulin protein com-

plex is called the g-tubulin ring complex (g-TuRC). Two

further proteins, Mto1 and Mto2, were discovered

through the analysis of polarity mutants (Snaith and

Sawin, 2003; Sawin et al., 2004). Both are required for

the recruitment of the g-TuRC to cytoplasmic MTOCs

(Samejima et al., 2005, 2010). Likewise, in the plant

pathogen Ustilago maydis, several cytoplasmic MTOCs

were identified (Straube et al., 2003). The composition

of non-SPB MTOCs, their activity, regulation and organi-

zation have not yet been resolved in any fungus.

In filamentous fungi, such as A. nidulans, cytoplasmic

MTs are very prominent. They serve as tracks for secre-

tion vesicles, endosomes, peroxisomes and other organ-

elles (Riquelme et al., 2011; Pe~nalva et al., 2012;

Steinberg, 2012, 2014; Takeshita et al., 2014; Ishitsuka

et al., 2015; Yao et al., 2015; Salogiannis and Reck-

Peterson, 2017). During mitosis most cytoplasmic MTs

disassemble but often one MT, or a bundle of MTs, per-

sists during mitosis to allow continuous transportation

processes during this stage of the cell cycle (Riquelme

et al., 2003; Zekert and Fischer, 2009). g-tubulin was

discovered in A. nidulans and has been studied exten-

sively since, but only a few other fungal MTOC compo-

nents have been characterized so far in A. nidulans

(Oakley and Oakley, 1989; Horio et al., 1991; Oakley,

1992, 1995; Xiong and Oakley, 2009; Zekert et al.,

2010; Oakley et al., 2015). The A. nidulans SPBs poly-

merize mitotic spindle and astral MTs during mitosis

(Manck et al., 2015). In addition, SPBs are very active

MTOCs during interphase and generate overlapping MT

arrays within hyphal compartments because each com-

partment contains several nuclei (Morris and Enos,

1992; Suelmann et al., 1997) with each having active

MTOCs at their SPBs. However, the analysis of a MT

plus-end tracking kinesin 7 motor protein, KipA, revealed

the emergence of MTs from septa (Konzack et al.,

2005). Furthermore, the analysis of a developmental

mutant (apsB 5 anucleate primary sterigmata) revealed

a protein that localized to SPBs as well as to septa

(Suelmann et al., 1998; Veith et al., 2005). When Mto1

was discovered as a MTOC component in S. pombe its

similarity to ApsB was also described (Samejima et al.,

2005, 2008). This similarity suggested a role of ApsB in

MTOCs. Later it was shown that ApsB interacts with g-

tubulin at SPBs and at septa (Zekert et al., 2010). A

systematic analysis of g-tubulin ring complex proteins

(Gcp) revealed the presence of orthologues of all human

GCP2–6 proteins at septa (Xiong and Oakley, 2009).

Recently, MT asters produced from septa were observed

after re-polymerization of cytoplasmic MTs (Shukla

et al., 2017). There is therefore good evidence for active

septal MTOCs in A. nidulans. However, the composition

and structural organization of septal MTOC structures

remains enigmatic.

Here, we studied the role of two septal pore-associ-

ated proteins, Spa10 (Shen et al., 2014) and Spa18,

and show that they are required for the assembly and

the functioning of sMTOCs. Whereas Spa18 is likely the

orthologue of S. pombe Mto2, a Spa10 orthologue

apparently does not exist in S. pombe. This may reflect

the fact that MTOCs in the division plane of S. pombe

are temporary structures, whereas MTOCs are perma-

nently associated with septa in A. nidulans.

286 Y. Zhang et al. �

VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303

Results

The septum-associated intrinsically disordered proteinSpa18Mto2 is a component of nuclear and septal MTOCs

The composition and organization of non-conventional

MTOCs, such as fungal septal MTOCs, is poorly under-

stood. In A. nidulans several g-TuRC components

(GcpB-F) and the centrosomin-containing protein ApsB

were found at SPBs as well as at sMTOCs suggesting

that some main components are conserved among dif-

ferent MTOCs (Xiong and Oakley, 2009; Zekert et al.,

2010). To further characterize the composition of

sMTOCs, we aimed at analyzing other proteins found at

the SPB of S. cerevisiae to determine which orthologues

of A. nidulans are shared between SPBs and sMTOCs

(Fig. 1, Supporting Information Fig. S1A) (Table 1). We

localized GFP-tagged orthologues of the half-bridge

proteins Sfi1 (Kilmartin, 2003; Lee et al., 2014; Bouhlel

et al., 2015; Burns et al., 2015), Cdc31 (Osmani et al.,

2006), the inner plaque protein Pcp1 (Flory et al., 2002;

Chen et al., 2012) and the outer plaque protein SepK

(Tomlin et al., 2002; Kim et al., 2009). All four proteins

exclusively localized to SPBs and were absent from

sMTOCs (Fig. 1). Because SfiA had not been studied in

A. nidulans before, we tested the functionality of the

GFP-SfiA fusion protein. Whereas down-regulation of

sfiA resulted in inhibition of sporulation induction led to

wild-type sporulation (Supporting Information Fig. S1B).

In previous work the Ndc1 transmembrane nuclear pore

complex protein was not detected at septa but was seen

to locate to SPBs during mitosis (Osmani et al., 2006).

These results, and the published data on Gcp proteins

(Xiong and Oakley, 2009), suggest that sMTOCs share

g-TuRC components whereas other components, such

Fig. 1. Localization of MTOCcomponents at the Spindle Pole Body(SPB) but not at septal MTOCs(sMTOCs) in A. nidulans.A–D. Localization of SfiA, Cdc31, PcpAand SepK in hyphae of A. nidulans. Thearrows and asterisks indicate the SPBsand septa respectively. Strains SMS3(alcA(p)::GFP::sfiA), SMS6(alcA(p)::GFP::Ancdc31) and CPA02(alcA(p)::GFP::pcpA) were incubated inMM (2% glycerol, alcA promoter de-repressed) at 288C overnight andimaged. Strain LO1591 (sepK::GFP) wasincubated in MM (2% glucose) at 288Covernight. Nuclei were stained withDAPI. Note that nuclei and septa werenot from the same cell in B, C and D.Scale bar, 2 mm.

MTOCs in Aspergillus 287


as proteins required for SPB duplication, appear to be

specific for SPBs.

In Neurospora crassa, 17 intrinsically disordered sep-

tal pore associated proteins (Spa proteins) have been

identified (Lai et al., 2012) and the orthologues of Spa3,

10 and 13 were also found at septa in A. nidulans

(Shen et al., 2014). During this analysis it was observed

that A. nidulans Spa18 (AN4545) localized not only to

septa but also to defined SPB-like foci at nuclei (unpub-

lished data, and (Fig. 2A). Notably Spa18 located to two

foci during mitosis appearing at the two ends of the

mitotic spindle structures further indicating location at

SPBs (Fig. 2A). The dual location of Spa18 at SPBs

and septa indicates that it might have MTOC functions

at these locations. spa18 is a 2591 bp gene on chromo-

some III with an intron at 1075–1172 bp (confirmed by

RNAseq data), encoding a 950 aa (MW 102.5 kDa) pro-

tein. Spa18 is conserved among the ascomycota

although there is no clear orthologue in S. cerevisiae.

As observed for other spa proteins, Spa18 contains a

high-predicted probability of disorder throughout most of

its sequence and the region conserved amongst its

orthologues is the least disordered.

To determine if the Spa18 dots at interphase nuclei

and at septa represent the SPBs and sMTOCs, we stud-

ied MT polymerization in a strain with Spa18-GFP and

GFP tagged alpha-tubulin. Because Spa18 is visible as

dots at septa while MTs form long filaments, the two

structures are distinguishable even when tagged with

the same fluorescent protein. MT filaments were

observed to emerge from the Spa18 foci at nuclei and

septa (Fig. 2B, Supporting Information Movie S1a and

b). These data support the idea that Spa18 locates both

at SPBs and septal MTOCs.

ApsB interacts with Spa18 and localizes to sMTOCsand SPBs partially dependent on Spa18

The localization pattern of Spa18 resembled the distri-

bution of ApsB (Zekert et al., 2010). ApsB is the ortho-

logue of S. pombe Mto1 (Sawin et al., 2004; Samejima

et al., 2005, 2008). The apsB gene was cloned originally

by complementation (Suelmann et al., 1998) and the

gene model predicted a protein of 1051 amino acids.

However, recent RNAseq data and gene predictions in

other Aspergilli suggested 1569 additional nucleotides at

the 50 region. The modified derived protein is comprised

of 1574 amino acids (MW 177 kDa), with an intron at

2051–2098 bp. The N-terminal region consists of a cen-

trosomin domain (amino acid 525–599), the middle

region forms several coiled coil structures and the C-

terminal region contains a MASC domain (Mto1 and

Spc72p C-terminus, amino acid 1476–1524, also called

Microtubular organiser Mto1 C-term Mto2-binding

region). The MASC domain was first characterized in S.

pombe Mto1 and found to be important for targeting

Mto1 to multiple distinct MTOCs (Samejima et al.,

2010).

To test for co-localization of ApsB and Spa18 in A.

nidulans, the two proteins were tagged with GFP and

mCherry respectively. The two proteins co-localized at

specific foci associated with septa and at nuclei (Fig.

3A). We tested potential interaction between the two

proteins by Bimolecular Fluorescence Complementation

(BiFC or Split YFP). ApsB and Spa18 were tagged with

the C-terminal (YFPC-ApsB) and N-terminal (YFPN-

Spa18) halves of YFP respectively. Reconstituted YFP

fluorescence was observed at SPBs and septal foci indi-

cating protein-protein interaction at these specific sites

(Fig. 3B). No signal was observed when only one of the

Table 1. Conserved microtubule-organizing center proteins in S. cerevisiae, S. pombe and A. nidulans and their localization at spindle pole

bodies or sMTOCs of A. nidulans.

S. cerevisiae S. pombe A. nidulans A. nidulans SPB localization A. nidulans sMTOC localization

Tub4 Gtb1 MipA Oakley et al. (1990) and Zekert et al. (2010) Zekert et al. (2010)Spc97 Alp4 GcpB Xiong and Oakley (2009) Xiong and Oakley (2009)Spc98 Alp6 GcpC Xiong and Oakley (2009) Xiong and Oakley (2009)– Gfh1 GcpD Xiong and Oakley (2009) Xiong and Oakley (2009)– Mod21 GcpE Xiong and Oakley (2009) Xiong and Oakley (2009)– Alp16 GcpF Xiong and Oakley (2009) Xiong and Oakley (2009)– Mzt1 MztA Our unpublished data Our unpublished dataSpc72 Mto1 ApsB Zekert et al. (2010) Zekert et al. (2010)– Mto2 Spa18 This work This workNud1 Cdc11 SepK Kim et al. (2009) No (this work)Cmd1 Cam1 Calmodulin Chen et al. (2012) No (Chen et al., 2012)Spc110 Pcp1 PcpA Chen et al. (2012) No (this work)Ndc1 Cut11 Ndc1 (Osmani et al., 2006) No (Osmani et al., 2006)Cdc31 Centrin Cdc31 This work No (this work)Sfi1 Sfi1 SfiA This work No (this work)

Proteins, which were found at SPBs and sMTOCs are shaded in green. Proteins, which are specific for SPBs and not found at septa areshaded in orange. Proteins written in bold letters in A. nidulans are the ones which we studied in this work.



two fusion proteins was expressed. As a further nega-

tive control we used YFPC-ApsB and the kinesin motor

protein YFPN-UncA and did not observe any reconsti-

tuted fluorescence (Supporting Information Fig. S2A).

The interaction between ApsB and Spa18 was further

investigated using the yeast-two hybrid assay and the

results further indicate ApsB and Spa18 are able to

interact (Fig. 3C). Importantly the test for self-activation

of either of the two constructs was negative (Supporting

Information Fig. S2B).

To study the function of Spa18, we generated a

spa18-deletion strain and compared its phenotype with

the wild type and an apsB-deletion strain (Supporting

Information Fig. S3A). In contrast to the apsB-deletion

strain, colonies of the spa18-deletion mutant grew like

wild type and asexual sporulation was not affected (Sup-

porting Information Fig. S3A). An apsB/spa18 double-

mutant strain resembled the apsB mutant. However, in

apsB mutants nuclear distribution is affected in hyphae

and during conidiophore development. spa18 mutants

did not show this phenotype, and the apsB/spa18 dou-

ble mutant appeared identical to the apsB mutant (Sup-

porting Information Fig. S3B).

Next we analyzed if Spa18 was required for recruit-

ment of ApsB to SPBs or sMTOcs. In a spa18-deletion

strain ApsB was still found at both MTOCs, but the

intensity was decreased (Fig. 3D). Thus, ApsB localizes

partly dependent on Spa18 to MTOCs.

Spa18 and ApsB are essential for sMTOC activity

Although the phenotypes of the apsB- and the spa18-

deletion strains differed at the colony level, we investi-

gated if MT formation was affected in the spa18

mutant as was previously described for the apsB-dele-

tion strain (Zekert et al., 2010). First, we visualized

astral MTs during mitosis and counted the number of

astral MTs formed from the SPBs. In wild type cells

between five and six astral MTs were counted per

mitotic spindle whereas, on average, only two were

seen in the DapsB strain (Fig. 4A). However, no statis-

tically significant difference was observed in the

Dspa18 mutant compared to wild type. To investigate

potential effects on interphase MTOC function the

number of cytoplasmic MTs was calculated and found

to be reduced from 5.8 in wild-type cells to 4.6 in the

spa18 mutant and to 3.2 in the apsB mutant. The

number of MTs in the double mutant was the same as

in the apsB-single mutant (Fig. 4B). Because astral

MTs are involved in the dynamics of mitotic spindles,

we analyzed their behavior in wild type and the differ-

ent mutants. Whereas in the apsB mutant mitotic spin-

dles were largely immobile and mitosis prolonged,

spindles in wild type (not shown) or the spa18 mutant

were more dynamic. In addition to the reduced

dynamics in the double mutant strain, spindle mor-

phology appeared to be affected with abnormally bent

mitotic spindles (Fig. 4C).

To further analyze the MTOC activity of SPBs and

sMTOCs, we visualized the MT-plus end tracking kinesin

7 motor, KipA (Konzack et al., 2005) which appears as

mobile comets representing the growing plus ends of

MTs. The number of GFP-KipA comets generated from

Fig. 2. Localization of Spa18 at SPBs and sMTOCs.A. Localization of Spa18 during interphase and during mitosis(upper three panels) at SPBs and at septa (lower panels). StrainSO1312 (Spa18::GFP) was grown in MM plus urea overnight at288C. Strain SXL21 (alcA(p)::mCherry::tubA; spa18::GFP) wasincubated in MM (2% glycerol) at 288C overnight. The arrowsmark the position of DAPI stained nuclei. Scale bar, 2 mm.B. Microtubule emergence from an sMTOC (left) and a SPB(right). To observe the dynamics of MTs, strain SXL17(spa18::GFP, alcA(p)::GFP::tubA) was incubated in an 8 wellu-slide with MM plus urea (2% glycerol) at 288C. After overnightincubation additional medium was added at room temperature tofill up the well. Images were taken at 15 second intervals (seeSupporting Information Movie SI). Maximum projection of adeconvolved Z-stack was applied. Deconvolution was performedwith Zen 2012 Blue Edition v1.20 (Zeiss, Jena, Germany). Scalebar, 2 mm.



SPBs was reduced by 0.7%, 21.7% and 18.8% in

Dspa18, DapsB and the double mutant respectively. This

result indicates that ApsB and Spa18 are largely dispen-

sable for nucleation of cytoplasmic MTs from interphase

SPBs but suggest they might play a minor role. More dra-

matically however, Spa18 and ApsB were found to almost

essential for MT formation from sMTOCs. In the absence

of Spa18 or ApsB, or of both, almost no GFP-KipA com-

ets emerged from sMTOCs while in wild type an average

of 5.6 comets were detected per 100 s (Fig. 4D) (Sup-

porting Information Movies SII, SIII). We confirmed the

failure of sMTOCs to produce MTs in the absence of

ApsB or Spa18 by inspection of MTs close to septa.

Whereas in wild type several MTs emerged from a point

at the septum, MTs only arose from SPBs and polymer-

ized towards the septa in the mutant cells (Supporting

Information Fig. S4).

The cTuRC is recruited to sMTOCs by ApsB/Spa18

To investigate further potential functions of Spa18 and

ApsB, we tested whether they might be required for the

Fig. 3. Co-localization and interaction of ApsBMto1

and Spa18Mto2.A. Co-localization of Spa18 and ApsB at SPBs andsMTOCs. The arrow points to the SPB, the asteriskindicates the septal signals. Strain SYZ47(spa18::GFP, alcA(p)::mCherry::apsB) wasincubated in MM with urea (2% glycerol) at 288Covernight. Nuclei were stained with DAPI. Scalebar, 2 mm.B. Interaction of ApsB and Spa18 shown in aBimolecular Fluorescence Complementation assay.Spa18 was N-terminally tagged with YFPN whileApsB was tagged with YFPC. The arrows and theasterisks indicate the YFP signals at SPBs and atthe sMTOCs. At septa the signal occurred as single(left) or two dots (right). Strain SYZ61(alcA(p)::YFPN::spa18, alcA(p)::YFPC::apsB) wasincubated in MM (2% glycerol) overnight at 288C.Scale bar, 2 mm.C. Yeast-two-Hybrid assay of ApsB and Spa18. S.cerevisiae strain YSYZ3 (Gal4 AD-ApsB; Gal4 BD-Spa18) was used for the analysis. Positive andnegative controls were provided in theMatchmakerTM Gold Yeast Two-Hybrid System byClontech Laboratories. Dilution series of respectivestrains were grown on SD-LW (selective dropoutleucine and tryptophan) and SD-LWHA (selectivedropout leucine, tryptophan, histidine and alanine)agar plates at 308C for 3 days.D. Images and quantification of ApsB fluorescenceintensity at SPBs and sMTOCs in Dspa18. StrainsSYZ2 (alcA(p)::GFP::apsB) and SYZ46 (Dspa18,alcA(p)::GFP::apsB) were incubated in MM (2%glycerol) with appropriate supplements at 288Covernight and observed. The arrows and theasterisks indicate SPBs and sMTOCs. Images of15–20 sections were taken along the z-axis at0.27-lm increments. The projection images ofmaximum intensity were obtained and maximumfluorescence intensities over the backgroundintensity were used for statistical analysis. Theexposure time and shutter level were set to beidentical for each strain. The number of SPBs andsepta checked in each strain was 30. Mann–Whitney U-test was performed with GraphPadPrism 7. Box diagrams are plotted in Spear style, inwhich the plus indicate the average, boxes indicatethe interquartile range and whiskers mark theminimal and maximal values. Different letters abovethe graph indicate significant differences (p <0.01). Scale bar, 2 mm.



recruitment of other MTOC-associated proteins to SPBs

or sMTOCs. Because sMTOC function was affected by

the lack of either of the two proteins, we hypothesized

that the recruitment of the g-TuRC components to

sMTOCs might depend on ApsB or Spa18 but not, or to

a lesser extent, their recruitment to the SPBs. We found

that in the absence of Spa18 or ApsB or both, the g-

TuRC component GcpC was unable to locate to

sMTOCs but was able to locate to SPBs (Fig. 5A and

Supporting Information Fig. S5), indicating that Spa18

and ApsB play a specific role in recruiting gTuRC pro-

teins to sMTOCs.

In this context we also investigated the role of the

centrosomin motif of ApsB. Centrosomin Motifs (CM1)

are conserved in S. pombe Mto1 and Pcp1 as well as in

S. cerevisiae Spc110 and Spc72 (Samejima et al.,

2008) being required for proper recruitment of the g-

TuSC to MTOCs. The strain with the modified ApsB

(DCM1) protein displayed the same phenotype at the

colony level as the apsB-deletion strain and produced

almost no asexual spores (Fig. 5B). This shows that the

centrosomin motif is essential for the function of ApsB.

mCherry-ApsB lacking the CM1 motif (mCherryDCM1),

still localized to SPBs and sMTOCs indicating that CM1

is not required for ApsB targeting to MTOCs. However,

the g-TuRC component GcpC was only detected at

SPBs but not at septa (Fig. 5C). At SPBs GcpC is prob-

ably also recruited by PcpA and therefore less depend-

ent of ApsB.

Spa10 is required for ApsB/Spa18 targeting

to sMTOCs

Because AspB and Spa18 are required for recruitment

of g-TuRC proteins to sMTOCs, we next asked which

proteins are required for ApsB/Spa18 recruitment. In A.

nidulans and in S. pombe the g-TuRC consists of g-

tubulin and the full set of GCPs (GCP2–6), whereas the

complex of S. cerevisiae lacks several GCPs and is

called g-TuSC (g-tubulin small complex). At the inner

plaque of the S. cerevisiae SPB, Spc110 (Pcp1 in S.

pombe) is the recruiter of the g-TuSC, which then nucle-

ates the mitotic spindle MTs. An orthologue of Spc110,

PcpA (AN3062), has been studied in A. nidulans (Flory

et al., 2002; Chen et al., 2012). Like ApsB it contains a

CM1 region (amino acid 194–267). PcpA is highly con-

served in eukaryotes and is variously named kendrin

(Homo sapiens), pericentrin (Mus musculus), Spc110

(S. cerevisiae), Pcp1p (S. pombe) and centrosomin

(Drosophila melanogaster). We observed that PcpA

localized to SPBs but not to septa (Fig. 1C) indicating

PcpA can be excluded as a receptor for ApsB and

Spa18 at septa.

Because in S. cerevisiae Spc72 (ApsB) is recruited to

the outer plaque of the SPB by Nud1 (Cdc11 in S.

pombe) (Brachat et al., 1998; Adams and Kilmartin,

1999; Elliott et al., 1999; Samejima et al., 2010), we

studied an orthologue of this protein named SepK

(AN2459) in A. nidulans (Fig. 1D). In a sepK-deletion

strain GFP-ApsB exhibited the same signal intensity at

septa but had much weaker signals at SPBs compared

to wild type (Fig. 6A). This indicates that SepK is not

required to target ApsB to septa. Considering that

Spa18 is also involved in ApsB localization, we investi-

gated GFP-ApsB localization in a sepK/spa18-double

mutant. Surprisingly, the weak ApsB signal was still

visualized at SPBs (Fig. 6A).

Since SepK and PcpA were not detected at septa, it

remained open how gTuRCs are recruited to sMTOCs.

Therefore, the orthologues of several other intrinsically

disordered septal pore associated proteins of N. crassa

were investigated as candidates (Lai et al., 2012). In the

absence of Spa10 (AN1948), ApsB and Spa18 still

localized to SPBs but not to sMTOCs (Fig. 6B). GcpC

was also absent (data not shown) from septa and MT

polymerization from sMTOCs was blocked. Only a few

MTs generated from SPBs polymerized towards the

septal membrane and shrunk back (Supporting Informa-

tion Movies SVI and SVII). Mitosis was not affected in

the spa10 mutant compared to wild type (Supporting

Information Movies SIV and SV). Deletion of spa10 did

not cause any significant growth defects at the colony

level (Supporting Information Fig. S6A). Hyphae of the

spa10-deletion strain displayed a higher frequency of

tip-splitting, which was not observed in a spa18-deletion

strain (Supporting Information Fig. S6A). In N. crassa,

spa10 mutants developed abnormally high numbers of

septa at subapical regions (Lai et al., 2012). Likewise, in

A. nidulans the length of subapical compartments was

significantly smaller in the spa10 mutant compared to

wild type or the spa18 mutant (Supporting Information

Fig. S6B).

In conclusion, SepK is involved in the recruitment of

ApsB to SPBs, while Spa10 is essential for the recruit-

ment of ApsB and Spa18 to sMTOCs.

Spa10 is targeted to sMTOCs earlier than

ApsB and Spa18

To study the timing of sMTOC assembly during the sep-

tation process, Spa10 and ApsB were GFP tagged and

introduced into a strain with mCherry-tagged tropomyo-

sin TpmA, which binds specifically to F-actin. TpmA

localizes very early at the septation site and follows the

constricting ring (Bergs et al., 2016) (Fig. 7A and B).

GFP-Spa10 appeared at the septation site together with



TpmA (Fig. 7A), and the signal intensity increased dur-

ing septation. At mature septa TpmA was absent (Fig.

7A), whereas Spa10 was still associated with septa and

formed a more condensed structure at the center of

mature septa (Fig. 7A) (Shen et al., 2014). GFP-ApsB

and GFP-Spa18 were only visible at mature septa and

never co-localized with TpmA (Fig. 7B and C). Thus the

recruitment of Spa10 to septa was much earlier than

ApsB and Spa18 and Spa10 localization at early or

mature septa was independent of Spa18 (Supporting

Information Fig. S7A). Spa10 could therefore act as an

anchor for sMTOCs.



To further clarify the arrangement of Spa10, ApsB,

Spa18 and TpmA, three-dimensional images of the pro-

teins in different septation phases were generated

based on maximum projection of deconvolved Z-stack

images (Fig. 8). At the beginning of septation (early),

Spa10 co-localized with TpmA in a full size ring (Ring

diameter 5 hyphal diameter), later at the middle phase it

constricted with TpmA to a smaller ring (Ring

Fig. 4. Comparison of the effect on MTOC activity and MT organization in the absence of ApsB or Spa18 or both.A. Images of astral MTs during mitosis in WT, a Dspa18, a DapsB and the double-mutant strain. Strains SJW02 (alcA(p)::GFP::tubA), SYZ34(Dspa18, alcA(p)::GFP::tubA), SYZ11 (DapsB, alcA(p)::GFP::tubA) and SYZ49 (DapsBDspa18, alcA(p)::GFP::tubA) were grown in MM (2%glycerol) with appropriate supplements at 288C overnight. Astral MTs are indicated with arrows. The quantification (right) is based on countingastral MTs from 50 spindles for each strain and displayed as box plots. The statistical analysis was performed with the Mann–Whitney U-test.Different letters above the graph indicate significant differences (p < 0.01). Box diagrams are plotted in Tukey’s style, in which the lines displaythe median, the full dots are the mean and whiskers indicate statistical outliers. Scale bar, 5 mm.B. Comparison of the number of cytoplasmic MTs during interphase in WT, DapsB, Dspa18 and the double-mutant strain. The same strainsand culture conditions were used as in (A). 100 hyphae were counted for each strain. Statistical analysis as in (A).C. Comparison of mitosis in a DapsB, Dspa18 and a double mutant strain. Strains SYZ11 (DapsB, alcA(p)::GFP::tubA), SYZ34 (Dspa18,alcA(p)::GFP::tubA) and SYZ49 (DapsBDspa18, alcA(p)::GFP::tubA) were grown in 8 well u-slides at 288C overnight for long term live cellobservation. Time-lapse images were taken every 1 min at room temperature. In the Dspa18 strain mitosis finished after 10 min. In the DapsBand the double mutant strain, mitosis was delayed and spindles were less dynamic. Bent spindles were observed in the double mutant. Scalebar, 2 mm.D. Analysis of the activity of SPBs and sMTOCs in WT, DapsB, Dspa18 and the double-mutant strain analyzed with the MT-plus-end markerKipA. Time-lapse analyses were performed to quantify the GFP-KipA signals emerging from MTOCs. KipA is known to track the plus ends ofgrowing MTs (Konzack et al., 2005). Strains SSH27 (alcA(p)::GFP::kipA), SYZ43 (Dspa18, alcA(p)::GFP::kipA), SYZ9 (DapsB,alcA(p)::GFP::kipA) and SYZ51 (DapsBDspa18, alcA(p)::GFP::kipA) were grown in MM (2% glycerol) with appropriate supplements at 288Covernight. GFP::KipA signals from 50 SPBs and 50 sMTOCs were counted for each strain. Each MTOC was monitored for 100 s.Representative movies see Supporting Information Movies SII and SIII. Statistical analysis as in (A).

Fig. 5. Targeting the gTuRC component GcpC to sMTOCs depends on ApsB and Spa18.A. Localization of GcpC at sMTOC depends on ApsB and Spa18. Strains SNZ-SH80 (gcpC::GFP), SYZ39 (DapsB, gcpC::GFP), SYZ42(Dspa18, gcpC::GFP) and SYZ44 (DapsBDspa18, gcpC::GFP) were grown in MM with appropriate supplements at 288C overnight. Nucleiwere stained with DAPI. Scale bar, 2 mm.B. Colonies of DapsB, apsBDCM1 and wild type. Strains SYZ3 (DapsB), SYZ56 (DapsB, gcpC::GFP, mCherry::apsBDCM1) and TN02A3 weregrown on a MM (2% glycerol) agar plate with appropriate supplements at 378C for 3 days. Scale bar, 1 cm.C. Analysis of the role of the CM1 domain for recruitment of GcpC to nuclei (upper row) and to sMTOCs (lower row). Strain SYZ56 (DapsB,gcpC::GFP, alcA(p)::mCherry::apsBDCM1) was grown in MM (2% glycerol) at 288C overnight. Scale bar, 2 mm.



diameter 5 1/2 hyphal diameter) and eventually at

mature septa was concentrated in a central-disc struc-

ture and TpmA was absent (Fig. 8A). The disc-like

Spa10 structure had in some cases a central area with

less or no Spa10 protein (Supporting Information Fig.

S7B). Using the same methods 908 views of ApsB and

Spa18 were constructed. Different from Spa10, both

appeared as a ring structure with several foci (Fig. 8B

and C). Co-localization of ApsB and Spa10 indicate that

ApsB foci attach to the Spa10 disc at its periphery (Fig.

8D).

In S. pombe eMTOCs are transient structures at form-

ing septa (Heitz et al., 2001). In A. nidulans septa are

stable structures, but the pore in the center still allows

exchange of material and even organelles between adja-

cent compartments. Using a Spa10-GFP mCherry-TubA

strain we could show that at early stages during con-

striction, MTs still passed through the forming septum,

whereas in mature septa MTs emanated from the cen-

tral pore region away from the septum (Fig. 9A; Sup-

porting Information Movies SVIII and SIX). Interestingly,

septal pores appear to be sealed during mitosis, sug-

gesting cell-cycle dependent dynamics (Shen et al.,

2014). Analysis of germlings undergoing mitosis

revealed that Spa10 and Spa18 remained at septa dur-

ing mitosis (Fig. 9B).

Discussion

Microtubules cannot be generated de novo but require a

seeding complex for their initial assembly. The best-

studied examples are probably the centrosomes of

higher eukaryotes and the nuclear MTOCs of yeast,

also known as spindle pole bodies (SPB). However,

other types of MTOCs exist which have been less well

studied and their roles are not well understood. In many

cases not even the molecular composition is known (Kil-

martin, 2014). One well-studied example is the fission

yeast S. pombe. After the discovery of g-tubulin at

SPBs, new MTOC components were identified in non-

targeted approaches to characterize MT-dependent

Fig. 6. Analysis of the recruitment function of SepK and Spa10.A. Localization of ApsB in WT, and a DsepK and a DsepKDspa18 double-mutant strain. Arrows indicate the GFP-ApsB signals. Strains SYZ2(alcA(p)::GFP::apsB), SYZ60 (DsepK, alcA(p)::GFP::apsB) and SYZ70 (DsepKDspa18, alcA(p)::GFP::apsB) were incubated in MM (2%glycerol) at 288C overnight. Scale bar, 2 mm.B. Localization of ApsB and Spa18 in wild type (upper row), and in the absence of Spa10 (lower row). Strains SYZ2 (alcA(p)::GFP::apsB),SYZ69 (Dspa10, alcA(p)::GFP::apsB), SYZ75 (alcA(p)::GFP::spa18), SYZ74 (Dspa10, alcA(p)::GFP::spa18) were incubated in MM (2%glycerol) with appropriate supplements at 288C overnight. Scale bar, 2 mm.



processes (Pereira and Schiebel, 1997; Snaith and

Sawin, 2003). One novel candidate was Mto1 (Mod20),

which has similarity to the A. nidulans ApsB protein and

is important for MT formation not only from SPBs but

also from iMTOCs and eMTOCs (Suelmann et al., 1998;

Sawin et al., 2004). Mto1 (Microtubule organizer 1) was

the first protein identified with a direct role in recruiting

the g-tubulin complex to non-centrosomal MTOCs (Suel-

mann et al., 1998; Sawin et al., 2004). Shortly after this

discovery, a second component, Mto2, was identified

using a mutant of the same insertional mutagenesis

screen and independently as an interacting protein of

Mto1 (Samejima et al., 2005; Venkatram et al., 2005).

Later it was shown that the Mto1/2 protein complex is

not only responsible for the recruitment of the g-tubulin

complex to MTOCs, but that it also activates the poly-

merization machinery (Lynch et al., 2014).

In the filamentous fungus A. nidulans, SPBs seed

MTs of the mitotic spindle, astral microtubules and –

during interphase – cytoplasmic MTs. In addition,

Fig. 7. Analysis of the timingof the recruitment of Spa10,ApsB and Spa18 to sMTOCs.A. Co-localization of Spa10with TpmA. The image showsa septum very early during itsformation (red asterisk) duringseptation where TpmA andSpa10 co-localize, a septumat a later stage (yellowasterisk) and a matureseptum (green asterisk)where TpmA disappeared andSpa10 became moreconcentrated. Strain SXL6(alcA(p)::GFP::spa10;alcA(p)::mCherry::tpmA) wasincubated in MM (2%glycerol) at 288C overnight.Scale bar, 2mm.B. Analysis of the localizationof ApsB and TpmA in early,and in middle phases offormation and at maturesepta. StrainSXL11(alcA(p)::GFP::apsB;alcA(p)::mCherry::tpmA) wasincubated in MM (2%glycerol) at 288C overnight.Scale bar, 2mm.C. Same analysis as in (B)but with Spa18. Strain SXL12(alcA(p)::GFP::spa18;alcA(p)::mCherry::tpmA) wasincubated in MM (2%glycerol) at 288C overnight.Scale bar, 2mm.



MTOCs were discovered at septa in this fungus. In this

work we compared the molecular composition of SPBs

and sMTOCs and started to analyze the biogenesis of

sMTOCs. The two MTOCs are fundamentally different

when it comes to their biogenesis. Whereas SPBs

duplicate prior to mitosis, sMTOCs need to be

assembled de novo after septum formation. In this

regard, we were able to assign a novel function for the

septum-associated protein Spa10 as an anchor for

sMTOCs (Fig. 10). Spa10 is recruited to the constricting



cytokinetic ring and follows the rim of the ring during its

constriction. However, at mature septa the protein forms

a central disc to which the sMTOCs appear to be

attached. It seems that the disc still has a central area

with less Spa10 protein. Spa10 appears to be an excel-

lent candidate as an anchor protein, because it localized

Fig. 8. Analysis of the timing of sMTOC assembly and spatial organization of Spa10, ApsB, Spa18 and TpmA during septum formation.A. At the beginning of septation Spa10 colocalized with TpmA in a full-size ring (left). At the middle phase of septation Spa10 constricted withTpmA to a small ring (right) while at mature septa Spa10 became more concentrated to a central disc structure where TpmA was absent. Thestructure was constructed with maximum projection of deconvolved Z-stack images. The 908 enlargement pictures indicate an observationangle of 908. Strain SXL13 (spa10(p)::spa10::GFP; alcA(p)::mCherry::tpmA) was incubated in 8 well u-slide at 288C overnight. Scale bar,2 mm.B and C. ApsB and Spa18 at mature septa. Maximum projection of a deconvolved Z-stack showed both ApsB and Spa18 display adiscontinuous (several foci) ring structure at septa. Normal and 908 view. Strains SYZ2 (alcA(p)::GFP::apsB) and SO1332 (spa18::GFP) wereincubated in MM (2% glycerol) with appropriate markers at 288C overnight. Scale bar, 2mm.D. Spinning disc image of Spa10 and ApsB. Strain SXL9 (alcA(p)::GFP::apsB; spa10::GFP) was incubated in MM (2% glycerol) at 288Covernight and imaged using spinning disc confocal microscopy. Maximum projection of Z-stacks is shown. The 908 view indicate anobservation angle of 908 for Spa10-GFP. Scale bar, 2 mm.

Fig. 9. sMTOC are stable structures at septa and remain intact during mitosis.A. Visualization of Spa10 and MTs at a forming (early) and a mature septum. Strain SXL20 (alcA(p)::mCherry::tubA; spa10(p)::spa10::GFP)was incubated in an 8 well u-slide at 288C overnight. Scale bar, 2 mm.B. Spa10 and Spa18 localize to septa during mitosis. Strains SXL20 (alcA(p)::mCherry::tubA; spa10::GFP) and SXL21(alcA(p)::mCherry::tubA; spa18::GFP) were incubated in MM (2% glycerol) at 288C overnight and imaged. The asterisks mark the septumposition. Scale bar, 2 mm.



in a stable manner at mature septa. Whereas other sep-

tal protein signals recovered at the septum after photo-

bleaching, Spa10 did not, suggesting that there is no

further exchange between this structure and the cyto-

plasmic pool of Spa10 (Shen et al., 2014). Protein local-

ization studies in several mutants allowed us to propose

a hierarchy for the recruitment of the sMTOC proteins.

We speculate that Spa18 and ApsB attach first to

Spa10 and they then recruit g-TuRC proteins, including

g-tubulin and GcpC. The experiments do not allow us to

decipher the assembly process with high spatial or tem-

poral resolution. Therefore, it could well be that protein

complexes assemble in the cytoplasm which then sub-

sequently are recruited together to form sMTOCs. In S.

cerevisiae Sfi1 is required for the recruitment of new g-

TuRC proteins and the activity is coupled to the regula-

tion of the cell cycle. One hypothesis – derived from our

findings – is that Spa10 serves an analogous function

as Sfi1 in S. cerevisiae but at septa. Sfi1 proteins are

anchored at the SPB and extend with their C-termini

into the half bridge (Elserafy et al., 2014; Seybold et al.,

2015). Positive and negative signals regulate the dimeri-

zation of Sfi1 molecules in early G1, leading to antipar-

allel C-to-C dimerization and exposure of free N-termini

at the side of the half bridge (R€uthnick and Schiebel,

2016). At the free Sfi1 N-termini the new SPB can

assemble. It will be interesting to see if posttranslational

regulation of Spa10 changes its activity and couples g-

TuRCs recruitment to septation. One candidate for such

modification could be the septum-associated kinase

KfsA, which appears also to be quite stable at the sep-

tum and in close proximity to Spa10 (Takeshita et al.,

2007; Shen et al., 2014). In the case of S. pombe Mto2

the activity is indeed controlled by phosphorylation

(Borek et al., 2015). An interesting area of research will

also be to solve the question why an anchor for an

sMTOC should be an intrinsically disordered protein.

Such proteins normally enable weak but reversible bind-

ing of proteins. They normally fold upon binding and

thereby enable specific interactions with different part-

ners (Wright and Dyson, 2009; Berlow et al., 2015).

One strategy to decipher the exact molecular function of

Spa10 will be therefore the identification of Spa10-

interacting proteins.

Experimental procedures

Strains, plasmids and culture conditions

Supplemented minimal media (MM) and complete medium

(YAG) for A. nidulans were prepared as previously

described, and standard strain construction procedures

were used (Hill and K€afer, 2001). Expression of tagged

genes under control of the alcA-promoter was regulated by

the carbon source: repression on glucose, derepression on

glycerol (2%) and induction on threonine (2%) (Waring

et al., 1989). A list of A. nidulans strains used in this study

is given in Supporting Information Table S1. S. cerevisiae

cells were grown in YPDA complete medium, or in minimal

medium (SD) supplemented with the dropout-mix neededfor selection, as described in the Clontech MatchmakerTM

GAL4 Two-Hybrid System 3 Manual (http://www.clontech.

com). S. cerevisiae strains used in this study are listed in

Supporting Information Table S2. Standard laboratory Esch-

erichia coli strains (XL-1 blue, Top 10 F0) were used. Plas-

mids are listed in Supporting Information Table S3.

Molecular techniques

Standard DNA transformation procedures were used for A.

nidulans (Yelton et al., 1984), E. coli (Sambrook and

Fig. 10. Scheme of septal microtubule organizing centers(sMTOCs) of A. nidulans. In the center of a mature septum Spa10concentrates into a central disc structure and acts as an anchor/scaffold for sMTOC components.



http://www.clontech.com

http://www.clontech.com

Russel, 1999) and S. cerevisiae (Clontech). For PCR

experiments, standard protocols were applied using a Bio-

metra Personal Cycler (Biometra, G€ottingen, Germany) for

the reaction cycles. DNA sequencing was done commer-

cially (MWG Biotech, Ebersberg, Germany). Total DNA was

extracted from A. nidulans according to (Zekert et al.,

2010). Southern hybridizations were performed according

to the DIG Application Manual for Filter Hybridization

(Roche Applied Science, Technical Resources, Roche Diag-

nostics GmbH, Mannheim, Germany). The first-strand

cDNA synthesis was carried out with a SuperScript III

Reverse Transcriptase kit (Invitrogen).

Construction of deletion strains and double mutants. The

full apsB open reading frame (ORF) was deleted in the wild

type (WT) strain TN02A3 with pyroA as selection marker.

The flanking regions of apsB were amplified by PCR with

genomic DNA as template, and primers apsB-LB_fwd/

apsB-LB-linker_rev (see Supporting Information Table S4)

for the upstream region of apsB and apsB-RB-linker_fwd/

apsB-RB_rev for the downstream region. pyroA was ampli-

fied with pyroA-linker_fwd/pyroA-linker_rev. Then the left

border (LB), pyroA and right border (RB) were fused

together by fusion PCR with nested primers apsB-LB-

N_fwd and apsB-RB-N_rev. The construct was ligated to

pJET1.2/blunt, yielding vector pYZ11. After transformation

of pYZ11 to TN02A3 and selection by diagnostic PCR

(Supporting Information Table S4 primers:apsB-LB_fwd/

apsB-ORF_rev) and Southern blot, the apsB-deletion strain

SYZ3 was selected.

The CM1 domain of ApsB was deleted. The 50 region of

apsB was amplified with apsB_AscI_fwd and

DCM1_L_linker_R, and the 30 region part was amplified with

DCM1_R_F and apsB-4.7_PacI_rev. The two parts were

fused together by fusion PCR, followed by digestion with

restriction enzymes AscI and PacI and ligation into the

pMCB17apx-mCherry vector, yielding pYZ65. The GcpC C-

terminal tagging GFP cassette gcpC::GFP::AfpyrG::RB-

gcpC was amplified with Alp6_Nprimer_fwd and Alp6_Npri-

mer_rev (Zekert et al., 2010) using genomic DNA of strain

SNZ-SH80 as template. The PCR product and pYZ65 were

co-transformed into SYZ3 (DapsB), yielding strain SYZ56

with mCherry::apsB and gcpC::GFP. gcpC homologous

integration was checked with primers pyrG check fwd/

GcpC-GFP check rev (Supporting Information Table S4).spa10 and spa18 deletion: Deletion constructs (De

Souza et al., 2013) were obtained from the FGSC (http://

www.fgsc.net/Apergillus/KO_Cassettes.htm) to delete spa10

and spa18 in strain SO451. Diagnostic PCR confirmed

deletions using primers as listed in Supporting Information

Table S4.To generate a spa18/apsB double mutant, SO1334 was

crossed with RMS011, yielding a para-aminobenzoic acid

(pabaA) and pyridoxine auxotrophic (pyroA) mutant strain

SYZ33, an arginine (argB) auxotrophic mutant strain

SYZ32 and a pyridoxine and arginine auxotrophic mutant

strain SYZ31. Subsequently, we crossed SYZ33 with SYZ3

(DapsB). After checking the deletion event by PCR, the

spa18 and apsB double deletion strain SYZ45 were

selected with the pabaA auxotrophy marker. We also cre-

ated an apsB and spa18 double-deletion strain SYZ35

(argB auxotrophic) by crossing SYZ31 with SYZ3. The

sepK and spa18-double mutant SYZ70 was created by

crossing SYZ32 with LO1906. Crossings were done as

described (Todd et al., 2007).

C-terminal tagging of Spa10 with GFP expressed underthe native promoter. We used 1.0 kb from the 30-end of

the gene for PCR amplification with the primer pair

Spa10 C-ter fwd/Spa10 C-ter rev, and the 1 kb terminator

region of the gene was amplified with the primer pair

Spa10 RB fwd/Spa10 RB rev. A fragment of the GFP::pyrG

cassette was amplified from pFNO3 (Yang et al., 2004)

using primers GA_linker/pyrG_cas_rev. Subsequently, the

three fragments were fused together by fusion PCR with

nested primers spa10 nest fwd/spa10 nest rev (Supporting

Information Table S4). The construct was ligated to

pJET1.2/blunt, resulting in plasmid pRM124. The plasmid

was transformed into TN02A3, and the strain SRM212

where the construct replaced the endogenous 30-end of the

gene, was checked with primers pyrG check fwd/Spa10-

GFP check rev. The strain expresses Spa10-GFP from the

natural promoter and Spa10-GFP was the only source of

Spa10.

N-terminal tagging of proteins with a fluorescent proteinunder alcA promoter control. About 1.0 kb of the 50 region

of the 4.7 kb apsB gene starting from the start codon was

amplified with primers apsB_AscI_fwd and apsB-1_PacI_rev

(AscI and PacI restriction sites are used and in italics on

the list), cloned into the pMCB17apx vector, resulting in

pYZ6. The plasmid was subsequently transformed into

TN02A3. After homologous recombination of the circular

plasmid this yields the N-terminally fused protein expressed

under the control of the alcA promoter and a truncated 50-end fused to the natural promoter. Using primers alcA

check fwd/GFP-ApsB check rev (outside of 1kb ORF),

homologous recombination was confirmed.Besides a C-terminally tagged version of Spa10, we also

created a N-terminal GFP fusion protein using the same

pMCB17apx vector. The constructed plasmid and strain are

pXL2 and SXL1 respectively.The N-terminal fusion of Spa18 with GFP was created

similarly except replacing pyr4 with pyroA in the

pMCB17apx vector. The corresponding plasmids and

strains are pYZ66 and SYZ75. Both Spa10 and Spa18

were homologously integrated at the gene locus as con-

firmed by PCR. Primers were alcA check fwd/GFP-Spa10

check rev and alcA check fwd/GFP-Spa18 check rev

respectively.

Two potential orthologues of Sfi1 and Cdc31 in S. cerevi-

siae and S. pombe were fused to GFP at the N-terminus in

A. nidulans. A protein with similarity (13%) to Sfi1 was iden-

tified in A. nidulans and named SfiA (AN0704). It contains

the Sfi1-specific domain pfam08457 (Bit Score: 694.92, E-

value: 0e 1 00). A. nidulans sfiA is a 3319 bp gene residing

on chromosome VIII with a predicted intron at 65–140 bp

(confirmed by RNAseq data), encoding a 1080 aa (MW

126.1 kDa) protein. Using the same N-terminal tagging

strategy, 1kb of sfiA was cloned into the pMCB17apx vec-

tor, resulting in pMS3. The homologous integration was

confirmed with primers alcA check fwd/GFP-SfiA check rev.



http://www.fgsc.net/Apergillus/KO_Cassettes.htm

http://www.fgsc.net/Apergillus/KO_Cassettes.htm

The strain was named SMS3. Instead of a 1.0 kb fragment,

full-length cdc31 (0.8 kb) was ligated into the pMCB17apx

vector. The resulting plasmid pMS5 was transformed into

TN02A3 and integrated into the genome, yielding strain

SMS6.

Co-localization of two proteins with GFP andmCherry. To investigate co-localization of Spa18 and ApsB,

ApsB was tagged with mCherry at the N-terminus in the

Spa18-GFP strain SO1312. About 1.0 kb of the 50 region of

the apsB ORF was digested with AscI and PacI from pYZ6,

and ligated into the pMCB17apx-mCherry vector containing

mCherry instead of GFP. The resulting plasmid pYZ27 was

transformed into SO1312 and integrated into the genome

by homologous recombination confirmed by primers alcA

check fwd/GFP-ApsB check rev resulting in strain SYZ47.

Similarly, co-localization of Spa10 and ApsB was done by

transforming pYZ27 into the Spa10-GFP strain SRM212

yielding homologous integration strain SXL9.To investigate the timing of the recruitment of Spa10,

Spa18 and ApsB at septa, they were co-localized with the

actin ring marker TpmA (tropomyosin). TpmA was tagged

with mCherry at the N-terminus in strains where either of

the other three proteins were N-terminally tagged with GFP.

The full-length tpmA gene was amplified from genomic

DNA with the primers TpmA_fwd and TpmA_rev. The PCR

fragment was digested with AscI and PacI then cloned into

AscI-PacI digested pCMB17apx-mCherry vector yielding

pXL9. The plasmid pXL9 was transformed into GFP-

Spa10(SXL1) and GFP-ApsB(SYZ2) yielding strains SXL6

and SXL11. pXL9 was also co-transformed with pAB4–1

into GFP-Spa18 (SYZ75) resulting in strain SXL12. N-

terminally tagged mCherry-TpmA was introduced into a

strain with C-terminally GFP tagged Spa10 (SRM212) or

Spa18 (SO1312), yielding strains SXL13 and SXL16. All

the homologous integration events of TpmA were confirmed

with primers alcA check fwd/GFP-TpmA check rev.

To investigate if sMTOCs remain associated with septa

during mitosis, TubA was tagged with mCherry in a strain

expressing Spa10-GFP and Spa18-GFP by transforming

pSH44 into SRM212 and SO1312 respectively. The result-

ing strains are SXL20 and SXL21. To observe microtubule

emergence from SPBs and sMTOCs, both TubA and Spa18

were tagged with GFP by transforming pNZ64 into the

Spa18-GFP strain (SO1312) yielding strain SXL17.

Bimolecular fluorescence complementation assay. To

analyze the interaction of ApsB and Spa18, a Bimolecular

Fluorescence Complementation assay (BiFC or Split YFP)

was performed. Full-length ApsB was amplified with primers

apsB_AscI_fwd and apsB-4.7_PacI_rev, cloned into

pMCB17apx-YFPC vector, which contained YFPC instead of

GFP, yielding pYZ47. Similarly, full-length spa18 was ampli-

fied with primers SPA18_AscI_fwd and SPA18-f_PacI_rev,

cloned into pMCB17apx-YFPN vector, giving pYZ56. Then

pYZ56 and pYZ47 were co-transformed into TN02A3. After

screening, the resulting strain SYZ61 was examined for

YFP signals. As a negative control we checked for interac-

tion between ApsB and the motor protein UncA by trans-

forming pRM23 (YFPN-uncA) and pYZ47 (YFPC-apsB) into

TN02A3 resulting in strain SXL58. As additional controls,

the individual plasmid pYZ47 or pYZ56 was transformed

into TN02A3 resulting in strains SXL61 and SXL62.

Yeast two hybrid assay. The MatchmakerTM Gold Yeast

Two-Hybrid System (Clontech Laboratories) was used in

this work following the User Manual. Full-length spa18 and

apsB cDNA fragments were amplified by PCR with total

cDNA of TN02A3 as the template (Primers: Y2H_SPA18_N-

deI_F/Y2H_SPA18_BamHI_R and Y2H_ApsB_NdeI_F/

Y2H_ApsB_BamHI_R) and respectively ligated into

pGBKT7 and pGADT7-Rec using NdeI and BamHI restric-

tion sites, resulting in plasmids pYZ51 and pYZ61. Strains

AH109 and Y187 were transformed with pYZ61 and pYZ51

separately using the lithium chloride method and transform-

ants were selected on selective synthetic dropout media as

described in the MatchmakerTM GAL4 Two-Hybrid System

Manual. The resulting strains YSYZ2 and YSYZ1 were

mated yielding YSYZ3. To test the self-activation of BD-

Spa18 and AD-ApsB alone, pYZ51 and pYZ61 were trans-

formed into AH109 separately resulting in strains YSXL1

and YSXL2. To analyze the interaction of the proteins, a

5 ml overnight culture of YSYZ3 in SD medium was incu-

bated overnight at 308C/230 rpm. The culture was diluted to

an OD600 of 1, as well as a dilution series (1:10, 1:100,

1:1000). Then 5 ml of each suspension (1:1–1:1000) was

dropped onto the SD-LW and SD-LWHA agar plates

respectively with positive (SRM49) and negative (SRM50)

controls. YSXL1 and YSXL2 were dropped onto the SD-

WH and SD-LH agar plates including positive and negative

controls.

Light and fluorescence microscopy. For live-cell imaging,

fresh spores were inoculated in 0.5 ml MM (2% glycerol for

alcA promoter induction) with appropriate supplements on

18 x 18 mm cover slip (Roth, Karlsruhe). The samples were

incubated at 288C overnight and then 2 h at RT before

microscopy. VECTASHIELD Mounting Medium with DAPI

(Vector Laboratories) was used for staining nuclei in cells.

Light and fluorescence images were taken with the Zeiss

Microscope “AxioImager Z1” (Carl Zeiss, Jena, Germany)

and the software ZEN pro 2012, using the Planapochromatic

633 or 1003 oil immersion objective lens, and the Zeiss

AxioCam MR camera. Image and video processing was

done with ZEN pro 2012, Adobe Photoshop or ImageJ

(National Institutes of Health, MD, USA). Spinning disc

microscopy was performed with a conventional AxioObserver

Z1 inverted microscope employing a Plan-Apochromat

633/1.40 N.A. oil Ph3 M27 (ZEISS) objective lens, a ZEISS

Multi Laser module with a 488 Diode Laser and a 561nm

OPSL Laser and a spinning disc module CSU-X1M 5000.

Images were captured with a Evolve 512 Camera (photomet-

rics). Images were collected and analyzed using the ZEN

software (ZEISS).Alternatively, for in vivo time-lapse microscopy, cells were

incubated in u-slide 8 well Glass Bottom dishes from Ibidi

(cells in focus) in 0.5 ml minimal medium 1 2% glycerol and

appropriate supplements, and an additional 2 ml of medium

added after overnight incubation. Movies were taken at a

time-lapse interval and maximum projection of a decon-

volved z-stack images were applied. For quantification of

fluorescence intensity, images of 15–20 sections were



taken along the z-axis at 0.27 mm increments. After decon-

volution, projection images of maximum intensity were

obtained, and maximum fluorescence intensities over the

background intensity were used for statistical data analysis.

A 5 3 5-pixel region of interest (ROI) was selected and two

20 3 20-pixel regions around the ROI were used for back-

ground subtraction.

Acknowledgements

This work was supported by the German Science Founda-

tion (DFG, FOR1334) and JST ERATO (Grant Number

JPMJER1502). Y. Zhang and X. Gao were fellows of the

Chinese Scholarship Council (CSC). We would like to thank

B. Oakley (University of Kansas, Lawrence, USA) for sending

us strains LO1591 and LO1906 and Ling Lu (Nanjing Normal

University, Nanjing, China) for strain CPA02. We thank Birgit

Schreckenberger and Elke Wohlmann for excellent technical

assistance.

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Microtubule‐organizing centers of Aspergillus nidulans are … Zhang et al.pdf · required for sMTOC activity. This is the first evidence for a septum-specific protein, Spa10, as