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
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
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
288 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
MTOCs in Aspergillus 289
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
290 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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
MTOCs in Aspergillus 291
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
292 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
MTOCs in Aspergillus 293
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
294 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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 in Aspergillus 295
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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
296 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
MTOCs in Aspergillus 297
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
298 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
MTOCs in Aspergillus 299
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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
300 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
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.
References
Adams, I.R., and Kilmartin, J.V. (1999) Localization of core
spindle pole body (SPB) components during SPB duplica-
tion in Saccharomyces cerevisiae. J Cell Biol 145: 809–823.Avena, J.S., Burns, S., Yu, Z., Ebmeier, C.C., Old, W.M.,
Jaspersen, S.L., and Winey, M. (2014) Licensing of yeast
centrosome duplication requires phosphoregulation of
sfi1. PLoS Genet 10: e1004666.Becker, B.E., and Cassimeris, L. (2005) Cytoskeleton:
microtubules born on the run. Curr Biol 15: R551–R554.Bergs, A., Ishitsuka, Y., Evangelinos, M., Nienhaus, G.U.,
and Takeshita, N. (2016) Dynamics of actin cables in
polarized growth of the filamentous fungus Aspergillus
nidulans. Front Microbiol 7: 682.Berlow, R.B., Dyson, H.J., and Wright, P.E. (2015) Func-
tional advantages of dynamic protein disorder. FEBS Lett
589: 2433–2440.Borek, W.E., Groocock, L.M., Samejima, I., Zou, J., de
Lima Alves, F., Rappsilber, J., and Sawin, K.E. (2015)
Mto2 multisite phosphorylation inactivates non-spindle
microtubule nucleation complexes during mitosis. Nat
Commun 6: 7929.Bouhlel, I.B., Ohta, M., Mayeux, A., Bordes, N., Dingli, F.,
Boulanger, J., et al. (2015) Cell cycle control of spindle
pole body duplication and splitting by Sfi1 and Cdc31 in
fission yeast. J Cell Sci 128: 1481–1493.
Brachat, A., Kilmartin, J.V., Wach, A., and Philippsen, P.
(1998) Saccharomyces cerevisiae cells with defective
spindle pole body outer plaques accomplish nuclear
migration via half-bridge-organized microtubules. Mol Biol
Chem 9: 977–991.Burns, S., Avena, J.S., Unruh, J.R., Yu, Z., Smith, S.E.,
Slaughter, B.D., et al. (2015) Structured illumination with
particle averaging reveals novel roles for yeast centro-
some components during duplication. eLife 4: e08586.Chen, P., Gao, R., Chen, S., Pu, L., Li, P., Huang, Y., and
Lu, L. (2012) A pericentrin-related protein homolog in
Aspergillus nidulans plays important roles in nucleus
positioning and cell polarity by affecting microtubule orga-
nization. Eukaryot Cell 11: 1520–1530.
De Souza, C.P., Hashmi, S.B., Osmani, A.H., Andrews, P.,
Ringelberg, C.S., Dunlap, J.C., and Osmani, S.A. (2013)
Functional analysis of the Aspergillus nidulans kinome.
PLoS One 8: e58008.
Dhani, D.K., Goult, B.T., George, G.M., Rogerson, D.T.,
Bitton, D.A., Miller, C.J., et al. (2013) Mzt1/Tam4, a fis-
sion yeast MOZART1 homologue, is an essential compo-
nent of the gamma-tubulin complex and directly interacts
with GCP3(Alp6). Mol Biol Cell 24: 3337–3349.Ding, R., West, R.R., Morphew, D.M., Oakley, B.R., and
McIntosh, J.R. (1997) The spindle pole body of Schizo-
saccharomyces pombe enters and leaves the nuclear
envelope as the cell cycle proceeds. Mol Biol Cell 8:
1461–1479.Efimov, A., Kharitonov, A., Efimova, N., Loncarek, J., Miller,
P.M., Andreyeva, N., et al. (2007) Asymmetric CLASP-
dependent nucleation of noncentrosomal microtubules at
the trans-Golgi network. Dev Cell 12: 917–930.Elliott, S., Knop, M., Schlenstedt, G., and Schiebel, E.
(1999) Spc29p is a component of the Spc110p subcom-
plex and is essential for spindle pole body duplication.
Proc Natl Acad Sci USA 96: 6205–6210.Elserafy, M., Saric, M., Neuner, A., Lin, T.C., Zhang, W.,
Seybold, C., et al. (2014) Molecular mechanisms that
restrict yeast centrosome duplication to one event per
cell cycle. Curr Biol 24: 1456–1466.
Flory, M.R., Morphew, M., Joseph, J.D., Means, A.R., and
Davis, T.N. (2002) Pcp1p, an Spc110p-related calmodulin
target at the centrosome of the fission yeast Schizosac-
charomyces pombe. Cell Growth Differ 13: 47–58.
Hamada, T. (2014) Microtubule organization and
microtubule-associated proteins in plant cells. Int Rev
Cell Mol Biol 312: 1–52.
Heitz, M.J., Petersen, J., Valovin, S., and Hagan, I.M.
(2001) MTOC formation during mitotic exit in fission
yeast. J Cell Sci 114: 4521–4532.
Hill, T.W., and K€afer, E. (2001) Improved protocols for
Aspergillus minimal medium: trace element and minimal
medium salt stock solutions. Fungal Genet Newsl 48:
20–21.Horio, T., Uzawa, S., Jung, M.K., Oakley, B.R., Tanaka, K.,
and Yanagida, M. (1991) The fission yeast g-tubulin is
essential for mitosis and is localized at microtubule
organizing centers. J Cell Sci 99: 693–700.Ishitsuka, Y., Savage, N., Li, Y., Bergs, A., Gr€un, N., Kohler,
D., et al. (2015) Superresolution microscopy reveals a
dynamic picture of cell polarity maintenance during direc-
tional growth. Sci Adv 1: e1500947.Jaspersen, S.L., and Winey, M. (2004) The budding yeast
spindle pole body: structure, duplication, and function.
Annu Rev Cell Dev Biol 20: 1–28.Kilmartin, J.V. (2003) Sfi1p has conserved centrin-binding
sites and an essential function in budding yeast spindle
pole body duplication. J Cell Biol 162: 1211–1221.Kilmartin, J.V. (2014) Lessons from yeast: the spindle pole
body and the centrosome. Philos Trans R Soc Lond B
Biol Sci 369: 20130456.Kim, J.M., Zeng, C.J., Nayak, T., Shao, R., Huang, A.C.,
Oakley, B.R., and Liu, B. (2009) Timely septation requires
MTOCs in Aspergillus 301
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
SNAD-dependent spindle pole body localization of
the septation initiation network components in the fila-
mentous fungus Aspergillus nidulans. Mol Biol Cell 20:
2874–2884.
Kollman, J.M., Merdes, A., Mourey, L., and Agard, D.A.
(2011) Microtubule nucleation by gamma-tubulin com-
plexes. Nat Rev Mol Cell Biol 12: 709–721.Konzack, S., Rischitor, P., Enke, C., and Fischer, R. (2005)
The role of the kinesin motor KipA in microtubule organi-
zation and polarized growth of Aspergillus nidulans. Mol
Biol Cell 16: 497–506.Lai, J., Koh, C.H., Tjota, M., Pieuchot, L., Raman, V.,
Chandrababu, K.B., et al. (2012) Intrinsically disordered
proteins aggregate at fungal cell-to-cell channels and reg-
ulate intercellular connectivity. Proc Natl Acad Sci USA
109: 15781–15786.Lee, I.J., Wang, N., Hu, W., Schott, K., Bahler, J., Giddings,
T.H. Jr, et al. (2014) Regulation of spindle pole body
assembly and cytokinesis by the centrin-binding protein
Sfi1 in fission yeast. Mol Biol Cell 25: 2735–2749.L€uders, J. (2012) The amorphous pericentriolar cloud takes
shape. Nat Cell Biol 14: 1126–1128.L€uders, J., and Stearns, T. (2007) Microtubule-organizing
centres: a re-evaluation. Nat Rev Mol Cell Biol 8:
161–167.Lynch, E.M., Groocock, L.M., Borek, W.E., and Sawin, K.E.
(2014) Activation of the gamma-tubulin complex by the
Mto1/2 complex. Curr Biol 24: 896–903.Manck, R., Ishitsuka, Y., Herrero, S., Takeshita, N.,
Nienhaus, G.U., and Fischer, R. (2015) Genetic evidence
for a microtubule-capture mechanism during polarised
growth of Aspergillus nidulans. J Cell Sci 128: 3569–3582.Masuda, H., Mori, R., Yukawa, M., and Toda, T. (2013)
Fission yeast MOZART1/Mzt1 is an essential gamma-
tubulin complex component required for complex recruit-
ment to the microtubule organizing center, but not its
assembly. Mol Biol Cell 24: 2894–2906.Morris, N.R., and Enos, A.P. (1992) Mitotic gold in a mold:
Aspergillus genetics and the biology of mitosis. Trends
Genet 8: 32–37.Oakley, B.R. (1992) Gamma-tubulin: the microtubule orga-
nizer? T Cell Biol 2: 1–5.
Oakley, B.R. (1995) g-Tubulin and the fungal microtubule
cytoskeleton. Can J Bot 73: S352–S358.Oakley, C.E., and Oakley, B.R. (1989) Identification of
gamma-tubulin, a new member of the tubulin superfamily
encoded by mipA gene of Aspergillus nidulans. Nature
338: 662–664.Oakley, B.R., Oakley, C.E., Yoon, Y., and Jung, K.M. (1990)
g-Tubulin is a component of the spindle pole body in
Aspergillus nidulans. Cell 61: 1289–1301.Oakley, B.R., Paolillo, V., and Zheng, Y. (2015) gamma-
Tubulin complexes in microtubule nucleation and beyond.
Mol Biol Cell 26: 2957–2962.Osmani, A.H., Davies, J., Liu, H.L., Nile, A., and Osmani,
S.A. (2006) Systematic deletion and mitotic localization
of the nuclear pore complex proteins of Aspergillus nidu-
lans. Mol Biol Cell 17: 4946–4961.Pe~nalva, M.A., Galindo, A., Abenza, J.F., Pinar, M.,
Cacagno-Pizarelli, A.M., Arst, H.N., and Pantazopoulou,
A. (2012) Searching for gold beyond mitosis: mining
intracellular membrane traffic in Aspergillus nidulans. Cell
Logist 2: 2–14.
Pereira, G., Hofken, T., Grindlay, J., Manson, C., and
Schiebel, E. (2000) The Bub2p spindle checkpoint links
nuclear migration with mitotic exit. Mol Cell 6: 1–10.Pereira, G., and Schiebel, E. (1997) Centrosome-microtu-
bule nucleation. J Cell Sci 110: 295–300.Petry, S., Groen, A.C., Ishihara, K., Mitchison, T.J., and
Vale, R.D. (2013) Branching microtubule nucleation in
Xenopus egg extracts mediated by augmin and TPX2.
Cell 152: 768–777.Piel, M., and Tran, P.T. (2009) Cell shape and cell division
in fission yeast. Curr Biol 19: R823–R827.Riquelme, M., Fischer, R., and Bartnicki-Garcia, S. (2003)
Apical growth and mitosis are independent processes in
Aspergillus nidulans. Protoplasma 222: 211–215.Riquelme, M., Yarden, O., Bartnicki-Garcia, S., Bowman,
B., Castro-Longoria, E., Free, S.J., et al. (2011) Architec-
ture and development of the Neurospora crassa hypha –
a model cell for polarized growth. Fungal Genet Biol 115:
683–695.Rose, M.D., and Fink, G.R. (1987) KAR1, a gene required
for function of both intranuclear and extranuclear microtu-
bules in yeast. Cell 48: 1047–1060.R€uthnick, D., and Schiebel, E. (2016) Duplication of the
yeast spindle pole body once per cell cycle. Mol Cell Biol
36: 1324–1331.
Salogiannis, J., and Reck-Peterson, S.L. (2017) Hitchhiking:
a non-canonical mode of microtubule-based transport.
Trends Cell Biol 27: 141–150.
Sambrook, J., and Russel, D.W. (1999) Molecular Cloning:
A Laboratory Manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York.Samejima, I., Laurenco, P.C.C., Snaith, H.A., and Sawin,
K.E. (2005) Fission yeast mto2p regulates microtubule
nucleation by the centrosomin-related protein mto1p. Mol
Biol Cell 16: 3040–3051.Samejima, I., Miller, V.J., Groocock, L.M., and Sawin, K.E.
(2008) Two distinct regions of Mto1 are required for normal
microtubule nucleation and efficient association with the
gamma-tubulin complex in vivo. J Cell Sci 121: 3971–3980.Samejima, I., Miller, V.J., Rincon, S.A., and Sawin, K.E.
(2010) Fission yeast Mto1 regulates diversity of cytoplasmic
microtubule organizing centers. Curr Biol 20: 1959–1965.Sanchez-Huertas, C., Freixo, F., Viais, R., Lacasa, C.,
Soriano, E., and L€uders, J. (2016) Non-centrosomal
nucleation mediated by augmin organizes microtubules in
post-mitotic neurons and controls axonal microtubule
polarity. Nat Commun 7: 12187.Sawin, K.E., Lourenco, P.C.C., and Snaith, H.A. (2004) Micro-
tubule nucleation at non-spindle pole body microtubule-
organizing centers requires fission yeast centrosomin-
related protein mod20p. Curr Biol 14: 763–775.Seybold, C., Elserafy, M., Ruthnick, D., Ozboyaci, M.,
Neuner, A., Flottmann, B., et al. (2015) Kar1 binding to
Sfi1 C-terminal regions anchors the SPB bridge to the
nuclear envelope. J Cell Biol 209: 843–861.Shen, K.F., Osmani, A.H., Govindaraghavan, M., and
Osmani, S.A. (2014) Mitotic regulation of fungal cell-to-
cell connectivity through septal pores involves the NIMA
kinase. Mol Biol Cell 25: 763–775.
302 Y. Zhang et al. �
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303
Shukla, N., Osmani, A.H., and Osmani, S.A. (2017) Micro-tubules are reversibly depolymerized in response tochanging gaseous microenvironments within Aspergillusnidulans biofilms. Mol Biol Cell 28: 634–644.
Snaith, H.A., and Sawin, K.E. (2003) Fission yeast mod5p
regulates polarized growth through anchoring of tea1p atcell tips. Nature 423: 647–651.
Steinberg, G. (2012) The transport machinery for motility offungal endosomes. Fungal Genet Biol 49: 675–776.
Steinberg, G. (2014) Endocytosis and early endosome
motility in filamentous fungi. Curr Opin Microbiol 20:10–18.
Straube, A., Brill, M., Oakley, B.R., Horio, T., andSteinberg, G. (2003) Microtubule organization requirescell cycle-dependent nucleation at dispersed cytoplasmic
sites: polar and perinuclear microtubule organizing cen-ters in the plant pathogen Ustilago maydis. Mol Biol Cell14: 642–657.
Suelmann, R., Sievers, N., and Fischer, R. (1997) Nuclear
traffic in fungal hyphae: in vivo study of nuclear migrationand positioning in Aspergillus nidulans. Mol Microbiol 25:757–769.
Suelmann, R., Sievers, N., Galetzka, D., Robertson, L.,Timberlake, W.E., and Fischer, R. (1998) Increased
nuclear traffic chaos in hyphae of apsB mutants of Asper-gillus nidulans: molecular characterization of apsB and invivo observation of nuclear behaviour. Mol Microbiol 30:831–842.
Takeshita, N., Manck, R., Gr€un, N., de Vega, S., and
Fischer, R. (2014) Interdependence of the actin and themicrotubule cytoskeleton during fungal growth. Curr OpinMicrobiol 20: 34–41.
Takeshita, N., Vienken, K., Rolbetzki, A., and Fischer, R.(2007) The Aspergillus nidulans putative kinase, KfsA
(kinase for septation), plays a role in septation and isrequired for efficient asexual spore formation. FungalGenet Biol 44: 1205–1214.
Todd, R.B., Davis, M.A., and Hynes, M.J. (2007) Genetic
manipulation of Aspergillus nidulans: meiotic progeny forgenetic analysis and strain construction. Nat Protoc 2:811–821.
Tomlin, G.C., Morrell, J.L., and Gould, K.L. (2002) The spindlepole body protein Cdc11p links Sid4p to the fission yeast
septation initiation network. Mol Biol Cell 13: 1203–1214.Veith, D., Scherr, N., Efimov, V.P., and Fischer, R. (2005)
Role of the spindle-pole body protein ApsB and the
cortex protein ApsA in microtubule organization and
nuclear migration in Aspergillus nidulans. J Cell Sci 118:
3705–3716.Venkatram, S., Jennings, J.L., Link, A., and Gould, K.L.
(2005) Mto2p, a novel fission yeast protein required for
cytoplasmic microtubule organization and anchoring of
the cytokinetic actin ring. Mol Biol Cell 16: 3052–3063.Waring, R.B., May, G.S., and Morris, N.R. (1989) Charac-
terization of an inducible expression system in Aspergillus
nidulans using alcA and tubulin coding genes. Gene 79:
119–130.
Wright, P.E., and Dyson, H.J. (2009) Linking folding and
binding. Curr Opin Struct Biol 19: 31–38.
Xiong, Y., and Oakley, B.R. (2009) In vivo analysis of the
functions of gamma-tubulin-complex proteins. J Cell Sci
122: 4218–4227.Yang, L., Ukil, L., Osmani, A., Nahm, F., Davies, J., De
Souza, C.P.C., et al. (2004) Rapid production of gene
replacement constructs and generation of a green fluo-
rescent protein-tagged centromeric marker in Aspergillus
nidulans. Eukaryot Cell 3: 1359–1362.Yao, X., Arst, H.N. Jr, Wang, X., and Xiang, X. (2015) Dis-
covery of a vezatin-like protein for dynein-mediated early
endosome transport. Mol Biol Cell 26: 3816–3827.
Yelton, M.M., Hamer, J.E., and Timberlake, W.E. (1984)
Transformation of Aspergillus nidulans by using a trpC
plasmid. Proc Natl Acad Sci USA 81: 1470–1474.Zekert, N., and Fischer, R. (2009) The Aspergillus nidulans
kinesin-3 UncA motor moves vesicles along a subpopula-
tion of microtubules. Mol Biol Cell 20: 673–684.Zekert, N., Veith, D., and Fischer, R. (2010) Interaction of
the Aspergillus nidulans microtubule-organizing center
(MTOC) component ApsB with gamma-tubulin and evi-
dence for a role of a subclass of peroxisomes in the for-
mation of septal MTOCs. Eukaryot Cell 9: 795–805.
Zimmerman, S., Tran, P.T., Daga, R.R., Niwa, O., and Chang, F.
(2004) Rsp1p, a J domain protein required for disassembly
and assembly of microtubule organizing centers during the
fission yeast cell cycle. Dev Cell 6: 497–509.
Supporting informationAdditional supporting information may be found in the
online version of this article at the publisher’s web-site.
MTOCs in Aspergillus 303
VC 2017 John Wiley & Sons Ltd, Molecular Microbiology, 106, 285–303