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The Spitzenkörper: a choreographer of fungal growth and
morphogenesis
Meritxell Riquelme and Eddy Sánchez-León
The Spitzenkörper (SPK) is a multicomponent pleomorphic
structure found at hyphal apices. It is necessary to maintain
hyphal growth and morphogenesis in numerous fungal species,
including plant and human pathogens. At the turn of the 21st
century extraordinary advances in protein tagging technology
and live microscopy allowed uncovering the main molecular
constituents of the SPK. Distinct layers of macrovesicles and
microvesicles, each carrying different cell wall synthetic
enzymes, along with the actin cytoskeleton and related
proteins are some of the components that make up the SPK.
One of the biggest current challenges is to decipher the
functional relationship between the SPK components and
macromolecular complexes, such as the polarisome and the
exocyst, which partially co-localize within the hyphal dome.
Addresses
Department of Microbiology, Center for Scientific Research and Higher
Education of Ensenada (CICESE), Carretera Ensenada-Tijuana No. 3918,
Ensenada, Baja California 22860, Mexico
Corresponding author: Riquelme, Meritxell (riquelme@cicese.mx)
Current Opinion in Microbiology 2014, 20:27–33
This review comes from a themed issue on Host–microbe interac-
tions: fungi
Edited by Jay C Dunlap and Jean Paul Latgé
http://dx.doi.org/10.1016/j.mib.2014.04.003
1369-5274/# 2014 Elsevier Ltd. All rights reserved.
Introduction
The Spitzenkörper (SPK) was first identified almost a
century ago as an apical body stained with iron hematox-
ylin in fixed hyphae of Coprinus narcoticus and Coprinus
sterquilinus [1] and was later recognized as a phase-dark
body at tips of actively growing hyphae of Polystictus
versicolor [2]. Several decades after these observations,
research conducted in a few labs revealed that the SPK
plays a pivotal role in maintaining apical growth and
determining hyphal growth direction and morphology
[3–5]. It was suggested that the SPK functions as a Vesicle
Supply Center (VSC) to where vesicles accumulate, and
from where they migrate in any random direction toward
the apical cell surface, delivering cell wall-building
enzymes [6]. By programming the VSC to advance while
delivering units of growth to the plasma membrane, the
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Fungus Simulator, a computer program that gave rise to
the hyphoid equation for fungal morphogenesis, allowed
scientists to duplicate morphogenetic events in hyphae of
Neurospora crassa, Rhizoctonia solani, and Aspergillus niger
[3,4,7].
Given the vulnerable nature of the SPK, any method that
perturbs hyphal growth when samples are not treated
gently, can impact its presence and behavior. At the turn
of the 21st century, the improvement of new molecular
tools and the advancement of live imaging technology
allowed a growing number of Fungal Biology labs to
revive the research on this structure [8,9]. The use of
fluorescent dyes, such as FM4-64 (N-(3-triethylammo-
niumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl)
pyridinium dibromide) and the development of fluor-
escent protein tagging have allowed analyses of some
of the key components of the SPK in living growing cells
[10]. Still, there are only a handful of reviews on the SPK
[11–15].
Architectural design of the SPK
Although not an organelle in the strictest sense, the SPK
is often regarded as such given its capacity to tightly hold
all its components together and move as a single unit
within the apical dome, as seen by phase-contrast micro-
scopy (Figure 1). Transmission electron microscopy
(TEM) revealed the SPK as a conspicuous accumulation
of vesicles, together with ribosomes, actin microfilaments
and an amorphous or granular material of undefined
nature [16–20]. The vesicles occupying the SPK were
predicted to carry the enzymes required for cell wall
synthesis as well as the enzymes secreted to the extra-
cellular space [21]. The localization of chitin synthases
(CHS) at the tips of several fungal species [22,23] pro-
vided the decisive proof that the cell wall building process
is a polarized mechanism with a maximum at the tip.
However, the precise localization of CHS within the apex
was not discerned. Hyphae of the ascomycete N. crassa
display one of the most prominent SPK (Figure 1), which
makes this species an excellent model system to study in
great detail the ontogenesis, composition and mode of
operation of the SPK. By using fluorescent protein tag-
ging, it was possible to identify CHS at the core of the
SPK, where microvesicles concentrate as observed by
TEM [24,25]. The joint presence of CHS and microve-
sicles in the SPK core supports the role of chitosomes, as
the microvesicular carriers of CHS activity to the growing
tip. In contrast, GS-1, a protein necessary for glucan
synthase activity, and RGF-1, a RHO-1 specific GEF
Current Opinion in Microbiology 2014, 20:27–33
28 Host–microbe interactions: fungi
Figure 1
N. crassa
T. viride
A. niger
Current Opinion in Microbiology
Hyphal apices of Neurospora crassa, Trichoderma viride and Aspergillus
niger imaged by light phase-contrast microscopy. Note phase-dark
Spitzenkörper at the foremost point of the apical dome (red arrow). Scale
bars 5 mm.
(GDP-GTP exchange factor) were both found in the
macrovesicle-rich region surrounding the CHS core
(Figure 2) [26,27]. Remarkably, in N. crassa the local-
ization of the putative catalytic subunit of the glucan
synthase complex, FKS-1, partially coincides with that
displayed by the above-mentioned markers at the SPK
Current Opinion in Microbiology 2014, 20:27–33
outer layer (Sánchez-León E et al., unpublished). These
exciting results revealing key aspects of the biochemical
nature of the vesicle cargo in the SPK, also unveiled the
morphological–functional differences that exist among
those vesicles [26].
Microtubules (MTs) can be found converging at, and in
some cases even traversing, the SPK [28,29] and are
thought to serve as tracks for vesicle delivery to the
SPK. The cell end marker protein TeaA, found at
hyphal tips in A. nidulans, is important not only to
maintain the shape of the hyphal tip but also to main-
tain the convergence of MTs and for proper localization
of components of the growth machinery at the tips [30].
The detection of actin at the SPK of different fungal
species by immunolabeling, phalloidin staining and
electron microscopy, suggested that it is the most
widely distributed structural cytoskeleton by which
integrity of the apical vesicles might be controlled at
the SPK [18,19,31]. More recently, F-actin and actin
binding proteins, such as tropomyosin, have been ident-
ified at the SPK in living cells of A. nidulans and N.
crassa [32–35]. In the latter, by using Lifeact-GFP, it
was possible to clearly observe F-actin confined to the
core of the SPK (Figure 2) [33]. The homologues of
Saccharomyces cerevisiae Myo2p (myosin V), and its regu-
latory light chain Mlc1p, have been localized at the
SPK in A. nidulans, Candida albicans and the rice blast
fungus Magnaporthe oryzae [36,37,38]. Furthermore, in
an A. nidulans MyoE deletion strain, the vesicular-
associated v-SNARE synaptrobrevin SynA no longer
localized to the SPK [36], thus, supporting the idea
that the actin cytoskeleton is mediating the flow of
vesicles in and out of the SPK. Additionally, F-actin
polymerization nucleating agents, such as the formins
SepA in A. nidulans [39] and Bni1 in C. albicans [37] and
N. crassa [40], have been localized at the SPK. This
organization of the cytoskeleton suggests the SPK to be
a transfer station, where vesicles trafficking on MTs are
discharged and loaded onto an array of actin microfila-
ments that emanate from the SPK, to be delivered to
the expanding plasma membrane [21].
Diversity of SPK across the fungal taxa
Within a given fungal species, the SPK is a highly
dynamic and pleomorphic multicomponent complex that
changes in shape, size and position within the hyphal
apex, although maintaining a basic structural organization
[41]. At a broader level, diverse SPK patterns have been
observed among some of the different fungal taxa stu-
died, which include important plant and animal fungal
pathogens. Many members of the Ascomycota and Basi-
diomycota, including plant and human pathogens, display
a conspicuous SPK at their hyphal tips. Light and electron
microscopy analyses revealed several SPK patterns
among the dikarya fungi [17,41]. A SPK has also been
recognized in A. macrogynus [20], previously classified in
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 The fungal Spitzenkörper Riquelme and Sánchez-León 29

Figure 2

Lifeact-GFP (a) GS-1-GFP (b) CHS-1-mChFP (c)

SEC-6-GFP
Cell Wall

F-actin
PM
Macrovesicles

CHS-1-mChFP CHS-1-mChFP
EXO-70-GFP (d)
Microvesicles
Chitosomes

(f)
SPK

MERGED MERGED
FM4-64 (e)
SPK core

Mitochondria

Current Opinion in Microbiology

Hyphal apices of Neurospora crassa showing the main components of the Spitzenkörper (SPK). (a–d) Confocal laser scanning microscopy showing the
localization of different fluorescently tagged proteins at the SPK; (a) chitin synthase 1 tagged with mChFP (CHS-1-mChFP) and the actin reporter
Lifeact-GFP co-localize at the SPK core. Note the endocytic subapical collar labeled by Lifeact-GFP (arrow). (b) CHS-1-mChFP occupies the core of
the SPK, whereas the glucan synthase related protein GS-1 tagged with GFP occupies the outer layer of the SPK. (c) The exocyst component SEC-6-
GFP (arrow) is found at the hyphal apical dome, in the region of the plasma membrane immediately in front of the SPK core elucidated by CHS-1-
mChFP. (d) The exocyst component EXO-70-GFP localizes at the SPK outer layer. Note the highest gradient of EXO-70-GFP at the most proximal
region of the SPK, close to the apex. (e) The lipophilic dye FM4-64 [10 mM] labels the outer layer of the SPK region and the apical plasma membrane.
Scale bar 5 mm. (f) Ultrastructure of a hyphal tip of N. crassa imaged by Transmission Electron microscopy. Scale bar 500 nm. Magnifications (white
boxes) shown at the top. PM, plasma membrane. Lifeact-GFP N. crassa strain was kindly provided by R Mouriño-Pérez lab, CICESE, Ensenada, Baja
California, Mexico. Electron micrograph was a generous gift of R Roberson, Arizona State University, Tempe, USA.

the Chitridriomycota, and currently in the Blastocla-
diomycota, and in Basidiobolus sp. [42], from the Ento-
mophtoromycotina (incertae sedi). It is somewhat
controversial whether members of the Mucoromycotina
(previously recognized as Zygomycota), such as Rhizo-
pus, Phycomyces, Mucor and Gilbertella, and Gigaspora, an
obligate symbiont belonging to the Glomeromycota,
the vesicular arbuscular mycorrhyzae, have a SPK in
the strict sense. Some authors [17,43,44], while describ-
ing an accumulation of vesicles at the hyphal apex of
these taxa, do not recognize it as a SPK as it had been
previously suggested [16]. Similarly, within the fungal
plant pathogens, germ tubes of the rusts Uromyces
phaseoli and Puccinia graminis fsp. Tritici [10,45], and
dikaryotic hyphae of the smut Ustilago maydis [46]
display an apical cluster at their hyphal tips that could
correspond to a SPK-like structure.
Despite earlier TEM observations showing an accumu-
lation of vesicles (65 nm in diameter) at mating projections
tips of S. cerevisiae, no SPK was recognized in this organism
[47]. Recently, live imaging of mating projections of S.
cerevisiae and C. albicans expressing GFP-tagged proteins
and stained with FM4-64, has led some authors to recog-
nize the existence of a SPK-like structure [48].
Thus far the stratified distribution of vesicles with distinct
biochemical and morphological characters described
above for the SPK of N. crassa, i.e. with macrovesicles
surrounding chitosomes (Figures 2 and 3), has not been
confirmed in other fungal species. However, it would be
remarkable if a similar division of function associated to
the distribution of differently sized vesicles did not exist
in the variety of SPK patterns identified across the fungal
kingdom.

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30 Host–microbe interactions: fungi

Figure 3

(a) (b)
Cell Wall
Macrovesicles Chitosomes

SPK
Ribosomes
outer layer

SPA-2
Actin
Microfilaments EXO-70
Microtubule EXO-84
GS-1
SPK core CHS RGF-1
Plasma
YPT-1 FKS-1
Membrane
YPT-3

Current Opinion in Microbiology

Schemes showing the proposed distribution of the main components of the Spitzenkörper (SPK) in Neurospora crassa. (a) Macrovesicles of different
nature surround a core occupied by chitosomes, ribosomes and F-actin. Microtubules reach the SPK and are presumably the tracks for delivering
vesicles to the actin microfilaments. (b) Spatial distribution of molecular components at the SPK. The polarisome component SPA-2 adopts a hand fan
shape that partially co-localizes with the chitin synthases (CHS) at the SPK core; exocyst components EXO-70 and EXO-84 occupy the anterior half of
the SPK outer layer, partially coinciding with the localization of GS-1, a protein needed for glucan synthase activity, FKS-1, the catalytic subunit of the
1,3-Beta-glucan synthase complex and RGF-1, a RHO-1-specific GEF. Rab GTPases YPT-1 and YPT-3 are distributed at distinct SPK layers.

Dynamic interplay of SPK, polarisome and
exocyst
The SPK together with the polarisome and the exocyst
has been considered part of the tip growth apparatus
[12,14,34]. The polarisome complex is part of the machin-
ery that mediates the nucleation of actin microfilaments,
and may link polarity components and signaling pathways
during polarized growth. In C. albicans it has been pro-
posed that the SPK and the polarisome are distinct
structures [37]. In contrast, polarisome components seem
to co-localize partially with the SPK at the hyphal dome in
U. maydis, A. nidulans, M. oryzae, in fast growing cells of
Ashbya gossypii and in N. crassa [38,49–51]. Specifically, in
N. crassa the polarisome component SPA-2 accumulates
at apices of mature hyphae adopting a hand fan shape,
whose base co-localizes partially with the SPK core [52].
Recent analyses of bni1 deletion mutants of C. albicans
and N. crassa have revealed the importance of formins
(considered to be components of the polarisome) in the
thigmotropic response [53,54]. On the basis of these
results, it has been proposed that the thigmotropic beha-
vior of hyphae is determined by polarisome components,
which ultimately regulate the vectorial flow of SPK
vesicles to the hyphal tip [53].
After leaving the SPK and before v-SNARE/t-SNARE
recognition and fusion with the plasma membrane,
vesicles are presumably tethered to sites of exocytosis
by the exocyst, a conserved octameric complex identified
first in S. cerevisiae. Most fungi analyzed have homologs
for all the exocyst components. In A. gossypii Exo70, Sec3
and Sec5 were identified in the SPK in fast growing
hyphae [55]. In N. crassa, independently of growth rate,
EXO-70 and EXO-84 have been found at the proximal
part of the outer layer of the SPK coinciding partially with
the region where macrovesicles have been identified by
TEM [56]. In contrast, SEC-3, SEC-5, SEC-6, SEC-8,
and SEC-15 were found at the apical plasma membrane
(Figure 2), which confirmed the role of the SPK as
distributing center of vesicles that are exocytosed in
delimited regions of the apical plasma membrane. Yet
TEM analysis has not shown but in very few instances
that secretory vesicles fuse with the plasma membrane at
the apex [56]. SEC-5 mutants of N. crassa showed
accumulation of vesicles in the cytoplasm and lacked a
visible SPK, a clear manifestation of secretion impairment
[56]. The accumulation of the v-SNARE SynA at the
SPK and apical plasma membrane in A. nidulans supports
the existence of a regulated vectorial flux of vesicles from
the SPK to specific regions of the plasma membrane [34].
Multiple lines of evidence indicate that the endosomal
pathway couples exocytosis and endocytosis, the yin and
yang of cellular transport [57]. The co-accumulation of
FM4-64 and exocytic markers at the SPK could suggest
its role as a hub regulating a correct balance between
exocytosis and endocytosis. Thus, the subapical endocy-
tic collar described for different fungal species [33–35]
could be preventing exocytosis occurring at sites other
than the apex, by impeding the diffusion of apical

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proteins to regions below the subapical collar and there-
fore delimiting the fusion of vesicles trafficking out of the
SPK to the apical plasma membrane. In C. albicans, a 3D
model for fungal hyphal growth has been recently pro-
posed. The model suggests that the shape of a hypha can
only be generated if a mechanism exists to remove or
inactivate cell wall synthesizing enzymes from the plasma
membrane in the region of the subapical actin collar
[58]. However, other authors have recently estimated
that membrane retrieval via endocytosis is indeed not as
significant as previously suggested [59]. To validate
either model, more experimental data is probably needed
to calculate the actual amount of membrane being
retrieved by endocytosis, which ultimately will allow
elucidating whether a true spatial coupling exists be-
tween endocytosis and exocytosis, as previously proposed
[34].
Finally, the presence of ribosomes in the SPK core
suggests local protein synthesis at the tips. While local
translation of specific mRNAs at tips has yet to be proven
at tips of filamentous fungi, in the pathogenic fungus U.
maydis RNA binding proteins have been shown to have a
role in polarity and pathogenic development [60].
Modus operandi of the SPK
One of the main functions attributed to the SPK is to
act as a receptacle of secretory vesicles, and synchronize
their delivery to the plasma membrane, where they
fuse, to produce new cell wall surface. Therefore, the
understanding of the mechanisms involved in the traffic
regulation of vesicles directed to the SPK opens
another window to know better the SPK modus oper-
andi. Rab GTPases are well-known molecular switches,
which coordinate the budding, transport, tethering and
fusion of secretory vesicles assisted by their interaction
with coat complexes, molecular motors, tethering fac-
tors, and v-SNAREs and t-SNAREs. Although the role
of Rab GTPases has been extensively studied in mam-
malian cells and S. cerevisiae, their participation in the
traffic of the vesicles reaching the SPK is poorly under-
stood. The intracellular analysis of the Rab GTPase
Sec4, in the human pathogens C. albicans and A. fumi-
gatus, not only revealed its localization at hyphal tips
but allowed elucidation of the dynamic pattern of what
might be vesicles associated with Sec4 [61,62]. These
observations are consistent with the notion that Rab
GTPases are indeed somehow involved in the regula-
tion of the traffic of the SPK vesicles. Accordingly, the
orthologs of the mammalian Rab1 and Rab6 in A.
nidulans, RabO and RabC, respectively, were localized
at the SPK [63,64]. Interestingly, the apical distri-
bution of YPT-1 and YPT-3 in N. crassa, homologues
of Rab1 and Rab11, was restricted to the core and outer
layer of the SPK, respectively (Sánchez-León E et al.,
unpublished). This differential arrangement of Rab
GTPases at the SPK suggests that complex regulatory
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The fungal Spitzenkörper Riquelme and Sánchez-León 31
processes of vesicle organization might occur at this
structure in order to maintain the homeostasis of mem-
branous carriers. In an effort to define the flow of
secretory vesicles into and out of the SPK, empirical
and theoretical approaches have been undertaken. For
example, estimation of turnover times of the SPK
vesicles was found similar in R. solani and N. crassa,
despite differences in the hyphal extension rates of
these two fungal species and the type of vesicles
analyzed [24,59].
Recent studies have attributed to the SPK an additional
role as coordinator of developmental transitions that
involve switching between a polarized and a non-polar-
ized stage. Accordingly, the transcription factor FlbB,
involved in conidiophore development in A. nidulans, is
presumably translocated from the SPK to the apical
nuclei in order to form a transcriptional complex with
FlbD, needed for the expression of the conidiation-
specific factor BrlA [65]. Moreover, in spores of A. nidulans
the calmodulin (CaM) Ca2+ receptor AnCaM was
observed at regions of germ tube emergence, in what
appeared to be an incipient SPK, and also at active
growing sites of germ tubes and mature hyphae. The
regulatable expression of AnCaM contributed to revers-
ible morphogenetic changes, which were highly associ-
ated with the presence of this protein at the SPK [66].
Conclusions
Recent research has elucidated the major components of
the SPK. A collective effort is nonetheless needed to
achieve better temporal resolution of microscopic data so
as to elucidate the routes of vesicles in and out of the SPK
and the associated cytoskeletal tracks and motors. In the
long run, this will allow better discernment of whether all
vesicles undergo a full fusion process, during which the
vesicle collapses and the entirety of its membrane merges
with the target membrane, or if they follow a kiss-and-run
fusion mechanism.
Both crio-TEM and high-resolution electron tomography
have allowed a detailed two-dimensional and three-
dimensional analysis of hyphal tips, providing further
understanding of the organization of the SPK [67]. Never-
theless, to better link the information obtained by live
microscopy of fluorescently labeled proteins and the
ultrastructure of the SPK we need to develop correlative
light and electron microscopy for fungal hyphae.
Acknowledgements
MR has been funded by the Mexican National Council for Science and
Technology, CONACYT (U-45818Q, B0C022, and CONACYT-DFG
75306). ES is supported by CONACYT (PhD Scholarship 252181). We
thank R Roberson from Arizona State University, Tempe for generously
providing the TEM image of N. crassa in Figure 2 and RR Mouriño-Pérez
from CICESE, Ensenada for providing the N. crassa Lifeact-GFP
expressing strain. We are grateful to S Bartnicki-Garcı́a for critically reading
the manuscript.
Current Opinion in Microbiology 2014, 20:27–33
32 Host–microbe interactions: fungi
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Current Opinion in Microbiology 2014, 20:27–33