Downloaded from www.asmscience.org by
IP: 131.211.104.131
On: Mon, 25 Sep 2017 14:28:11
Cell Biology of Hyphal Growth
GERO STEINBERG,
1,2
MIGUEL A. PEÑALVA,
3
MERITXELL RIQUELME,
4
HAN A. WÖSTEN,
2
and STEVEN D. HARRIS
5
1
Department of Biosciences, College of Live and Environmental Sciences, University of Exeter, EX1 1TE Exeter,
United Kingdom;
2
Department of Biology, University of Utrecht, 3584 CH, Utrecht, The Netherlands;
3
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Madrid,
28040, Spain;
4
Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada,
CICESE, Ensenada, Baja California C.P. 22860, Mexico;
5
Center for Plant Science Innovation and
Department of Plant Pathology, University of Nebraska, Lincoln, NE 68588-0660
ABSTRACT Filamentous fungi are a large and ancient clade of
microorganisms that occupy a broad range of ecological niches.
The success of filamentous fungi is largely due to their elongate
hypha, a chain of cells, separated from each other by septa.
Hyphae grow by polarized exocytosis at the apex, which allows
the fungus to overcome long distances and invade many
substrates, including soils and host tissues. Hyphal tip growth is
initiated by establishment of a growth site and the subsequent
maintenance of the growth axis, with transport of growth
supplies, including membranes and proteins, delivered by
motors along the cytoskeleton to the hyphal apex. Among the
enzymes delivered are cell wall synthases that are exocytosed
for local synthesis of the extracellular cell wall. Exocytosis is
opposed by endocytic uptake of soluble and membrane-bound
material into the cell. The first intracellular compartment in the
endocytic pathway is the early endosomes, which emerge to
perform essential additional functions as spatial organizers of
the hyphal cell. Individual compartments within septated hyphae
can communicate with each other via septal pores, which allow
passage of cytoplasm or organelles to help differentiation within
the mycelium. This article introduces the reader to more detailed
aspects of hyphal growth in fungi.
INTRODUCTION
Filamentous fungi are a large and ancient clade of mi-
croorganisms that occupy a broad range of ecological
niches (
1, 2). Fungi are recyclers, being major decom-
posers of plant debris (
3); they form mycorrhizal sym-
biosis with 93% of all flowering plant families (
4), and
they serve in the industrial production of proteins (
5).
However, fungi pose a threat to public health, the eco-
system, and our food security (
6, 7). The success of fil-
amentous fungi is largely due to their elongate hypha,
a chain of cells separated from each other by septa (
8).
Hyphae grow rapidly by polarized exocytosis at the
apex (
9–11), which allows the fungus to extend over
long distances and invade many substrates, including
soils and host tissues. Hyphal tip growth is initiated
by establishment of a growth site and the subsequent
maintenance of the growth axis, with transport of
growth supplies, including membranes and proteins, de-
livered by motors along the cytoskeleton to the hyphal
apex (
12). Among the enzymes delivered are cell wall
synthases that are exocytosed for local synthesis of the
extracellular cell wall (
13). Exocytosis is opposed by
endocytic uptake of soluble and membrane-bound ma-
terial into the cell (
14). The first intracellular compart-
ment in the endocytic pathway is the early endosomes
(EEs), which emerge to perform essential additional
functions as spatial organizers of the hyphal cell (
15).
Individual compartments within septated hyphae can
communicate with each other via septal pores, which
allow passage of cytoplasm or organelles (
16) to help
differentiation within the mycelium (
17). This article in-
troduces the reader to more detailed aspects of hyphal
growth in fungi.
Received: 28 November 2016, Accepted: 8 December 2016,
Published: 21 April 2017
Editors: Joseph Heitman, Department of Molecular Genetics and
Microbiology, Duke University Medical Center, Durham, NC 27710;
Neil A. R. Gow, School of Medical Sciences, University of Aberdeen,
Fosterhill, Aberdeen, AB25 2ZD, United Kingdom
Citation: Steinberg G, Peñalva MA, Riquelme M, Wösten HA, Harris
SD. 2017. Cell biology of hyphal growth. Microbiol Spectrum 5(2):
FUNK-0034-2016.
doi:10.1128/microbiolspec.FUNK-0034-2016.
Correspondence: Gero Steinberg,
gsteinberg@exeter.ac.uk
© 2017 American Society for Microbiology. All rights reserved.
ASMscience.org/MicrobiolSpectrum 1
Downloaded from www.asmscience.org by
IP: 131.211.104.131
On: Mon, 25 Sep 2017 14:28:11
REGULATION OF HYPHAL MORPHOGENESIS
Hyphal morphogenesis refers to the complex biologi-
cal processes that directly contribute to the formation of
highly polarized hyphae by filamentous fungi. The two
fundamental processes that underlie hyphal morpho-
genesis are polarity establishment and polarity mainte-
nance. The regulation of these processes over time and
space presumably accounts for much of the variation in
hyphal morphology and growth patterns observed in the
filamentous fungi (
18). Additional processes that con-
tribute to the complexity of hyphal morphology in those
filamentous fungi that belong to the Dikarya include
septation and the formation of septal pores (
19). The
regulation of these processes must also play an impor-
tant role in specifying the overall organization of hyphae
in these fungi. Nevertheless, we are still at an early stage
in understanding the temporal and spatial regulation
of hyphal morphogenesis. Progress has been achieved in
determining how hyphae maintain a polarity axis (
20,
21), but we still know little about how polarization
sites are initially established in germinating spores and
during branch formation. In addition, much remains to
be learned about when and where septa are made. The
following sections will address these points, but before
doing so, it is perhaps useful to focus on the broader
regulatory issue of how morphogenesis is coordinated
with hyphal growth.
Experimentally, we typically grow filamentous fungi
and assess morphogenesis under conditions that are
largely homogeneous and in which all needed nutrients
are available. This is unlikely to be representative of
conditions that hyphae would normally experience in
their natural environment (though there are likely to be
important exceptions). In reality, most hyphae propa-
gate in a spatially heterogeneous environment that is
characterized by a patchy distribution of nutrients. Ac-
cordingly, two possible strategies could conceivably be
invoked to account for hyphal extension under variable
conditions. First, hyphal growth could be a “brute-
force” mechanism by which a hypha harnesses the mor-
phogenetic machinery (i.e., the cytoskeleton and vesicle
trafficking machinery) and turgor pressure to plough
ahead with colonization of the substrate. Extension rates
and the frequency of branching may only be minimally
adjusted to account for a changing environment. Alter-
natively, hyphae may fine-tune extension rates and
morphogenesis to reflect the local environment at the tip
and at incipient sites of branch formation. It seems
reasonable to speculate that some combination of both
strategies is deployed within a given mycelium. How-
ever, the latter strategy would seemingly rely more on
the ability of hyphae to adjust the timing and location
of polarization events, as well as to modulate the degree
of polarity maintenance, in response to local environ-
mental conditions. Insight into how this might occur
comes from the model yeasts Saccharomyces cerevisiae
and Schizosaccharomyces pombe. Both normally utilize
an internal program to specify the position of localized
cell surface expansion and cell wall deposition but are
able to override the program in response to external sig-
nals such as mating pheromones (
22). Accordingly, it is
tempting to speculate that the signaling pathways that
mediate growth and stress responses in filamentous
fungi (e.g., TOR, PKA, HOG) likely interface with the
mechanisms that temporally and spatially regulate hy-
phal morphogenesis. To date, little is known about how
these pathways might impact hyphal morphogenesis,
though there is growing evidence that they do mediate
tropic responses that influence the directionality of hyphal
extension (
23). Characterization of the links between
these pathways and the morphogenetic machinery would
appear to be fertile territory for future investigation.
Spatial Regulation of Hyphal Morphogenesis
The key events underlying the spatial regulation of hy-
phal morphogenesis are the initial specification of a po-
larity axis (polarity establishment) and the subsequent
stabilization of the axis (polarity maintenance). Two
types of polarity establishment events are commonly
observed in a typical hypha: spore polarization and
branch formation (
Fig. 1). In both cases, specification
of a polarity axis occurs in a cell that is otherwise dis-
playing isotropic or apolar growth (i.e., germinating
spores or an existing hyphal compartment). Stabiliza-
tion of the resulting axis requires the recruitment of the
morphogenetic machinery to the specified site. As a re-
sult, cell surface expansion and cell wall deposition are
subsequently confined to a discrete cortical site, which
ultimately leads to the formation of a new hypha.
Detailed studies of S. cerevisiae and S. pombe have
provided significant insight into the nature of the spa-
tial landmarks that direct polarity establishment (
22).
S. cerevisiae utilizes a set of cortical landmark proteins
(the Bud proteins) to specify new polarity axes (
24),
whereas S. pombe relies on microtubule-based delivery
of a set of marker proteins (the Tea proteins) to position
new growth sites (
25). By contrast, comparatively little
is known about mechanisms that specify new polarity
axes in filamentous fungi. The Tea proteins are reason-
ably well conserved (
26) and play a fundamental role
in regulating the directionality of hyphal extension (see
below). A subset of the Bud proteins is also present in
2 ASMscience.org/MicrobiolSpectrum
Steinberg et al.
Downloaded from www.asmscience.org by
IP: 131.211.104.131
On: Mon, 25 Sep 2017 14:28:11
filamentous fungi (27, 28), but instead they regulate
septum formation (see below). Accordingly, at this time,
the identities of any landmark/marker proteins that me-
diate polarity establishment in filamentous fungi remain
a mystery. There is even some question as to whether a
marking system is indeed necessary. Few examples of
spatially biased spore polarization have been observed
(e.g., reference
23), and when reported, it is in response
to signals emanating from a potential plant host. In-
deed, for those fungi whose spores are capable of pro-
ducing multiple hyphae, it appears to be more important
to ensure that the second polarity axis is opposite to
the first. This type of bipolar spore polarization pattern
is often observed and can be perturbed by disruption of
the vesicle trafficking machinery, microtubules, and Tea
marking system (
29, 30).
A unique feature of filamentous fungi that enables
the formation of mycelia is the ability to simultaneously
sustain multiple axes of hyphal polarity. These axes re-
sult in the formation of branches, either lateral branches
that emerge from subapical compartments or apical
branches that form by “splitting” of the hyphal tip (
31).
As with spore polarization, branch formation requires
the initial specification of a polarity axis followed by
its stabilization. To date, the extent to which branching
occurs in a defined pattern has not been fully explored.
Furthermore, relatively little is known about mecha-
nisms potentially involved in spatial regulation of branch
formation in filamentous fungi. Nevertheless, key clues
have emerged from a limited number of studies. For ex-
ample, in Aspergillus nidulans, the absence of the Cdc42
GTPase or the FadA heterotrimeric G protein complex
largely abolishes the formation of lateral branches and
suggests signaling pathways that might be required for
the specification of branch sites (
32) (S. Harris, unpub-
lished results). More intriguingly, the well-characterized
septins appear to play a critical role in the specifica-
tion and/or stabilization of new polarity axes at lateral
branch sites (
33). Elegant work using Ashbya gossypii
suggests that the pivotal function of septins at incip-
ient branch sites is to recognize membrane curvature
(
34). Lastly, evidence suggests that some fungi (e.g.,
A. nidulans, Epichloe festucae) actively suppress lateral
branch formation from regions adjacent to hyphal tips
through the localized accumulation of reactive oxygen
species mediated by NADPH oxidase complexes (
35,
36). This presumably ensures that growth is directed
toward the tip and facilitates migration away from de-
pleted substrate when nutrients are scarce.
A key feature that distinguishes hyphae from yeast
cells is their ability to sustain polarized growth over a
considerable distance. This implies that the regulatory
systems that stabilize polarity axes must be much more
stringent in filamentous fungi to support the long-range
vectorial delivery of secretory vesicles to the hyphal tip.
Although several components of the morphogenetic
machinery have been implicated in the maintenance of
hyphal polarity (
20, 21), the nature and identity of any
regulatory system that organizes the machinery and
marks the hyphal tip remain elusive. It has been firmly
established that spatially coupled exocytosis and endo-
cytosis are essential for the stabilization of polarity axes
(
37, 38), and it has been suggested that this could reflect
the need to recycle one or more cell surface markers
FIGURE 1 Growth patterns in fungal hyphae.
Growth occurs in an isotropic fashion during
spore germination. Specification of a polarity
axis ultimately results in the formation of a hy-
pha that continues to grow at the tip. While tip
growth is maintained, the specification of ad-
ditional polarity axes enables the formation of
septa and lateral branches. Whereas septum
formation is transient, branching results in the
formation of a secondary hypha that also con-
tinues to grow at the tip. Red arrows designate
polarity axes.
ASMscience.org/MicrobiolSpectrum 3
Cell Biology of Hyphal Growth
Downloaded from www.asmscience.org by
IP: 131.211.104.131
On: Mon, 25 Sep 2017 14:28:11
that identify the hyphal tip (39, 40). It should also be
added that a secondary role for the coupling of endo-
cytosis with exocytosis might be to enable the sponta-
neous generation of local asymmetries that are then
amplified to define a polarity axis if no functional
marker is present. To date, the most compelling candi-
date for a hyphal tip marker is the A. nidulans TeaR
protein (a homologue of the S. pombe mod5 cell end
marker). TeaR is associated with exocytic vesicles that
are transported on microtubules and delivered to the cell
surface at the hyphal tip, where it mediates the local
recruitment of the morphogenetic machinery (
41). Re-
cent evidence suggests that membrane-associated TeaR
is rapidly dispersed at hyphal tips, only to be replenished
again via microtubules (
42). This result supports the
existence of a dynamic feedback loop that continually
establishes transient polarity axes at the hyphal tip that
serve to maintain the overall direction of hyphal exten-
sion. The presence of lipid microdomains at hyphal tips
and their importance in the formation of stable polarity
axes raise the possibility that additional regulatory sys-
tems may operate in parallel with TeaR to mark the
hyphal tip (
43, 44).
Filamentous fungi exhibit tropic responses to chemi-
cal, mechanical, electrical, and other environmental
stimuli (see references
45 and 46). The dynamic nature
of polarity maintenance provides a satisfying explana-
tion for how the direction of hyphal extension can be
rapidly reoriented in response to external signals. More-
over, it seems likely that the perception and transduction
of external signals can override internally programmed
polarity systems such as that mediated by TeaR. The
demonstration that a chemotropic response is mediated
by a cell surface pheromone receptor and a mitogen-
activated protein kinase signaling pathway in the root
colonizing fungus Fusarium oxysporum (
23) provides
some insight into how an existing polarity axis can be
subverted by external signals.
Temporal Regulation of
Hyphal Morphogenesis
In uninucleate fungal cells, the maintenance of nu-
clear content requires precise coordination of cellular
morphogenesis with nuclear division. Mechanisms that
couple these two processes have been described in
some detail in S. cerevisiae and relatives. For example,
in S. cerevisiae, regulatory mechanisms focused on the
cyclin-dependent kinase (CDK) Cdc28 coordinate po-
larity transitions with nuclear division at multiple points
in the cell cycle (
47). Candida albicans is a polymorphic
fungus that forms uninucleate hyphal cells, and here
also, CDK plays a critical role in controlling the timing
of polarity establishment, septum formation, and the
transition from budding to hyphal growth (
48). Key
CDK targets include the septins and polarisome pro-
tein Spa2 (
49, 50). The importance of CDK for the
temporal coordination of morphogenesis with the cell
cycle has also been documented in the plant pathogen
Ustilago maydis (
51).
In multinucleate fungal hyphae, the need to coordi-
nate the timing of polarity establishment with nuclear
division is not so apparent. Indeed, A. nidulans mutants
incapable of nuclear division are able to form elongated
hyphae (
52), and mutants that fail to undergo spore
polarization are able to complete multiple rounds of
nuclear division (
29). Despite this apparent lack of de-
pendency, there is some evidence in Aspergillus species
that polarity establishment and septum formation are
under normal circumstances coordinated with nuclear
division (
29, 53, 54). This presumably reflects the need
to maintain a preferred ratio of cytoplasmic volume
per nucleus in growing hyphae. The underlying pro-
cess, which has been termed the duplication cycle (
53),
remains poorly characterized, and it is not even clear
whether the phenomenon is broadly conserved. For ex-
ample, hyphal compartments in Neurospora crassa and
A. gossypii can possess >100 asynchronously dividing
nuclei, and there is no apparent evidence that hyphal
morphogenesis is temporally coupled to the division of
these nuclei (e.g., reference
55). One final observation
potentially refutes the notion that hyphal morphogene-
sis is not dependent on nuclear division in filamentous
fungi. In particular, the temperature-sensitive A. nidulans
nimL, nimM,andnimN (never-in-mitosis) fail to un-
dergo nuclear division and fail to establish polarity (un-
like other nim mutants; references
29, 52, 56). These
mutants are very sensitive to the DNA synthesis inhibi-
tor hydroxyurea and share their polarization phenotype
with wild-type conidia exposed to hydroxyurea (
29).
These observations suggest that completion of an as yet
to be defined step in the S phase of the cell cycle might be
necessary for spore polarization to proceed.
FUNGAL EXOCYTOSIS
Hyphal growth occurs by apical extension. Cell wall-
modifying enzymes, substrates, and the membrane re-
quired for cell expansion are delivered to the apex by
exocytosis, which suggests that the filamentous habit of
growth is strongly dependent on the secretory pathway.
Such an apparently simple fact has major implications
both in the fungal pathogenicity field (host invasion is
4 ASMscience.org/MicrobiolSpectrum
Steinberg et al.
Downloaded from www.asmscience.org by
IP: 131.211.104.131
On: Mon, 25 Sep 2017 14:28:11
dependent on apical extension) and in the field of bio-
technology (filamentous fungi are major producers of
industrial enzymes). The hyphal mode of growth and its
dependence on exocytosis represent an experimental
advantage, because mutations impairing exocytosis even
to a minor extent often result in reduced colony growth,
making them easily scorable (
57). Moreover, muta-
tions impairing exocytic regulators delay or preclude
polarity establishment (
58, 59) or, when inactivated by
conditional mutations in rapidly growing hyphae, often
result in a tip swelling (
60), which can be used as a cell-
autonomous indicator of exocytic deficit.
The Golgi and Its Regulation
Although an increasing body of evidence supports the
contention that unconventional protein secretion path-
ways do exist in fungi (
61), these are relatively poorly
understood. Therefore, this article will focus on the
Golgi as the central hub in the conventional secre-
tory pathway that sorts protein cargoes to their final
destination, be it the extracellular milieu, the plasma
membrane, or the endovacuolar system. Several re-
cent reports testify to the interest that the mechanistic
understanding of biosynthetic traffic is spawning. For
brevity we will consider here reports using the genetic
models A. nidulans and N. crassa. Pioneering work on
motors that move carriers toward their destinations,
carried out with U. maydis, will be considered below.
As in S. cerevisiae, in the filamentous ascomycetes
A. nidulans and N. crassa the Golgi consists of isolated
membrane-bound structures that do not pile up to form
the characteristic stacks of mammalian cells (
62, 63).
Even though these isolated membranous structures were
initially denoted Golgi “equivalents,” they are bona fide
Golgi “cisternae,” equivalent to those in other eukary-
otes. Their nonstacked organization represents a major
experimental advantage for live microscopy studies be-
cause cisternae can be resolved by diffraction-limited
optical microscopy (
64–66). When imaged with fluores-
cent proteins, the extensively studied Golgi of A. nidulans
is seen as a dynamic network of ring-shaped and fenes-
trated cisternae, often interconnected by tubular struc-
tures (
64, 67, 68). This morphology is consistent with
electron microscopy studies (summarized in reference
63).
The Golgi is an intrinsically transient and composi-
tionally heterogeneous membranous entity that is con-
stantly fed by anterograde coat protein complex II
(COPII) carriers budding from transitional endoplasmic
reticulum (ER) domains/ER exit sites (
Fig. 2). An unan-
swered question in cell biology is how the protein cargo
that exits the ER traffics across the Golgi apparatus and
is sorted into plasma membrane- and endosome-bound
carriers (
69). It is widely accepted that this traffic occurs,
at least in part, by cisternal maturation rather than by
vesicle-mediated connections between stable cisternae.
According to this view, early cisternae, which are formed
by ER-derived traffic, progressively change their lipid
and protein content, becoming not only gradually en-
riched in cargo but also, in the end, compositionally
competent (see below) to break up into carrier vesicles
destined for the plasma membrane and endosomes
(
Fig. 2). Cisternae at this final stage of maturation are
denoted “late” or “TGN” cisternae (see below). The
progressive attainment of late composition is thought
to be mediated by retrograde COPI vesicle traffic re-
trieving to cisternae in earlier stages of maturation those
components that do not belong to mature (i.e., later)
stages (
Fig. 2).
Strong support for the cisternal maturation model
comes from ground-breaking studies of S. cerevisiae (
70,
71) and from our own work in A. nidulans (Pantazopoulou
and Peñalva, unpublished observations; see also b elow).
However, readers should note that the maturation model
very likely requires some modification to accommodate
the involvement of tubular connections that are often ob-
served between metazoan cisternae (and between fungal
ones [
68]). These connections could represent highways for
certain types of cargo. The model also needs to ac com-
modate the experimentally supported possibility that rapid
partitioning mediated by domains of different lipid com-
position enables the budding of carriers at all levels (
72;for
overview see references
69, 71). In a useful attempt to relate
the compositional and morphological variety of the Golgi
with specific roles, Glick and coworkers (
73)havepro-
posed that the Golgi consists of cisternae in three different
functional stages (
Fig. 2). We will use this scheme for fur-
ther discussion.
In stage I, denoted “cisternal assembly,” ER-derived
COPII carriers coalesce to form cisternae (
Fig. 2). Dur-
ing this stage, ER protein residents that escape to the
Golgi incorporated into COPII carriers recycle to their
normal destination using COPI-coated retrograde vesi-
cles (
74). This is the case, for example, for TM receptors
such as A. nidulans RerA
Rer1
, which sorts soluble cargo
at the ER into anterograde COPII carriers, and for the
syntaxin SedV
Sed5
, a key component of the SNARE
machinery mediating membrane fusion at the Golgi (
60,
64, 75). Membranes in stage I contain a network of
peripheral proteins recruited to them by Golgi-specific
GTPases that tether membranes, thereby facilitating
fusion. Among them are A. nidulans GrhA
Grh1
(68) and
N. crassa USO-1 (
65). As cisternae increase in size at the
ASMscience.org/MicrobiolSpectrum 5
Cell Biology of Hyphal Growth
