EUKARYOTIC CELL, Feb. 2005, p. 225–229
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 4, No. 2
Polarisome Meets Spitzenko ¨rper: Microscopy, Genetics, and
Steven D. Harris,1* Nick D. Read,2Robert W. Roberson,3Brian Shaw,4
Stephan Seiler,5Mike Plamann,6and Michelle Momany7
Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska1; Fungal Cell Biology
Group, Institute of Cell Biology, University of Edinburgh, Edinburgh, Scotland, United Kingdom2; School of Life Sciences,
Arizona State University, Tempe, Arizona3; Department of Plant Pathology and Microbiology, Texas A&M University,
College Station, Texas4; Institut fur Mikrobiologie und Genetik, Georg-August-Universita ¨t Go ¨ttingen, Go ¨ttingen,
Germany5; School of Biological Sciences, University of Missouri—Kansas City, Kansas City, Missouri6;
and Department of Plant Biology, University of Georgia, Athens, Georgia7
The impact of filamentous fungi on human welfare has never
been greater. Fungi are acknowledged as the most economi-
cally devastating plant pathogens (1) and are attaining increas-
ing notoriety for their ability to cause life-threatening infec-
tions in humans (57, 71), and fungal products sustain a billion-
dollar manufacturing industry (70). The tools available to study
filamentous fungi are more sophisticated than ever and include
the complete annotated genome sequences of multiple fila-
mentous fungi (12), resources being made available through
various functional genomics projects, and advanced bioimag-
ing methods, including high-resolution live-cell imaging (20,
32) and electron tomography (19, 50). The increasing impact of
filamentous fungi, along with the rediscovery of pseudohyphal
growth in yeast (22), has focused attention on the molecular
mechanisms underlying hyphal morphogenesis.
Attempts to understand hyphal morphogenesis have histor-
ically followed two different lines of investigation. Microsco-
pists have defined, with increasing detail, the subcellular orga-
nization of the hyphal tip. This led to the description of the
Spitzenko ¨rper, an apical cluster of vesicles, cytoskeletal ele-
ments, and other proteins, which plays a crucial role in hyphal
extension (4). Geneticists have identified gene products re-
quired for hyphal morphogenesis by characterizing morpho-
logical mutants (51, 52). Initial studies in the laboratories of
Beadle, Tatum, and colleagues attempted to link morphogen-
esis to specific biochemical pathways. More recent screens
have identified a multitude of signaling and cytoskeletal func-
tions required for hyphal extension (62, 72).
In the past few years, comparative genomics efforts have
allowed fungal biologists interested in hyphal morphogenesis
to exploit the wealth of knowledge about polarized growth in
the yeast Saccharomyces cerevisiae. Many informative homol-
ogies between filamentous fungi and yeast have been uncov-
ered. Notably, this includes several components of a multipro-
tein complex termed the polarisome (28), which regulates
microfilament formation at polarized growth sites in yeast (61).
Perhaps more importantly, several gene products involved in
hyphal morphogenesis have been shown to have no homologue
outside of the filamentous fungi. This emphasizes the potential
novelty of the mechanisms underlying hyphal morphogenesis.
In this review, we summarize past efforts to understand hyphal
morphogenesis and pose a series of questions designed to
focus future efforts in this area.
HYPHAL MORPHOGENESIS: A BRIEF OVERVIEW
Fungal hyphae originate from either a germinating spore or
another hypha (i.e., during branch formation). Initially, an axis
of polarity is established from a symmetrically expanding spore
or hyphal compartment. Subsequently, cell surface expansion
is restricted to the specified axis, thereby leading to the for-
mation of a polarized hypha that displays a gradient of expan-
sion that peaks at the tip (2, 15, 25, 30, 52). Maintenance of the
polarity axis allows hyphae to achieve a linear extension rate
that can approach 7.5 mm/h (45). Successive hyphal branching,
together with hyphal fusion in the colony interior (24), results
in a complex coenocytic mycelial network in which nutrients
acquired at the colony periphery can be effectively distributed
to distal regions and used to support reproductive development.
The pioneering work of Girbardt (23) laid the foundation of
research on the biology of the Spitzenko ¨rper (? apical body).
Using phase-contrast light microscopy, Girbardt provided the
first description of the Spitzenko ¨rper as a phase-dark structure
located in tips of growing hyphae of higher fungi. His meticu-
lous observations of living hyphae demonstrated that the Spit-
zenko ¨rper (i) is present only in growing vegetative hyphal tips,
(ii) forms at sites of spore germination and branch formation,
and (iii) is located at a position within the hyphal tip that
correlates with the direction of hyphal growth. Girbardt was
thus the first to show the intimate association that exists be-
tween Spitzenko ¨rper behavior and hyphal morphogenesis.
With the extraordinary insights into fungal subcellular struc-
ture made possible by the use of transmission electron micros-
copy (TEM) during the 1960s, various research groups focused
* Corresponding author. Mailing address: Plant Science Initiative,
University of Nebraska, N234 Beadle Center, Lincoln, NE 68588-0660.
Phone: (402) 472-2938. Fax: (402) 472-3139. E-mail: sharri1@unlnotes
on trying to correlate what Girbardt had observed in living
hyphae with what could be detected in chemically fixed hyphae
at the ultrastructural level. Of these early studies, that of Grove
and Bracker (26) stands out in providing the most detailed
analysis of the ultrastructural organization of hyphal tips, as
well as illustrating the variable morphology of the Spitzenko ¨r-
per among different groups of fungi. Ultrastructural preserva-
tion took a quantum leap forward with the development and
use of rapid cryofixation and freeze-substitution protocols to
prepare hyphal cells for TEM (36, 37, 38), which set the bench-
mark for subsequent ultrastructural studies (33, 34, 49, 58, 59,
60, 68). All studies of hyphal tip ultrastructure by TEM have
demonstrated that the Spitzenko ¨rper is a complex, multicom-
ponent structure dominated by vesicles (Fig. 1e). These studies
have also shown that vesicles are organized around a promi-
nent core region that is enriched in a dense meshwork of mi-
crofilaments (36; R. Roberson, unpublished results). Interest-
ingly, polysomes are often closely associated with the posterior
boundary of the Spitzenko ¨rper core (36; Roberson, unpub-
lished). In addition, microtubules extend into and often through
the Spitzenko ¨rper (Fig. 1c and g) (36, 60). Woronin bodies are
also found in the apical region near the Spitzenko ¨rper (Fig. 1e
and f), although these peroxisome-related structures are not
thought to have any role in hyphal growth (39, 48, 54). Vesicles
of the Spitzenko ¨rper can be divided into two populations on
the basis of size (Fig. 1e to g), the large so-called apical vesicles
(70 to 90 nm in diameter) and the small microvesicles (30 to 40
nm in diameter). Although the concept that many, if not all, of
these vesicles are secretory vesicles involved in delivering ma-
terials required for hyphal elongation is entrenched in the
literature, little is known regarding vesicle composition and
whether there are biochemically distinct populations of vesicles
that contribute specifically to tip growth. In support of the
latter possibility, Bracker, Ruiz-Herrera, and Bartnicki-Garcia
(5, 10) isolated a population of microvesicles termed chito-
somes from fungal hyphae and showed that they possessed
chitin synthase activity. It is not clear if these vesicles corre-
spond to the population of chitosomes of endocytic origin that
were subsequently characterized in yeast (75).
Beginning in the 1990s, there has been a significant return to
live-cell analysis (Fig. 1a to d). Using video-enhanced light
microscopy, Lopez-Franco et al. (45, 46, 47) described the
diversity of the Spitzenko ¨rper among the fungi and discovered
a structure referred to as the “satellite Spitzenko ¨rper.” This is
a small cluster of vesicles and associated proteins that form de
novo at the plasma membrane near the hyphal apex that, upon
fusing with the Spitzenko ¨rper, may be involved in generating
growth pulses. Further work in Bracker’s laboratory on living
hyphae provided the most compelling evidence that the Spit-
zenko ¨rper is responsible for growth directionality and hyphal
tip morphogenesis through the use of laser tweezers (11; see
also reference 4). In more recent years, live-cell analysis has
profited from the use of fluorescent probes that label the Spit-
zenko ¨rper. An important breakthrough was the observation
that the endocytosis marker dye FM4-64 rapidly stains vesicles
within the Spitzenko ¨rper (Fig. 1b to d) (18, 32, 35), not only
providing evidence for the interconnection of endocytic and
secretory pathways but demonstrating the dye’s utility for vi-
sualizing and analyzing Spitzenko ¨rper behavior in wild-type
and mutant strains (31, 32).
The central importance of the cytoskeleton in hyphal mor-
phogenesis is well established (4). It is thought that microtu-
bules are primarily responsible for the long-distance transport
of secretory vesicles to the Spitzenko ¨rper, while actin micro-
FIG. 1. (a to d) Live-cell imaging of growing hyphal tips of N. crassa (a to c) and A. nidulans (d). Scale bars ? 2.5 ?m. (a) Digitally enhanced
phase-contrast image of an unstained hypha showing Spk composed of a phase-dark cloud of secretory vesicles (arrow) and a phase-bright core
(arrowhead) (M. Uchida and R. W. Roberson, unpublished data). (b) Confocal image of a hypha stained with FM4-64 showing pronounced
staining of vesicles (arrow) within the Spk (P. C. Hickey and N. D. Read, unpublished data). (c) Confocal image colabeled with ?-tubulin–GFP
(green, black arrow) and FM4-64 (red, white arrow) showing microtubules extending into Spk. (d) Confocal image colabeled with SepA-GFP and
FM4-64 (red, arrow) showing colocalization of SepA (yellow, arrowhead). In the central region of Spk is the region occupied by microvesicles (see
panels e to g) (E. R. Kalkman and N. D. Read, unpublished data). (e to g) TEM of a hyphal tip of A. nidulans. A cluster of microvesicles within
the Spk core (asterisks) surrounded by apical vesicles (white arrows), Woronin bodies (white arrowheads), and microtubules (black arrows) are
indicated in these images. (e) Standard transmission electron micrograph (60-nm-thick section). (f) Electron tomographic section (?3-nm z-axis
resolution) of a dual-axis tomogram taken at 200 kV. (g) Model of panel f showing the 3D distribution of apical cytoplasm through an
?170-nm-thick section (Uchida and Roberson, unpublished). Scale bar ? 250 nm.
226 MINIREVIEWSEUKARYOT. CELL
filaments primarily control vesicle organization within the Spit-
zenko ¨rper and transport to the plasma membrane. If this is
correct, then the Spitzenko ¨rper may be viewed as a switching
station from microtubule-based to microfilament-based vesicle
transport. These concepts emerged in part from an insightful
vesicle-based mathematical model of fungal morphogenesis,
which predicts that the Spitzenko ¨rper acts as a vesicle supply
center (i.e., a moveable distribution center for vesicles involved
in cell surface expansion) (3, 4). Immunolocalization studies
and phalloidin staining have emphasized the presence of actin
in the Spitzenko ¨rper and hyphal apex (8, 66, 69), supporting
TEM data and suggesting that the Spitzenko ¨rper may func-
tion as a microfilament-organizing center. ?-Tubulin, which
is normally associated with microtubule organizing centers
(MTOCs) such as spindle pole bodies, was immunolocalized
within the Spitzenko ¨rper of the chytrid Allomyces macrogynus
(49). The idea that the Spitzenko ¨rper might be an MTOC is a
very attractive one (4), but attempts to localize ?-tubulin within
the Spitzenko ¨rper of higher fungi have failed (Roberson, un-
published). Furthermore, results of recent live-cell studies of
microtubule dynamics after tagging ?- or ?-tubulin with green
fluorescent protein (GFP) have provided no indication that
microtubules emanate from the Spitzenko ¨rper (20, 27), which
would be expected from an MTOC.
The first attempt at using a genetic approach to understand
hyphal morphogenesis evolved directly from the Neurospora
crassa mutagenesis program initiated by Beadle and Tatum.
Because N. crassa hyphae extend at such a rapid rate, morpho-
genetic mutants typically form compact colonies that can be
easily distinguished from the wild type. Accordingly, a few
obvious morphogenetic mutants were recovered and described
(6). Later, Garnjobst and Tatum (21) undertook the first sys-
tematic attempt to identify and characterize morphological
mutants, which resulted in the identification of 90 mutants that
defined at least 58 loci. These mutants were sorted into phe-
notypic classes (i.e., cot [temperature-sensitive colonial], col
[colonial], spco [spreading colonial], ro [ropy], etc.) on the basis
of colony morphology. As a result of these and related efforts
(summarized in references 13 and 51), several key mutations
such as cot-1, mcb, and cr-1 were initially characterized. Using
the tools that were then available, attempts were made to link
the new morphological mutations to specific biochemical de-
fects. This approach was successful in some cases, such as
linking cr-1 to a severe defect in adenylyl cyclase activity (67).
More generally, however, these studies highlighted the contri-
bution of the cell wall to the maintenance of normal hyphal
With the advent of molecular genetics, it became possible to
clone and characterize several genes identified in the earlier
genetic screens, including cot-1 (73) and mcb (14), both of
which encode protein kinases. In addition, Plamann and col-
leagues showed that the ro genes encode components of the
cytoplasmic dynein motor complex (56). Simultaneously, re-
verse genetic approaches were used to characterize the mor-
phogenetic functions played by diverse components of the cy-
toskeleton, cell wall, and signaling pathways (reviewed in
references 52 and 71). Notably, these studies demonstrated
that the establishment and maintenance of hyphal polarity are
compromised by mutations affecting, among other functions,
microtubule- and microfilament-based motor proteins, chitin
deposition, and both cyclic AMP and mitogen-activated pro-
tein kinase signaling. Although these studies have only pro-
vided fragmented insights into the molecular mechanisms un-
derlying hyphal morphogenesis, they have revealed sufficient
differences from the well-characterized yeast morphogenetic
model system to justify additional mutation hunts.
More recently, systematic attempts to identify a large frac-
tion of the genes involved in hyphal morphogenesis have been
initiated by using N. crassa and Aspergillus nidulans. The most
thorough screen identified 45 genes involved in diverse aspects
of morphogenesis in N. crassa (62). Remarkably, this collection
included several genes not previously implicated in polarized
growth on the basis of studies with yeast, as well as another
group of novel hypothetical proteins with morphogenetic func-
tions. Multiple temperature-sensitive mutations affecting po-
larity establishment and maintenance have also been recovered
in A. nidulans (29, 40, 53). Consistent with the results obtained
with N. crassa, characterization of the affected genes has iden-
tified several functions whose involvement in hyphal morpho-
genesis was not previously suspected (43, 44, 64, 65). Collec-
tively, these systematic studies underscore the complexity of
hyphal morphogenesis and emphasize several features that
distinguish it from the well-characterized yeast model.
The use of genetic approaches has provided initial insight
into the molecular composition of the Spitzenko ¨rper. Notably,
Sharpless and Harris (63) demonstrated that the formin SepA
is a component of the Spitzenko ¨rper of A. nidulans (Fig. 1d).
SepA is a homologue of yeast Bni1p, which is a key part of the
multiprotein polarisome complex responsible for nucleation of
microfilaments. Homologues of another polarisome protein,
Spa2p, localize to hyphal tips in Ashbya gosypii, Candida albi-
cans, and A. nidulans (41, 74; A. Virag and S. Harris, unpub-
lished results). Because the Spitzenko ¨rper may represent a
microfilament-organizing center, these results are consistent
with the idea that the polarisome is a component of the Spit-
zenko ¨rper. However, the full extent of the relationship be-
tween the Spitzenko ¨rper and the polarisome remains to be
determined. One attractive possibility is that the Spitzenko ¨rper
is a dynamic structure composed of many interacting protein
complexes, one of which is the polarisome.
GENOMICS: LESSONS FROM YEAST AND ANIMALS
The mechanisms that generate polarity in yeast cells have
been characterized in considerable detail. These studies have
led to the development of a model whereby divergent posi-
tional landmarks locally activate conserved signaling modules
that subsequently function via scaffold proteins to organize the
cytoskeleton and regulate protein transport (55). With this
model as a guide, it is reasonable to predict that a similar
hierarchy of functions underlies the establishment and main-
tenance of hyphal polarity (28). For example, as in yeast, po-
sitional landmarks presumably designate polarization sites
within hyphae. These landmarks may include internal cues
such as cell wall proteins or cell surface receptors that respond
to external factors. Notably, most of the landmarks character-
ized in yeast do not appear to be conserved in filamentous
VOL. 4, 2005MINIREVIEWS 227
fungi (7, 28). Once polarization sites are specified, the posi-
tional information is likely to be transduced by conserved
Cdc42 and Rho-related GTPase modules. As in animal cells, a
Rac GTPase module may also mediate this step in filamentous
fungi (9, 16). Effectors of the GTPases regulate organization of
the cytoskeleton and vesicle trafficking and are generally well
conserved in the genomes of filamentous fungi (7, 28). This
model provides a useful framework for assessing the function
of those morphogenetic proteins that are highly conserved among
animals, yeast, and filamentous fungi. However, the functional
role of the growing number of novel, filamentous-fungus-spe-
cific morphogenetic proteins remains unclear. These proteins
may contribute to the far greater complexity of hyphal mor-
phogenesis relative to that of yeast.
Hyphae have morphological features in common with other
highly polarized cells, such as animal neurons and plant pollen
tubes. While the mechanisms are not fully known, it is clear
that some of the same signaling molecules important in neu-
rotransmitter activity are up-regulated in the tip growth of
pollen tubes and that these molecules affect the actin cytoskel-
eton (17). It seems likely that animals, plants, and fungi use the
same core cellular machinery for polar growth but that they
organize this common polarity machinery in different ways. It
will be of great interest to see which Spitzenko ¨rper compo-
nents represent conserved core polarity machinery and which
are unique to filamentous fungi.
HYPHAL MORPHOGENESIS: THE KEY PROBLEMS
The significant impact of fungi on human welfare has helped
to focus attention on the role that hyphae play in fungal biol-
ogy. Because of this, and the recent development of genomic
and imaging tools that greatly enhance our ability to manipu-
late fungi, we propose that now is the time to initiate a systematic
analysis of the molecular mechanisms underlying hyphal mor-
phogenesis. Below, we propose several critical questions whose
answers will likely provide unparalleled insight into hyphal
morphogenesis and, in a more general sense, fungal biology.
(i) What is the composition of the Spitzenko ¨rper? The Spit-
zenko ¨rper is the organizing center for hyphal growth and mor-
phogenesis. Moreover, its behavior has been the subject of
extensive microscopic characterization and mathematical mod-
eling. Nevertheless, the molecular composition of the Spitzen-
ko ¨rper remains largely unclear. To fill this void, it will first be
necessary to identify and characterize its component parts.
This should include (i) characterization of the different popu-
lations of vesicles found within the Spitzenko ¨rper and the
identification of their contents, (ii) characterization of the res-
ident proteins that underlie Spitzenko ¨rper function, and (iii)
identification of the mRNA species that are transported to
ribosomes associated with the Spitzenko ¨rper.
(ii) How is the Spitzenko ¨rper assembled and disassembled?
The mechanisms underlying the de novo formation and divi-
sion of the Spitzenko ¨rper must be addressed. This includes (i)
investigating the possible role of landmark proteins in regulat-
ing the location and timing of assembly, (ii) characterizing the
interactions between proteins and complexes that underlie the
assembly pathway, (iii) determining how a satellite Spitzenko ¨r-
per is incorporated into a preexisting structure, and (iv) under-
standing the mechanisms that regulate Spitzenko ¨rper division.
(iii) How is Spitzenko ¨rper function regulated during hyphal
morphogenesis? The mechanisms that regulate Spitzenko ¨rper
behavior must be characterized. This includes (i) characteriz-
ing the dynamic interactions between proteins involved in Spit-
zenko ¨rper function, (ii) determining the nature of the physio-
logical and environmental signals that regulate Spitzenko ¨rper
function, and (iv) understanding how apical dominance, which
normally constrains hyphal branching to subapical regions, is
(iv) How is hyphal morphogenesis modified during patho-
genesis and development? Fungal hyphae can differentiate
elaborate structures that facilitate pathogenesis or the dissem-
ination of spores. The signaling pathways that regulate differ-
entiation are beginning to be understood (42). Nevertheless,
the mechanisms by which these signals affect Spitzenko ¨rper
function during infection structure differentiation and repro-
ductive development are not known.
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