Microbiology (2005), 151, 2685–2692
DOI 10.1099/mic.0.27947-0
Mass flow and pressure-driven hyphal extension in
Neurospora crassa
Roger R. Lew
Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3
Correspondence
Roger R. Lew
[email protected]
Mass flow of cytoplasm in Neurospora crassa trunk hyphae was directly confirmed by injecting oil
droplets into the hyphae. The droplets move in a manner similar to cytoplasmic particles and
vacuoles within the hyphae. The direction of mass flow is towards the growing hyphal tips at the
colony edge. Based on flow velocities (about 5 mm s”1), hyphal radius and estimates of
cytoplasm viscosity, the Reynolds number is about 10”4, indicating that mass flow is laminar.
Therefore, the Poiseulle equation can be used to calculate the pressure gradient required for
mass flow: 0?0005–0?1 bar cm”1 (depending on the values used for septal pore radius and
cytoplasmic viscosity). These values are very small compared to the normal hydrostatic pressure
of the hyphae (4–5 bar). Mass flow stops after respiratory inhibition with cyanide, or creation
of an extracellular osmotic gradient. The flow is probably caused by internal osmotic gradients
created by differential ion transport along the hyphae. Apical cytoplasm migrates at the same rate
as tip extension, as do oil droplets injected near the tip. Thus, in addition to organelle positioning
mediated by molecular motors, pressure-driven mass flow may be an integral part of hyphal
extension.
Received 4 February 2005
Revised
18 April 2005
Accepted 1 May 2005
INTRODUCTION
METHODS
Mass translocation is a natural process by which material
is transported to specific regions of a fungal organism.
Most measurements of mass flow have relied upon the
use of radioactive tracers. Rates of flow are in the range
0?3–7?061023 cm s21 (3–70 mm s21) in Basidiomycetes:
Armillaria mellea rhizomorphs and Serpula lacrimans
mycelia (Jennings, 1987). The driving force for mass translocation appears to be a pressure gradient, based upon
pressure-probe measurements of mycelia and sclerotia of
Morchella esculentum (Amir et al., 1995). The pressure
gradient between mycelia and sclerotia, and between primary hyphae and peripheral cells in the sclerotia is about
5 bar, consistent with mass flow supplying sclerotia and the
peripheral cells at the colony edge. Compared to the complex structures of differentiated tissues, the Ascomycete
Neurospora crassa offers a simpler system in which to
examine the physical characteristics of mass flow in single
hyphae.
Strain maintenance and preparation. The Neurospora crassa
In this paper, for the first time, the hydrodynamic nature
of mass flow, and the role of pressure-driven mass flow as
an integral part of fungal tip growth are assessed. Besides
microscopic analysis of cytoplasm movement, oil droplet
injection was used to distinguish mass flow from the role
of molecular motors and the cytoskeleton.
Abbreviation: BS, buffer solution.
0002-7947 G 2005 SGM
wild-type strain 74-OR23-1A (FGSC 987) was obtained from the
Fungal Genetics Stock Center (School of Biological Sciences,
University of Missouri, Kansas City, MO, USA) (McCluskey, 2003).
It was grown in Vogel’s minimal medium (Vogel, 1956) plus 1?5 %
(w/v) sucrose on 2 % (w/v) agar slants at room temperature.
For initial experiments measuring cytoplasmic movement, conidia
from slants were sown on Petri dishes containing Vogel’s minimal
medium plus 1?5 % sucrose on 2 % agar overlaid with scratched
cellophane. Portions of the mycelial mat, including the growing
edge, were cut along with the cellophane, which was taped down in
an upside-down lid of a 60 mm Petri dish and flooded with buffer
solution (BS) [(in mM): Mes (10), KCl (10), CaCl2 (1), MgCl2 (1)
and sucrose (133); pH adjusted to 5?8 with 10 M KOH; osmolarity
195–198 mOsmol kg21, measured with a Wescor (model 5100C)
vapour pressure osmometer]. For oil injections, cyanide treatments
and osmotic gradient experiments, the fungal colony growing on
agar was flooded with BS.
Mass flow measurements. After addition of BS, the culture was
allowed to acclimate for 30–60 min. Recovery was confirmed by
monitoring the resumption of tip growth at the growing edge of
the colony. Microscopy was performed using a Zeiss Axioskop. The
colony was scanned with a 610 objective to select hyphal compartments in which cytoplasm movement was detectable. Either 640 or
663 (water immersion) objectives were used for imaging of
cytoplasmic movement. For quantitative measurements, 64 or 128
digital images of the hyphal compartment were acquired using an
exposure time of less than 20 ms and a time-lapse interval of 0?5
or 1?0 s. The camera was a digital CCD camera (model C-474295, Hamamatsu Photonics KK). The software program Openlab
(Improvision Inc.) was used to control the camera and acquire
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R. R. Lew
digital images. The time-lapse movies were saved in TIF format.
Particles that moved along the hyphae were tracked using the public
domain ImageJ program (developed at the US National Institutes of
Health and available online at http://rsb.info.nih.gov/ij/). The distance the particles travelled per 0?5 or 1?0 s interval was converted
into a velocity (mm s21) in Excel (Microsoft).
For treatments with NaCN, time-lapse movies were taken before and
after the addition of 10 mM NaCN (final concentration) from a
100 mM stock in BS. The NaCN was added dropwise in a circle
around the objective, then mixed with the bath solution. Addition
and mixing took less than 60 s. For osmotic gradient treatments, the
fungi growing on agar were flooded with 5 ml BS. After acclimation,
cytoplasm movement was measured in a trunk hyphal compartment
about 500 mm behind the colony edge. Then, 0?5 ml BS+1 M sucrose
was added 1–1?5 cm from the hyphal compartment, near the centre
of the fungal colony, to create the osmotic gradient. In some experiments, based on a change in refractivity, the hyperosmotic solution
reached the hyphae that were being measured and caused shrinkage
of the hyphae. These experiments were discarded to ensure that it was
generation of an extracellular gradient behind the hypha that was
affecting the intrahyphal osmotic gradient and cytoplasm movement.
Oil injections. A pressure-probe apparatus (Lew, 1996) was used
to impale the hyphae and inject droplets of a low-viscosity silicone
oil (Dow Corning 200, 1?5 centistokes) into the hyphae. Briefly, the
micropipette was fabricated using a double-pull method from borosilicate tubing (1 mm external diameter, 0?58 mm internal diameter)
with an internal filament. The pressure transducer (XT-140–300G,
Kulite Semiconductor Products) was housed with the pressureprobe micropipette in a small brass holder. Pressure was applied
with a micrometer-driven piston connected to the brass holder with
thick-walled teflon tubing. The piston, tubing, holder and micropipette were filled with the low-viscosity silicone oil. After impalement with the pressure probe, turgor forced the oil back from the
micropipette tip. The silicone oil was brought back to the micropipette tip by applying pressure, then a slight additional pressure
was used to inject a droplet of oil into the cell.
RESULTS
To measure cytoplasm movement in hyphae, the hyphae
were first observed with a 610 objective, then hyphae
with easily discernible cytoplasmic movement were selected
for velocity measurements with a 640 or 663 objective.
These were usually large trunk hyphae behind the colony
edge. Particles, normally small (about 1 mm) and round,
were chosen based on the ease with which they could be
tracked over time; their identity is not known. The rate of
particle movement varied within a hypha and over time
Fig. 1. Examples of cytoplasm movement within a hyphal compartment of Neurospora crassa. The velocity of particle
movements (different symbols represent different particles) tracked for 72 or 128 s as they moved along the hypha. The faster
the velocity, the fewer the measurable time-lapse images (upper panel), since the particle moved out of the field of view. The
first measurements were performed soon after addition of BS and recovery of the colony (upper panel, photo at right). The
second measurements were performed about 60 min later (lower panel). Note that different particles tend to move at the same
velocity over time, expected for mass flow. Bars, 50 mm.
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Microbiology 151
Mass flow and tip growth in Neurospora
Fig. 2. Example of vacuole movement along a hypha and through a septal pore. Images were collected every 0?5 s. The arrow
in the first panel points to the septum, through which the vacuole is passing in the fourth panel. Bar, 10 mm.
(Fig. 1). In all cases, the direction of particle movement
was towards the growing edge of the colony. To ensure that
particle movement was not due to molecular motors and
the cytoskeleton, oil droplets were injected into the hyphae.
In a similar manner to vacuole movement through septal
pores and along the hyphae (Fig. 2), oil droplets also
‘blebbed’ through septal pores and travelled along the
hyphal trunk (Fig. 3). Since there is no reason to expect that
oil droplets can interact with molecular motors or the
cytoskeleton, their movement through the septal pores
and the hyphal trunk must be a consequence of mass flow of
cytoplasm.
Neurospora crassa hyphae regulate turgor to maintain a
normal hydrostatic pressure of 4–5 bar (4?8±1?2 bar)
(Lew et al., 2004). To calculate the pressure gradients
required to drive mass flow, it was first necessary to determine whether mass flow was turbulent or laminar. There
was no indication of visual turbulence. Furthermore, if
the flow occurs at a low Reynolds number, it would be
laminar flow (Purcell, 1977). The Reynolds number is
defined as the ratio of inertial to viscous forces (Nobel,
1991):
Re~
o.J v .d
g
where r is the density (approximately equal to that of water:
1 g ml21), Jv the velocity (561024 cm s21), d the diameter
of the hypha (1861024 cm), and g the viscosity [reported
to be similar to that of water: 0?01 g s21 cm21 (Fushimi
& Verkman, 1991)]. The calculated Reynolds number,
9?061025, is very low compared to the value at which flow
is turbulent [1–103 (Brody et al., 1996)]. Thus, smooth
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laminar flow is occurring, so the Poiseulle equation can be
used to calulate the pressure gradient required to cause the
observed velocity (Brody et al., 1996; Nobel, 1991):
dP J v .8.g
~ 2
dx
r
where Jv is the velocity (561024 cm s21), g the viscosity
(1027 N s cm22) and r the radius (961024 cm), yielding a
pressure gradient of 4?9361024 N cm23, equivalent to
4?961025 bar cm21, very low compared to the hydrostatic pressure of the hypha (4–5 bar). After accounting for
the smaller radius of the septa, the pressure differential
remains very low: if a septal radius of 2 mm is assumed, the
pressure gradient would be 0?01 N cm23, or 0?001 bar cm21.
Another factor that could affect the pressure gradient required
for mass flow is the viscosity of the solution. Cytoplasmic
viscosity is reported to be very similar to that of water: about
1027 N s cm22 (Fushimi & Verkman, 1991). Even if a viscosity 100-fold larger is assumed, and using the septal radius
of 2 mm, the pressure difference required for the measured
velocity of mass flow remains very low: 0?1 bar cm21. This is
less than 10 % of the hydrostatic pressure of the hyphae: about
4–5 bar (Lew et al., 2004).
Pressure-driven mass flow must rely upon small differences in the osmolarity of the cytoplasm along the hyphae,
extending to the colony edge. Ion transport and osmolyte
production are two possible mechanisms that would create
an intrahyphal osmotic gradient, and thus the pressure
difference that causes mass flow. The role of ion transport
was examined by treating the hyphae with cyanide to deplete
ATP and thus most ion transport, especially the plasma
membrane proton pump (Slayman et al., 1973). Treatment
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R. R. Lew
Fig. 3. Example of oil droplet movement along a hypha and through a septal pore. The images were taken at the times shown
(in minutes). A pressure probe was impaled into the hypha (0 and 0?6 min), and the silicone oil brought to the micropipette tip
(2?7 min) to obtain an estimate of the turgor (2?4 bar in this example). Oil droplets (marked by *) were introduced into the
hypha (4?4 min), then fused. A vacuole moving along the hypha fused with another vacuole at the septum (8?1 through
8?29 min), then part of the vacuole blebbed through the septal pore (not shown). The oil droplet subsequently blebbed
through the pore (9?4 through 10?59 min), and moved along the hypha in a similar manner to the vacuoles (Fig. 2). The similar
properties of vacuole and oil droplet movement prove that mass flow is the driving force, rather than molecular motors and the
cytoskeleton. Bar, 20 mm.
with 10 mM NaCN inhibited mass flow rapidly and completely (Fig. 4). To directly demonstrate that pressure gradients were responsible for the mass flow, an extracellular
osmotic gradient was created by adding BS plus 1 M sucrose
at the base of the colony, 1–1?5 cm behind the growing edge
of the colony, and mass flow measured near the growing
edge. The osmoticum treatment would create a low turgor
within the hyphae near the site of osmoticum addition,
causing a shift in the pressure differences within the hyphae.
The addition of osmoticum inhibited mass flow (Fig. 5).
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One role of pressure-driven mass flow could be supply of
cytoplasm to the extending hyphal tips at the growing edge
of the colony. Cytoplasm migrates in tandem with the tip
extension. This is observed very close to the extending tip,
as measured by the location of cytoplasmic particles during
tip growth: the distance between the tip and the particles
remains constant during tip growth (Fig. 6), To demonstrate
directly that this is due to mass flow, oil droplets were injected
into hyphae behind the growing tip; they migrated in tandem
with tip extension during continued hyphal growth (Fig. 7).
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Mass flow and tip growth in Neurospora
Fig. 4. The effect of respiration on mass flow along hyphae of Neurospora crassa. Three experiments are shown by different
symbols. Mass flow velocities were measured by tracking particle movement before and after the addition of NaCN (10 mM
final concentration) in BS, as shown.
DISCUSSION
There are two components of hyphal pressure. The first
is turgor, the internal hydrostatic pressure caused by
differences in the intra- and extracellular osmolarities. It
is an intrinsic property of the walled cell, pressing the plasma
membrane against the cell wall evenly throughout the cell.
However, in the long tubular cells of a fungus (or xylem
tubes of higher plants), the second component becomes
important: an intracellular pressure gradient which causes
mass flow of cytoplasm (or water) through the tubular
‘pipe’.
It is commonly accepted that cellular expansion of walled
cells is caused by an interplay between turgor and the
elastic/plastic properties of the wall (Boudaoud, 2003).
Localized changes in the extensibility of the wall cause
localized cell expansion, resulting in a well-defined cell size
and shape. Doubts have been raised about the role of
turgor in hyphal growth of water moulds (Money & Harold,
1993; Lew et al., 2004), but water moulds are known to have
contractile vacuoles (Heath & Harold, 1992) and thus an
osmoregulatory system commonly associated with animals,
rather than the regulation of turgor by changes in ionic
fluxes observed in plants (Shabala & Lew, 2002) and fungi
Fig. 5. The effect of an osmotic gradient on mass flow along hyphae of Neurospora crassa. Three experiments are shown by
different symbols. Trunk hyphae near the growing edge of the colony were chosen. An extracellular osmotic gradient was
created by adding BS+1 M sucrose 1–1?5 cm behind the colony edge, which should cause a change in the intrahyphal
osmotic gradient. Mass flow velocities were measured by tracking particle movement before and after the addition of the
hyperosmotic solution, as shown.
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Fig. 6. Cytoplasm migration within the growing tip. Particles were tracked during hyphal extension. Their position relative to
the tip is shown versus time. The distance between tip and particle remained constant during tip extension. Thus, cytoplasm
migrates in tandem with tip extension. Particle movements for three hyphae (circles, squares or triangles) are combined. The
micrograph shows one of the hyphae, scaled to the distance from the tip. Mean growth rates were 13?4 (circles), 29?7
(triangles) and 23?3 mm min”1 (squares).
(Lew et al., 2004). More pertinent for fungal organisms is
the wall-less ‘slime’ mutant of Neurospora crassa (Emerson,
1963). The mutant has three mutations (fz, sg, os–1). Of
these, only os–1 (nik–1) has been cloned; it is homologous with two-component histidine kinases, such as the
Saccharomyces cerevisiae Sln1p osmosensor of the HOG
pathway (Alex et al., 1996; Schumacher et al., 1997),
important in osmotic regulation. The mutant grows as
a protoplast in osmotically buffered media and exhibits
amoeboidal movement, pointing to a role for the cytoskeleton during growth (Heath & Steinberg, 1999). The
‘slime’ mutant is a clear example of the ability of fungal
organisms to grow in the absence of turgor, an example
which supports the idea that amoeboidal growth can be an
Fig. 7. Oil droplet movement in tandem with tip growth: evidence for pressure-driven growth from behind. The oil droplet was
injected into the hypha (time 0?2 min, arrow) by applying a pressure of 2?7 bar to the pressure probe. The oil droplet moved
towards the tip during continued growth (1?5 and 2?5 min, arrow), indicating that cytoplasm migrates in tandem with tip
extension. This is an example from a hypha embedded in agar. These experiments were replicated seven times, with both
hypha embedded in agar (n=5) and hyphae growing on the surface of the agar (n=2), using a 610 objective. Bar, 100 mm.
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Mass flow and tip growth in Neurospora
alternative mechanism of tip growth (Heath & Steinberg,
1999). Thus, turgor-driven growth is not obligatory,
although it is probably the norm. The second component
of pressure, intrahyphal pressure gradients, may also have
a role in fungal growth.
In the work presented here, pressure gradients are inferred
from observations of cytoplasm and vacuole movement
through the trunk hyphae, and more directly by observations of oil droplet movement. The latter observations
preclude a role for molecular motors and the cytoskeleton.
The magnitude of the pressure gradient can be estimated
from Poiseulle’s equation of hydraulic flow if smooth
laminar flow occurs, rather than turbulent flow. To ensure
that laminar flow was occurring, the Reynolds number was
calculated.
A number of authors have described the importance of a
low Reynolds number (Purcell, 1977; Berg, 1993). D’Arcy
Thompson’s oft-cited comments (Thompson, 1992) about
the ‘third world, where the bacillus lives’, that ‘we have come
to the edge of a world of which we have no experience, and
where all our perceptions must be recast’ refer in part to the
low Reynolds number, since viscosity rather than inertia
dominates the bacillus’s environment and the intracellular
milieu of the hypha because of their small dimensions. By
comparison, inertia is the dominant force for swimming
fish or even humans, for whom the Reynolds number is
much higher (102–103). Given the low Reynolds number
of cytoplasm movement through hyphae (1024–1025),
the pressure gradient required for mass flow is very small
(0?001–0?1 bar cm21) compared to the magnitude of the
other component of hyphal pressure, turgor (4–5 bar).
In microfluidics, the concepts of non-turbulence and low
pressure gradients are well-established (Brody et al., 1996).
In microchannels <100 mm wide, not only is fluid flow
non-turbulent and driven by very low pressure gradients
[pressures in the range 0?01–0?2 bar in microchannels create
velocities of 50–500 mm s21 (Brody et al., 1996)], but mixing
of the fluid flowing within the microchannel is dominated
by diffusion.
The physical forces underlying mass flow and cytoskeletonmediated organelle and vesicle movement are different.
Organelle and vesicle movement requires localized molecular motors directly energized by ATP. The kinesin molecular motor from N. crassa operates at a speed of about
2?5 mm s21, fivefold faster than other kinesins (Gilbert,
2001) and 7?5-fold faster than the usual rate of hyphal
extension (20 mm min21). The rate of mass flow (about
5 mm sec21) is similar to kinesin velocity, but varies. The
fastest cytoplasm flow observed in these experiments was
60 mm s21 (not shown), but there were also hyphal trunks
in which no visible movement was observed. Mutations
in kinesin and dyenin affect growth rates severely (7–22 %
of wild-type), as well as hyphal morphology (variable
hyphal diameter and curling compared to the straight
hyphae of wild-type) and positioning of nuclei and vacuoles
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(Seiler et al., 1999). The two motors may not be obligatory
requirements for hyphal growth, but are important in
morphogenesis, as is the cytoskeleton (Virag & Griffiths,
2004; Seiler & Plamann, 2003). In contrast to the localized control of molecular motors, mass flow requires an
intrahyphal osmotic gradient which must be maintained
over relatively long distances.
An immediate cause of the intrahyphal osmotic gradients
required for mass flow is likely to be ion transport. This
is based upon the rapid effect of cyanide, and the experimental creation of extracellular osmotic gradients that
would affect the intracellular pressure gradient and thus
mass flow. The inhibition of mass flow by these treatments
could be pleiotropic. For example, cyanide inhibits tip
growth (Lew & Levina, 2004); inhibition of mass flow may
be a secondary consequence. Although the hyperosmotic
treatment was imposed 1–1?5 cm from the hyphae in
which mass flow was being measured, it may be affecting
multiple aspects of hyphal physiology which in turn could
affect mass flow. The similar effects of the two different
treatments, which should modify intrahyphal osmotic
gradients by different mechanisms, does suggest that the
underlying cause of mass-flow inhibition is changes in
the intrahyphal osmotic gradients. The pressure differences required for mass flow are very low compared to the
hydrostatic pressure of the hyphae grown under the same
conditions, which is actively maintained at 4–5 bar (Lew
et al., 2004). Therefore the greater part of ion transport
and osmolyte production required to create and maintain
the intrahyphal osmotic gradient causing the pressure will
be allocated to hydrostatic pressure rather than the pressure differences required for mass flow.
At the growing edge of the colony, even though cytoplasm
and oil droplet migration towards the tip occurred in
tandem with tip extension, it would be unwise to claim that
there is an obligatory requirement for mass flow during tip
growth. Mass flow may not be actively maintained, but
simply the outcome of intrahyphal osmotic imbalances that
arise as a natural consequence of cellular volume increases
during hyphal tip expansion, in which water influx is an
explicit requirement, and may be supplemented by water
supplied by the hyphae behind the growing edge. De novo
cytoplasm synthesis near the tip would obviate a requirement for cytoplasm migration from behind the tip, as would
H+ influx at the apical region of growing hyphae that may
be due to an influx of co-transported solutes, such as glucose
and other nutrients (Lew, 1999). This process may rely
upon properties of the expanding tip, for example, the high
Ca2+ gradient in the tip (Silverman-Gavrila & Lew, 2003).
Ca2+ activates the H+ pump (Lew, 1989), which may
function in osmotic adjustment by generating the driving
force for influx of co-transported solutes. However, it
remains likely that pressure-driven cytoplasm flow is an
integral and normal component of fungal tip growth, and
represents an example of what could be described as ‘growth
from behind’, distinct from the vesicle-dense tip-region
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of the hypha. It is not clear how regulation of mass flow
and cytoskeleton-mediated organelle/vesicle movement at
the tip is coordinated. They are two very different processes operating during hyphal growth. Discovering the
signal transduction linkages between cytoskeleton-mediated
polarity and intrahyphal osmotic gradients will be a difficult
goal to attain.
Lew, R. R. (1999). Comparative analysis of Ca2+ and H+ flux
magnitude and location along growing hyphae of Saprolegnia ferax
and Neurospora crassa. Eur J Cell Biol 78, 892–902.
Lew, R. R. & Levina, N. N. (2004). Oxygen flux magnitude and
location along growing hyphae of Neurospora crassa. FEMS Microbiol
Lett 233, 125–130.
Lew, R. R., Levina, N. N., Walker, S. K. & Garrill, A. (2004).
Turgor regulation in hyphal organisms. Fungal Genet Biol 41,
1007–1015.
ACKNOWLEDGEMENTS
McCluskey, K. (2003). The Fungal Genetics Stock Center: from
This research was supported by a Discovery Grant from the Natural
Sciences and Engineering Research Council of Canada.
Money, N. P. & Harold, F. M. (1993). Two water molds can grow
molds to molecules. Adv Appl Microbiol 52, 245–262.
without measurable turgor pressure. Planta 190, 426–430.
Nobel, P. S. (1991). Plants and fluxes. Chapter 9 in Physicochemical
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Microbiology 151