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
planters{at}yorku.ca


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 µm 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.


Abbreviations: BS, buffer solution


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 0·3–7·0x10–3 cm s–1 (3–70 µm s–1) 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.

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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strain maintenance and preparation.
The Neurospora crassa 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 kg–1, 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 x10 objective to select hyphal compartments in which cytoplasm movement was detectable. Either x40 or x63 (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-4742-95, Hamamatsu Photonics KK). The software program Openlab (Improvision Inc.) was used to control the camera and acquire 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 (µm s–1) 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 µm 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 pressure-probe 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To measure cytoplasm movement in hyphae, the hyphae were first observed with a x10 objective, then hyphae with easily discernible cytoplasmic movement were selected for velocity measurements with a x40 or x63 objective. These were usually large trunk hyphae behind the colony edge. Particles, normally small (about 1 µm) 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). 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.



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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 µm.

 


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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 µm.

 


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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 µm.

 
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):

{mic1512685E001}
where {rho} is the density (approximately equal to that of water: 1 g ml–1), Jv the velocity (5x10–4 cm s–1), d the diameter of the hypha (18x10–4 cm), and {epsilon} the viscosity [reported to be similar to that of water: 0·01 g s–1 cm–1 (Fushimi & Verkman, 1991)]. The calculated Reynolds number, 9·0x10–5, is very low compared to the value at which flow is turbulent [1–103 (Brody et al., 1996)]. Thus, smooth 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):

{mic1512685E002}
where Jv is the velocity (5x10–4 cm s–1), {epsilon} the viscosity (10–7 N s cm–2) and r the radius (9x10–4 cm), yielding a pressure gradient of 4·93x10–4 N cm–3, equivalent to 4·9x10–5 bar cm–1, 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 µm is assumed, the pressure gradient would be 0·01 N cm–3, or 0·001 bar cm–1. 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 10–7 N s cm–2 (Fushimi & Verkman, 1991). Even if a viscosity 100-fold larger is assumed, and using the septal radius of 2 µm, the pressure difference required for the measured velocity of mass flow remains very low: 0·1 bar cm–1. 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 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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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.

 


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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.

 
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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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 µm min–1 (squares).

 


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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 x10 objective. Bar, 100 µm.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 (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 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 (10–4–10–5), the pressure gradient required for mass flow is very small (0·001–0·1 bar cm–1) 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 µm 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 µm s–1 (Brody et al., 1996)], but mixing of the fluid flowing within the microchannel is dominated by diffusion.

The physical forces underlying mass flow and cytoskeleton-mediated 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 µm s–1, fivefold faster than other kinesins (Gilbert, 2001) and 7·5-fold faster than the usual rate of hyphal extension (20 µm min–1). The rate of mass flow (about 5 µm sec–1) is similar to kinesin velocity, but varies. The fastest cytoplasm flow observed in these experiments was 60 µm s–1 (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 (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 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.


   ACKNOWLEDGEMENTS
 
This research was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 4 February 2005; revised 18 April 2005; accepted 1 May 2005.



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