Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3
Correspondence
Roger R. Lew
planters{at}yorku.ca
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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Mass flow measurements.
After addition of BS, the culture was allowed to acclimate for 3060 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 s1) 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 11·5 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-140300G, 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.
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RESULTS |
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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, 11·5 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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DISCUSSION |
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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, os1). Of these, only os1 (nik1) 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 (102103). Given the low Reynolds number of cytoplasm movement through hyphae (104105), the pressure gradient required for mass flow is very small (0·0010·1 bar cm1) compared to the magnitude of the other component of hyphal pressure, turgor (45 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 0·010·2 bar in microchannels create velocities of 50500 µm s1 (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 s1, fivefold faster than other kinesins (Gilbert, 2001) and 7·5-fold faster than the usual rate of hyphal extension (20 µm min1). The rate of mass flow (about 5 µm sec1) is similar to kinesin velocity, but varies. The fastest cytoplasm flow observed in these experiments was 60 µm s1 (not shown), but there were also hyphal trunks in which no visible movement was observed. Mutations in kinesin and dyenin affect growth rates severely (722 % 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 11·5 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 45 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 4 February 2005;
revised 18 April 2005;
accepted 1 May 2005.
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