1 Department of Chemistry and Biochemistry, University of Regina, 3737 Wascana Parkway, Regina, SK, Canada S4S 0A2
2 Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E2
Correspondence
Tanya E. S. Dahms
tanya.dahms{at}uregina.ca
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ABSTRACT |
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These authors contributed equally to the written manuscript.
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INTRODUCTION |
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Tip extension is the highly conserved polarized growth pattern characteristic of fungi (Bartnicki-García, 2003; Harold, 2002
; Momany, 2002
), oomycetes (Meyer et al., 1976
), plant pollen tubes (Yang, 1998
) and root hairs (Mathur & Hulskamp, 2002
). Tip-growing cells are structurally and functionally polarized, so that their growth direction is highly predictable, and polarity is essential for directed movement in all cell types (Small & Kaverina, 2002
; Wedlich-Soldner & Li, 2003
). Fungal hyphae provide an attractive model for studying tip growth, and the filamentous fungus Aspergillus nidulans is especially suitable. The fungal cytoplasm is surrounded by a cell membrane that in turn is encased in a wall, consisting in Aspergillus primarily of a carbohydrate matrix, composed of (1
3)- and (1
6)-
-glucans, reinforced by chitin microfibrils (Beckett et al., 1974
; Guest & Momany, 2000
; Lipke & Ovalle, 1998
; Vermeulen & Wessels, 1984
, 1986
). Internal hydrostatic pressure (turgor), typically several atmospheres [1·4 MPa (Beever & Laracy, 1986
)], presses the cell membrane tightly against the wall. Turgor is a non-polarized component of tip-growing cells, since its force is equal in all directions. Turgor can vary dramatically without substantial changes in growth rate, and vice versa, so local regulation of wall and cytoskeletal properties is proposed to influence tip growth rate (Money & Harold, 1992
; Money & Hill, 1997
). As reviewed by Bartnicki-García (2003)
, tip growth requires the insertion of new membrane and wall-forming materials (matrix components via exocytosis, and wall fibrils synthesized in situ by integral enzymes) at the apex (Bartnicki-García & Lippman, 1972
; Bartnicki-García et al., 2000
; Gooday, 1994
), so the tip wall must be able to accommodate new material during cell growth. Growing tips are protected against rupture from turgor through internal reinforcement by actin microfibril arrays that are secured to transmembrane proteins (Kaminskyj & Heath, 1995
; Torralba & Heath, 2001
). In sum, tip growth in fungal hyphae results from the interplay between localized wall deposition, localized internal cytoskeletal support, a gradient of wall maturation, and turgor that acts evenly along the cell to produce a tubular cell capable of invading nutritive substrates.
Mature Aspergillus conidia have a surface hydrophobin rodlet layer (Stringer et al., 1991) that is shed prior to germination. Following germination, cell growth is localized to hyphal tips, so that the distance behind the tip is correlated with the age of the wall. Growing tip walls incorporate new material for cell extension (reviewed by Bartnicki-García, 2003)
, whereas mature basal walls are fully cross-linked and inextensible (Gooday, 1994
). During hyphal-wall maturation, chitin microfibrils are synthesized by enzymes in the cell membrane and are then cross-linked to matrix components (Sietsma & Wessels, 1979
, 1981
). In A. nidulans, the major zone of hyphal expansion is in the apical 3 µm, and approximates to a hemispheric dome (Gierz & Bartnicki-García, 2000
). At typical growth rates (0·20·5 µm min1; Kaminskyj & Hamer, 1998
), hyphal-wall maturation is probably complete within the apical 6 µm of growth, coincident with the base of the internal actin reinforcement (Fig. 1B
in Shi et al., 2004
). Branching involves the formation of a new tip, typically from the mature wall, requiring that the branch site be made extensible, likely the result of the localized release of enzymes (Bartnicki-García, 2003
; Dynesen & Nielsen, 2003
; Harold, 2002
; Money & Hill, 1997
), some of which have been shown to be endoglucanases (Money & Hill, 1997
). Wall softness' has also been suggested to contribute to wall extensibility at the apex during tip growth (Bartnicki-García & Lippman, 1972
; Gooday, 1994
; Money & Harold, 1992
).
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Atomic force microscopy (AFM) can be used in microbiology (Dufrêne, 2004; Firtel & Beveridge, 1995
) to image surface detail at the nanometre scale, and to directly address the relationship between the viscoelasticity and extensibility of living tip-growing cells at high resolution by force spectroscopy (FS). Force curves record the deflection of a flexible cantilever as a function of distance when applying force to a specimen through contact with the cantilever-mounted tip, and can be converted to viscoelasticity (force compliance), for example, in specific regions of wall maturation. Theoretical models can then be used to provide quantitative estimates of specimen elasticity (e.g. Young's modulus).
We have used AFM, in combination with techniques such as scanning electron microscopy of frozen hydrated cells (cryoSEM), to examine the surface features of A. nidulans hyphae, and FS to probe relative wall rigidity at cell tips, branch junctions and basal regions in growing hyphae, or as a function of tip extension.
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METHODS |
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Samples for scanning electron microscopy (SEM) and AFM consisted of hyphae growing on the surface of a substrate, either under a coverslip submerged in liquid CM for fixed samples, or on dialysis tubing overlying solid CM for live and cryoSEM samples. Cell morphology and growth rate was wild-type under these constrained conditions, except that the hyphae grew along the substrate surface rather than in three dimensions.
Fresh spores were harvested from conidiating cultures, typically 23 days following inoculation. For AFM studies of conidia, glass coverslips were silanized (2 % octadecyltrichlorosilane/hexane), rinsed (hexane), air-dried overnight and touched to a conidiating culture so that a layer of conidia adhered. Non-adherent spores were removed with compressed air before imaging by AFM.
Preparation of fixed cells for AFM.
Hyphae grown overnight on dialysis tubing overlying CM agar were fixed by inverting the Petri dish over (not in) drops (200 µl) of OsO4 (4 % aqueous) placed in the lids. OsO4 is highly volatile, so cells were vapour-fixed from a source about 3 mm distant. Cells were fixed [at least 2 h at room temperature (RT)], until the underlying agar was dark from Os accumulation. Pieces of dialysis tubing with fixed cells were frozen by plunging into cold anhydrous acetone (60 ml, 80 °C), dehydrated (at least 4 h at 80 °C), warmed slowly to avoid condensation (2 h at 20 °C, 2 h at 4 °C, 2 h at RT), and critical-point dried. For the aforementioned procedure, the acetone is exchanged for liquid CO2 (in a pressure chamber), which is taken through a phase transition to gas as the temperature is raised through the critical point, circumventing surface-tension artifacts. Dried samples were mounted on aluminium stubs (for SEM) or glass coverslips (for AFM) with double-sided tape, and gold sputter coated. Some AFM samples were imaged without gold coating. Images of fixed, coated and uncoated AFM samples were comparable (data not shown), but gold coating facilitated fast-contact-mode imaging. Some SEM samples were later imaged by AFM for direct comparison.
Preparation for cryoSEM.
Cultures were grown (16 h) on dialysis tubing overlying CM agar, frozen in nitrogen slush (196 °C) and transferred to the SEM specimen chamber (80 °C). Surface water was removed by sublimation (10 min, 60 °C). Samples were gold coated and examined at 80 °C using a 1630C field emission scanning electron microscope (JEOL USA) equipped with digital image capture (Department of Earth Sciences, University of Alberta). Images were processed for optimal contrast.
AFM and FS of live cells.
Samples were grown overnight on dialysis tubing overlying CM agar. Subsequently, cells were hydrated and nourished by supplying CM liquid to a paper wick under the dialysis tubing (i.e. cells were not immersed), which was mounted on mica and secured with tape. Samples were transferred to the AFM stage and incubated (2 h, 28 °C) to allow hyphae to re-establish growing tips after handling. AFM images were collected in contact mode at high resolution (1000x1000 lines). Hyphal growth rate was estimated by stationary line scanning, beginning as a hyphal tip grew past the scan line and continuing until the hyphal width stabilized. Hyphal width reached 2·9±0·4 µm (n=20), similar to the mean width at the septa of A28 hyphae grown at 28 °C (Kaminskyj & Hamer, 1998), at approximately 3 µm behind the tip.
AFM imaging conditions.
An Explorer atomic force microscope (Veeco) with a dry scanner (Veeco, 10 µm in z and 100x100 µm in x and y, model 5460-00) was used for imaging and FS. The atomic force microscope was suspended by bungee cords on an iron plate for vibrational isolation, in a wooden cabinet insulated with Sonex foam for sound isolation, which was seated on an optical table (Coherent Ealing Optics) mounted on I-2000 pneumatic isolators (Newport). Hyphae were visually selected using a CCD camera (200xmagnification), followed by imaging at low resolution (200x200 lines per scan). Data were collected from high-resolution images (1000x1000 lines per scan, scan rate=1 Hz) of fixed cells using Si3N4 probe tips (Veeco model #1520-00, k=0·032 nN nm1, nominal resonance =17 kHz).
For living cells, intermittent contact and non-contact modes were sometimes used to collect images with Si probe tips (Veeco model #1650-00, k=2 nN nm1, =230280 kHz) to prevent samples from sticking to the probe during imaging. For images larger than 5x5 µm, a
Hz scan rate was used to avoid dislodging the hyphae. Images smaller than 5x5 µm were acquired at
Hz. All images represent typical results.
The varying geometry and characteristics of AFM probes may cause image distortion and overestimation of the lateral size of small features (x and y). Therefore, to minimize experimental error, we tested all AFM probes (Xu & Arnsdorf, 1994) using colloidal gold particles (0·01 % HAuCl4 in 0·02 % NaN3) with a radius of 5 nm, showing that our tips were capable of resolving these spheres within the experimental error [full width at half maximum (FWHM)=5·3 nm±0·4 nm, n=20). To test whether we could resolve fine features (10 nm) on a topographically complicated surface, we imaged dry-harvested conidia, which have been studied elsewhere at high resolution with SEM (Beever & Dempsey, 1978
).
Images were processed using horizontal levelling, with the maximal height adjusted for optimum contrast (SPMLab version 6.0 software, Veeco). Since images of the surface result from a convolution of the shapes of the AFM probe and surface features, widths were measured at the FWHM of the peak height, and both widths and heights were measured manually using the line measurement option. In some cases, images have been presented enlarged for clarity. Data are presented as mean±standard deviation. Differences in the mean subunit sizes in apical, subapical and branch-point junction regions were tested by a one-way ANOVA using InStat 3 (GraphPad).
Force spectroscopy (FS).
For FS, the AF microscope was equipped with hydrophilic Si4N3 probe tips. The interaction between the AFM probe and the sample was tracked by cantilever deflection (Zc) as a function of the piezo elongation (Zp) during probe approach and retraction. For soft materials, meaningful FS comparisons depend on the velocity of the surface approach. The approach velocity (10 µm s1) was kept constant to facilitate the comparison of relative viscoelasticity and adhesion forces measured at different regions of the sample. Repeated measurements of individual sites on mature walls gave consistent values and images obtained before and after FS were unchanged (data not shown), indicating that the walls were not damaged during data collection. FS measurements (46 per area on each hypha) were collected from the hyphal wall in the apical expansion zone (1 µm steps), and from the mature wall 20 and 40 µm from the tip. FS data were also collected at tips of branches about 2 µm long, at hypha-to-branch junctions, and on the mature wall of the subtending hypha.
During FS, the magnitude of AFM-probe deflection per unit of applied force is measured as the probe approaches and retracts from the sample. Probe responses during the approachretract cycle, summarized as force curves, can be used to measure the unit force required to indent a surface a given distance. The last portion of the approach cycle (Fig. 6B, line bc) and the first part of the retraction cycle (Fig. 6B
, line cd) can be used to examine the relative rigidity of the surface. For a hard surface such as glass, the slope of the line bc during the approach cycle will be identical to that (cd) during retraction (Fig. 6B
insert). In contrast, sample deformation will appear as a difference between the slopes bc and cd (Fig. 6B
). Adhesion values are measured from the last segment of the retraction cycle (Fig. 6B
, line ef), and if there is chemical attraction between the sample and the AFM probe, segment ef will be a measure of its intensity.
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Since the aim was to report relative changes in elasticity along hyphae, a single AFM cantilever (spring constant k=0·032 nN nm1, Veeco) and tip were used for all FS measurements. The force exerted by the piezo on the cantilever was 0·125 nN for each nA of deflection.
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RESULTS |
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Surface morphology
Fig. 1 shows cryoSEM (Fig. 1A
) and AFM (Fig. 1B
) images of fixed mature hyphal wall surfaces of A. nidulans, each taken
40 µm behind the tip. Both imaging methods show rounded features, the diameter (25±6 nm, n=60, range 1537 nm) of which was measured from AFM images. Critical-point drying maintains three-dimensional structures far better than freeze-drying or air drying (data not shown), and was therefore used for all specimens except those used for cryoSEM and AFM of live samples. There was no qualitative or quantitative difference between AFM images of fixed hyphae with or without gold coating (data not shown). Fig. 1(A)
also shows small areas in the cryoSEM image in which vitreous water had not been removed by sublimation before gold coating. As yet we have little evidence for the shape and/or packing of the surface features within the wall. We used SEM and AFM to image the hyphal wall surface, so our use of rounded (and later ellipsoid) refers only to the surface appearance of the features.
The surface of hyphal walls viewed with cryoSEM is distinctly different from the rodlets characteristic of conidial surfaces (Beever & Dempsey, 1978; Stringer et al., 1991
), which are 10±2 nm wide (n=60) and more than 100 nm long. AFM was able to image these details on topographically complex spore surfaces (Fig. 2
).
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The surface features at young branch tips were similar in size and shape to those at hyphal tips (data not shown), but larger, ellipsoid, and different from those of mature walls. Surface packing at branch points (Fig. 5A) suggested a mixture of mature wall features intercalated with newly deposited ones (Fig. 5B
).
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DISCUSSION |
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Samples for cryoSEM are rapidly frozen and remain at or below 60 °C during subsequent preparation and imaging, making cryoSEM the gold standard for preserving ultrastructural information (Howard & O'Donnell, 1987). However, SEM must infer that specific cells were alive at the moment they were frozen, whereas AFM can image cells known to be growing. In addition to imaging, FS can be applied to sample the cell wall properties on different regions of growing hyphae. Our high-resolution AFM images of the mature wall surfaces of fixed, dry hyphae are quantitatively similar to those from our best high-resolution cryoSEM, which gives us confidence that the differences reported by AFM of growing versus mature wall surfaces are accurate, consistent with the differences between hyphal and spore wall surfaces. Like SEM, AFM images of fixed (and fixed/gold-coated) hyphae produce high-lateral-resolution surface structural data. The manner in which an AFM image is acquired (essentially by touch) gives AFM better depth resolution than SEM, whereas SEM images have better depth of focus and field of view.
The hyphal wall surfaces in apical and mature regions are both composed of regular protrusions, but there are clear differences between their size, shape and packing for newly deposited and mature walls, consistent with models suggesting that wall maturation follows deposition (Sietsma & Wessels, 1979, 1981
; Vermeulen & Wessels, 1984
) and that hyphal wall remodelling can expose or mask epitopes (Momany et al., 2004
). The compact, rounded features on the surface of mature walls appear to develop from the rearrangement within and/or lateral packing of the larger ellipsoid features found at hyphal tips. The hyphal-wall surface does not change appreciably between 20 and 40 µm from the tip, and therefore maturation appears to be complete at or before the 20 µm mark.
The width of the rounded features (25 nm) is comparable to that of the macromolecular triple helix (triplex) of fungal (1
3)-
-D-glucan derivatives (e.g. scleroglucan: 18±2·5 nm) that have been characterized in situ by electron microscopy and AFM (Stokke et al., 2001
). Since the hyphal wall of A. nidulans includes glucan polymers (e.g. Guest & Momany, 2000
), we speculate that the features on the surface of mature walls could be glucans extending from the cell wall matrix [consistent with the orthogonal expansion pattern of hyphal growth proposed by Bartnicki-García and colleagues (Bartnicki-García et al., 2000
; Gierz & Bartnicki-García, 2000
)] and/or glycoproteins (Lipke & Ovalle, 1998
. The difference in subunit appearance at growing tips compared to mature regions suggests that the wall at the apex could have different mechanical properties, but images alone do not provide evidence for relative rigidity.
This is the first report of using AFM to image growing fungal hyphae. AFM has been used elsewhere to image the surface of live cells at relatively high resolution (Ahimou et al., 2003; Bushell et al., 1999
; Cho et al., 2002
; Gebeshuber et al., 2003
, 2004
; Ricci & Grattarola, 1994
; Schneider et al., 1997
; van der Mei et al., 2000
; Yang & Shao, 1995
), despite the difficulty in some cases of supporting cell growth during examination. Dufrêne and coworkers (Ahimou et al., 2003
) have examined the surface structure of living yeast cells and fungal conidia in liquid medium (Dufrêne, 2000
), but have not yet extended the work to hyphae. Wall undulations were visible for both fixed and live hyphae, with similar size and frequency. Topography images of surface features in living hyphae are not entirely consistent with those in chemically fixed cells. The discrepancy between AFM images of fixed and live hyphae likely results from the reversible deformation of the hyphal wall features in contact with the tip as a result of pressure from the AFM cantilever (Yang & Shao, 1995
) and/or subtle movements of the hypha during scanning. These differences could be compounded by cell growth (
250 nm min1) at the tip and by the mobility of living hyphal cells that could not be secured firmly to the substrate, since only conidia adhere to dialysis tubing. Nonetheless, the general rounded shape of the surface features and undulations observed in live A. nidulans are both consistent with those obtained with AFM for fixed, mature hyphal walls, and are distinctly different from the parallel linear arrangements of the hydrophobin rodlets on spore surfaces. The lateral force image represents high-resolution data for the surface of living hyphal cells that were growing during data capture. The features observed on the surfaces of fixed cells, possibly glycoproteins or glucans (Lipke & Ovalle, 1998
; Zhao et al., 2005
), can be resolved in vivo. We expect in the future to be able to extend this work to examine the dynamics of wall maturation and changes during branch formation, as well as the surface chemistry of specific surface regions, using a variety of chemically modified AFM tips and near-field scanning optical microscopy.
Measurements of hyphal rigidity are most relevant for growing hyphae, which were imaged by AFM and then analysed using FS, showing that walls were less rigid in regions of active extension (hyphal and branch apices) than in mature regions. Consistent with this finding, preliminary FS experiments, in which we were unable to demonstrate hyphal growth, showed no difference between the rigidity of tips and mature walls. Hyphal rigidity will be the sum of contributions from 1) wall elasticity and thickness, 2) internal reinforcement from the cytoskeleton, 3) internal support from turgor, and 4) radius of curvature. Turgor pressure will be evenly distributed along the hypha, while actin is concentrated in the apical 6 µm of the A. nidulans tip (Shi et al., 2004), where it is thought to support the extending region. There are only minor differences in thickness between apical (44±3 nm) and mature (48±4 nm) hyphal walls (Kaminskyj & Boire, 2004
), too small to explain the almost twofold difference in rigidity that we observe between the two areas of the cell wall. Therefore, differences observed in hyphal rigidity can be attributed to cell wall elasticity (Zhao et al., 2005
) and the radius of hyphal curvature.
Arnoldi et al. (2000) developed theoretical and experimental models to determine turgor pressure in the Gram-negative bacterium Magnetospirillum gryphiswaldense using FS, but the peptidoglycan walls of Gram-negative bacteria are thinner (38 nm) than the glucan/chitin walls of A. nidulans (
46 nm; Kaminskyj & Boire, 2004
). Nonetheless, applying their model to A. nidulans hyphae, using estimates for Young's modulus (
110 MPa; Zhao et al., 2005
), turgor pressure (
1·4 MPa; Beever & Laracy, 1986
) and hyphal width (3 µm), reveals a tension-dominated mode, as expected. According to shell theory (Landau & Lifshitz, 1959
) the viscoelasticity of the hypha should vary with its radius of curvature. Therefore, it is still important to demonstrate that the changes in rigidity observed from the growing apex to the mature wall are not solely a function of radius of curvature. Aspergillus hyphal tips are approximately parabolic in shape (Gierz & Bartnicki-García, 2000
), so that their radius of curvature will be directly proportional to hyphal diameter. If viscoelasticity varies solely with hyphal curvature, we would expect a linear relationship between our rigidity and hyphal diameter measurements, but we find a non-linear relationship (data not shown), implying a significant contribution from cell wall elasticity.
Touhami et al. (2003) used force mapping and simple Hertzian models to derive Young's moduli for yeast bud scars and cell surfaces. Their model is valid for elastic surfaces for which adhesion forces are negligible, and is therefore not applicable to hyphae, for which adhesive properties vary from tip to mature wall. Alternatively, Yao et al. (1999)
isolated cell walls to quantify their viscoelasticity, but this type of analysis, while important, is not relevant for describing dynamic maturation processes in live cells, nor is there a simple way to extract sufficient cell wall material specifically from hyphal tips. A recent study (Zhao et al., 2005
) derived Young's moduli for azide-treated (dead), dried, rehydrated hyphae from a theoretical model that relates force measurements directly to wall elasticity. Using their model, we estimate the Young's modulus of the mature wall to be 115±31 MPa, significantly different (P<0·01) than that of the tip, 75±27 MPa.
Branches were examined for localized changes in wall surface structure and viscoelasticity. Hyphal branching requires that the mature wall be altered to permit extension of the new tip (Bartnicki-García, 2003; Harold, 2002
), for which there is experimental evidence in oomycetes (Money & Hill, 1997
), and there are also candidate genes for endoglucanases in the A. nidulans and Aspergillus fumigatus genomes. Consistent with this, the wall structure at branch points was different from that of the subtending mature wall and the tip. Regardless of the mechanism for altering the wall structure, the walls of a growing hyphal tip and of a newly formed branch are expected to be less rigid than a mature wall, which is supported by our FS data. If differences in wall surface structure are related to wall maturation, we might also expect to find lower rigidity at a newly forming branch tip than at the subtending mature wall, and again we find these differences. The rigidity of branch tips was comparable to that of the primary hyphal tip (P>0·05), as expected, whereas FS values for the subtending mature wall are comparable to other mature wall regions (340 µm from the tip). Furthermore, the branch-point junction is much closer in curvature to the subtending mature wall, yet its rigidity is substantially different, supporting the idea that changes in rigidity at hyphal tips cannot simply be explained by curvature or wall thickness.
The change in hydrophilicity of the hyphal surface as a function of growth, maturation and branching mirrored hyphal viscoelasticity. These results indicate that the wall surface is altered during maturation, by rearrangement and/or through the addition of materials. Capillary forces are generally thought to be responsible for drawing the AFM tip into contact with the sample (point b, Fig. 6B), but as shown here, their magnitude does not overwhelm differences in the hydrophilicity of the wall regions. The adhesive forces measured by FS are the sum of the capillary force Fc, due to Laplace pressure from the water meniscus that forms between the AFM tip and the sample, and the direct adhesion between the two surfaces once in contact. Capillary effects should be relatively uniform along the hypha, whereas the cell wall structure changes with wall maturation, affecting the relative adhesive properties between the cell wall and the AFM tip. By 3 µm behind the tip, hyphal surface subunit diameter, hyphal wall viscoelasticity and surface hydrophilicity were similar to those of mature walls, suggesting that under these growth conditions, the surface of the hyphal wall has matured within approximately 12 min.
Our main findings show that A. nidulans hyphae have significant surface-structure and viscoelasticity differences between actively growing tips, branch junctions and mature walls. As the wall matures, the surface-feature size and packing changes, which correlates with hyphal viscoelasticity and surface chemistry, in keeping with previously described biochemical changes. These results are consistent with cross-linking during maturation and structural reorganization at branch points. Locating precise regions of living hyphae with AFM presented the opportunity to measure the relative stiffness of these regions by FS. Hyphal tips and branch points were significantly less rigid than the mature wall at least 3 µm behind the hyphal apex. Together, this is the first direct evidence for localized cell wall changes during hyphal tip growth, and demonstrates the power of combining AFM with FS for live specimens. We have shown a structurefunction relationship for A. nidulans hyphal walls that corresponds mechanistically with models of tip growth.
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ACKNOWLEDGEMENTS |
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Received 7 July 2005;
revised 30 July 2005;
accepted 2 August 2005.
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