3116
Langmuir 2001, 17, 3116-3119
Stretching Cell Surface Macromolecules by Atomic Force Microscopy Bruno C. van der Aa,† Romain M. Michel,†,‡ Marcel Asther,§ Marco Torrez Zamora,† Paul G. Rouxhet,† and Yves F. Dufreˆne*,† Unite´ de chimie des interfaces, Universite´ catholique de Louvain, Croix du Sud 2/18, B-1348 Louvain-la-Neuve, Belgium, De´ partement de me´ canique, Universite´ catholique de Louvain, Place du Levant 2, B-1348 Louvain-la-Neuve, Belgium, and Institut National de la Recherche Agronomique and Unite´ de Biotechnologie des Champignons Filamenteux, Faculte´ des Sciences de Luminy, 163, avenue de Luminy - C.P. 925, F-13288 Marseille, France Received November 13, 2000. In Final Form: February 13, 2001 We report the first quantitative measurement of macromolecular stretching at the surface of living cells using combined atomic force microscopy imaging and force spectroscopy. The surface of dormant spores of Aspergillus oryzae was covered with a layer of crystalline-like nanostructures (rodlets) and showed no/weak adhesion forces. By contrast, the surface of germinating spores consisted of soft granular material, attributed to cell surface polysaccharides, and elongation forces reflecting macromolecular stretching were observed in the force-extension curves. These elongation forces were well described by an extended freely jointed chain model with a Kuhn length of 3.2 ( 0.9 Å and a segment elasticity of 3.9 ( 1.8 N/m, which are consistent with values reported for the elastic deformation of single dextran and amylose polysaccharides. We therefore suggest that the elongation forces measured on germinating spores are due to the stretching of cell surface polysaccharides.
Introduction In recent years, single-molecule force spectroscopy experiments have provided valuable insight into intermolecular and intramolecular forces associated with biomolecules.1-10 In particular, recording force-extension curves by atomic force microscopy (AFM) has made it possible to directly measure the nanomechanical properties of DNA,2,9 proteins,6,8,10 and polysaccharides.5,7 So far, these measurements have been essentially performed on well-defined macromolecular systems, and the application to the stretching of cell surface macromolecules has not been reported. Fungal spores have a great importance in the natural environment because they are a means of vegetative multiplication, dispersal, and survival for fungi.11 On the other hand, fungi are used in a broad range of biotechnological applications, including the production of organic substances (enzymes, antibiotics) and the solution of * Corresponding author. Phone: (32) 10 47 35 89. Fax: (32) 10 47 20 05. E-mail:
[email protected]. † Unite ´ de chimie des interfaces, Universite´ catholique de Louvain. ‡ De ´ partement de me´canique, Universite´ catholique de Louvain. § Institut National de la Recherche Agronomique and Unite ´ de Biotechnologie des Champignons Filamenteux, Faculte´ des Sciences de Luminy. (1) Smith, S. B.; Finzi, L.; Bustamante, C. Science 1992, 258, 1122. (2) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science 1994, 266, 771. (3) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795. (4) Hinterdorfer, P.; Baumgartner, W.; Gruber, H. J.; Schilcher, K.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3477. (5) Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275, 1295. (6) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109. (7) Marszalek, P. E.; Oberhauser, A. F.; Pang, Y.-P.; Fernandez, J. M. Nature 1998, 396, 661. (8) Mu¨ller, D. J.; Baumeister, W.; Engel, A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13170. (9) Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346. (10) Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; Mu¨ller, D. J. Science 2000, 288, 143. (11) Wessels, J. G. H. Adv. Microb. Physiol. 1993, 34, 147.
environmental problems.12,13 Cell surface macromolecules, such as proteins and polysaccharides, play a central role in determining the surface properties of spores and, in turn, their interfacial interactions. Therefore, measuring intermolecular and intramolecular forces at the surface of fungal spores is a key to gaining a detailed understanding of their biological functions and to efficiently exploiting them in biotechnological processes. Until recently, these forces were not directly accessible to study. Here, we show that combined AFM imaging and force spectroscopy can be used to probe quantitatively the mechanical properties of macromolecules at the surface of native spores. The results point to the power of AFM for elucidating the physical properties of living cells at the molecular level and, in turn, the molecular basis of interfacial phenomena such as cell adhesion and cell aggregation. Materials and Methods Spores of Aspergillus oryzae LMTC 2.14 (Laboratoire de Microbiologie et Technologie Ce´re´alie`re, INRA, Nantes, France) were selected because of their great potential for the production of enzymes. A. oryzae spores were collected from mycelial mats14 grown on 15 g of agar (Biofit) per liter containing whole maize grains. For germination, the spores were grown in an agitated liquid medium15 for ∼10 h at 25 °C. Optical microscopy showed that although dormant spores were spherical particles ∼5 µm in diameter, germinating spores after 10 h of incubation were ∼10 µm in diameter. Spores were immobilized by mechanical trapping in Isopore polycarbonate membranes (Millipore).16 After a spore suspension (10 mL, 2 × 106 cells per mL) was filtered, the filter was carefully rinsed in deionized water (Millipore, Milli-Q), cut (1 cm × 1 cm), and attached to a steel sample puck (Digital (12) Huynh, V. B.; Chang, H.-M.; Joyce, T. W. Tappi J. 1985, 68, 98. (13) Carlsen, M.; Spohr, A. B.; Nielsen, J.; Villadsen, J. Biotechnol. Bioeng. 1996, 49, 266. (14) Gerin, P. A.; Dufrene, Y.; Bellon-Fontaine, M.-N.; Asther, M.; Rouxhet, P. G. J. Bacteriol. 1993, 175, 5135. (15) Record, E.; Asther, M.; Moukha, S.; Marion, D.; Burlat, V.; Ruel, K.; Asther, M. Can. J. Microbiol. 1998, 44, 945. (16) Dufreˆne, Y. F.; Boonaert, C. J. P.; Gerin, P. A.; Asther, M.; Rouxhet, P. G. J. Bacteriol. 1999, 181, 5350.
10.1021/la001573s CCC: $20.00 © 2001 American Chemical Society Published on Web 04/05/2001
Letters
Langmuir, Vol. 17, No. 11, 2001 3117
Figure 1. Changes of the surface ultrastructure of A. oryzae spores upon germination. The AFM deflection images (forward scanning, z-range ) 300 nm), recorded under water, show nanoscale rodlet structures at the surface of a dormant spore (A) and the rougher surface morphology of a germinating spore (B). The data shown are representative of results obtained on more than 10 spores, using different probes and independent preparations. Instruments, Santa Barbara, CA) using a small piece of adhesive tape, and the mounted sample was transferred into the AFM liquid cell. AFM measurements were made at room temperature (20 °C), under water, using an optical lever microscope (Nanoscope III, Digital Instruments) with an applied force maintained below 1 nN and a scan rate of 2-4 Hz unless stated otherwise. We used oxide-sharpened microfabricated Si3N4 cantilevers from Park Scientific Instruments (Mountain View, CA) with spring constants of 0.01 and 0.03 N/m and a probe curvature radius of typically 20 nm (according to manufacturer specifications). Force-extension curves were recorded at a rate of 1-2 µm/s, which is close to values used by others for stretching model polysaccharides.5 The slope of the retraction force curves in the region where probe and sample are in contact was used to convert the voltage into cantilever deflection. Least-squares fits to the experimental force-extension data were performed (Matlab) using three entropic elasticity models (see below), considering a temperature (T) of 293 K and allowing all the other parameters to vary.
Results and Discussion High-resolution images (Figure 1) show that dramatic changes of the spore surface ultrastructure occurred upon germination. The surface of dormant spores was covered with crystalline-like rodlet structures, 10 ( 1 nm in diameter, assembled in parallel to form supramolecular fascicles having different orientations (Figure 1A). The image resolution was not significantly affected by the imaging force (up to forces of a few nN), indicating that the sample had a fairly high mechanical stability. Along the same line, approaching force-distance curves showed no curvature in the contact region, indicating a fairly stiff sample surface (data not shown). Images obtained by forward and backward scanning were identical, indicating no significant contribution of lateral forces to the apparent topographic contrast. The observation of rodlets is in agreement with earlier electron microscopy studies of Aspergillus spores.17,18 Note, however, that AFM offers the unique possibility to observe these structures directly under aqueous conditions, without requiring chemical fixation or drying. Chemical analysis and enzymatic (17) Hess, W. M.; Stocks, D. L. Mycologia 1969, 61, 560. (18) Cole, G. T.; Sekiya, T.; Kasai, R.; Yokoyama, T.; Nozawa, Y. Exp. Mycol. 1979, 3, 132.
digestion have shown the outer rodlet layers of Aspergillus species to be essentially made of proteins.18,19 The surface morphology of germinating spores (Figure 1B) differed markedly from that of dormant spores in that soft granular material was observed instead of crystallinelike rodlets. On close examination, some areas showed streaks oriented in the scanning direction, that is, pointing toward the right-hand side of the image. When scanning backward (data not shown), streaks were oriented toward the left-hand side. These streaks were more pronounced when scanning the same area repeatedly, when increasing the imaging force (>1 nN), and when decreasing the scan rate (
