Atomic Force Microscopy in Cell Biology Episode 2 Part 11 ppt

386 Zuccheri and Samor ` ı itself approximately 18 times on average. When not complexed with proteins (like in nucleosomes), the supercoiled DNA should be in the interwound form, which is called “plectonemic.” What is almost constantly found in the SFM literature is the imaging of DNA plasmids that simply do not appear plectonemic, even if the authors describe them as supercoiled. Among other researchers, we have seen and reported plasmids that appeared too loosely coiled for their native superhelical density. A comparison between theoretical and com- putational evaluations would also suggest that plasmids with a native superhelical density should be significantly more coiled under the conditions of SFM imaging (Vologodskii, 1992; Vologodskii and Cozzarelli, 1994). The shape of supercoiled plasmids in SFM images is often very open, with only one or two apparent crossovers. Sometimes it is dif- ficult to distinguish the loosely coiled plasmids from the relaxed plasmids that can have “random flops,” especially if they are very long. At times, strange looking supercoiled plasmids have been presented. The superhelical density should be spread homogeneously along the entire length of the molecule, where no extended sections have a significantly looser appearance. On the contrary, many reports show plasmids with apparently highly coiled sections and other sections which are completely loose (Pope et al., 2000). It is our understanding that this could be due to local condensation conditions, which can drive some strand-to-strand contacts not only due to supercoiling. Under the conditions of DNA deposition and dehydration adopted for SFM imaging, some condensation could occur, especially if enabled by the superhelical tension. These conditions could even drive the molecules toward unnatural shapes. A seriously limited number of EM studies demonstrate nicely interwound plectonemic plasmids; among them, the cryo-EM studies demonstrate the nicest ones (Adrian et al., 1990). As mentioned earlier, the cryo-EM pic- tures generated much criticism (Gebe et al., 1996). Theoretical evaluations suggest that the motivation of the highly coiled forms is due to the very low temperature equilibration of the plasmids (Gebe et al., 1996). Evidence is building toward the idea that under the normal conditions of imaging the observed shapes are normally fairly loose. If the ionic strength of the solution to deposit is very high, then the deposited plasmids can appear as highly coiled. A good example is that of Lyubchenko and Shlyakhtenko (1997), who produced very nice images by depositing on AP-mica a plasmid solution with a very high concentration of NaCl. Supercoiled plasmids deposited on freshly cleaved mica are normally loosely coiled, unless uranyl-acetate is used in the dehydration phase to pre- serve the structure of the molecules (Cherny et al., 1998; F. Nagami et al., unpublished results). In this case (see Fig. 8), the plasmids can be highly coiled, and it is very easy to distinguish between relaxed and supercoiled plasmids in which case, the statistical evaluation of the two populations is very similar to the quantitative analysis by agarose gel electrophoresis. On the other hand, in a few cases, very coiled plasmids were imaged at low ionic strength on either freshly cleaved mica or oxidized silicon (Hansma et al., 1996; Yoshimura et al., 2000). Under high ionic strength conditions, plasmids are extremely coiled and can be seen as coiled on the surfaces especially if the experimental conditions (AP-mica or uranyl acetate) can limit their mobility before the ionic conditions are changed (e.g., in a rinsing step). Uranyl acetate might have the dual role of limiting the mobility and increasing the ionic strength. (Not all salts would work in this way.) 17. SFM of Single DNA Molecules 387 Fig. 8 SFM image of plasmid DNA deposited on freshly cleaved mica and dehydrated from a solution containing uranyl acetate. Highly supercoiled, completely relaxed, and fragmented linear DNA molecules are very easy to distinguish from the images under these conditions. Courtesy of Fuji Nagami (F. Nagami, G. Zuccheri, B. Samor`ı, and R. Kuroda (2002). Time-lapse imaging of conformational changes in supercoiled DNA by scanning force microscopy. Anal. Biochem. 300, 170 –176). Thus, why are the same plasmids so loosely coiled under conditions that should still preserve their coiling? We presently believe that the electrostatic interactions with the surface and the statistical effects of being confined in a very thin layer of solution strongly increase the intramolecular strand-to-strand repulsion. The electrostatic repulsion should keep the DNA strands apart, and thus uncoil them, except in the presence of a high con- centration of salt that screens the charges. The natural partition of a linking deficit in DNA between writhing and twisting of the chain might be altered in favor of twisting under these conditions. We are not aware of computer simulations that consider these factors to determine the partition of supercoiling under strongly spatially confined conditions. A partial B-to-A transition has been known to occur on DNA on the surface of mica (see Section III,E). Under these conditions, the superhelical tension should be greatly reduced (Krylov et al., 1990) and might help to bring the supercoiled DNA to a low state of coiling. It appears to us that the shape of supercoiled DNA evidenced from SFM images could be the result of multiple factors and further evidences are awaited to completely clarify the issue. Imaging supercoiled DNA in fluid at high ionic strength seems to be the only safe method to have nice plectonemic DNA on freshly cleaved mica under completely con- trolled conditions and without the aid of extraneous molecules. To image plectonemic DNA in air, strongly adhesive surfaces (like AP-mica) need to be coupled to the high- ionic-strength environment. Nicked plasmids appear open on freshly cleaved mica since they have the mobility to respond to the high electrostatic repulsion between strands. This same repulsion that opens them up like circles is also responsible for the self- avoiding that causes DNA strands to never overlap if imaged on mica under nontrapping 388 Zuccheri and Samor ` ı conditions (Rivetti et al., 1996). When imaged under trapping conditions, supercoiled DNA might appear more nicely plectonemic. Unfortunately, under the same conditions, nicked plasmids might display many chain crossovers, due to their random adsorption and trapping on the surface (such as for the many rosette-like shapes shown by Lyubchenko and Shlyakhlenko, 1997), and they will be more difficult to visualize and characterize. VIII. Conclusions and Perspectives After several years of SFM experiments on DNA, there are many questions that micro- scopists would still like to answer. Newer techniques and instruments are still emerging, often from the close collaboration of chemists and biologists with physicists. Newer technologies and interdisciplinary approaches will certainly expand the knowledge on the structure and behavior of DNA in the following years. A. SFM Single-Molecule Stretching Experiments on DNA: Only a Brief Note on a Booming Issue The last few years have seen the emergence of force spectroscopy as a new field of research. Using several kinds of force transducers, measured small forces can be applied to properly selected parts of a molecule in order to study its behavior and the forces that hold its structure together. The SFM cantilever is one of the most frequently used force transducers for force spectroscopy. Many groups are already working in SFM force spectroscopy of DNA, studying the forces that not only keep the double helix together but also might determine its behavior in the interaction with proteins or other molecules (Clausen-Schaumann et al., 2000; Lee et al., 1994; Noy et al., 1997; Strunz et al., 2000; Strunz et al., 1999). B. The Structure of ss-DNA Relatively few SFM studies have been done on single-stranded nucleic acids. Some possible reasons are the variability, complexity, and fragility of their structures. Recent studies have shown that some useful information can also be gathered from single- stranded DNA (Zuccheri et al., 2000). It can be easily predicted that some of the numerous interesting questions regarding single-stranded DNA and RNA will find answers as a result of SFM studies. Acknowledgments We are grateful to Pasquale De Santis (University La Sapienza, Rome, Italy) for the very helpful discussion on DNA curvature and flexibility, to Giuseppe Gargiulo (University of Bologna, Italy) for sharing his DNA constructs, and to Fuji Nagami (University of Tokyo, Japan) not only for helpful discussion but also for sharing his images of supercoiled plasmids. This work was supported by Programmi Biotecnologie Legge 95/95 (MURST 5%); MURST PRIN (Progetti Biologia Strutturale 1997–1999 and 1999–2001). 17. SFM of Single DNA Molecules 389 References Adrian, M., Ten Heggeler-Bordier, B., Wahli, W., Stasiak, A. Z., Stasiak, A., and Dubochet, J. (1990). Direct visualization of supercoiled DNA molecules in solution. EMBO J. 9(13), 4551–4554. Aich, P., Labiuk, S. L., Tari, L. W., Delbaere, L. J., Roesler, W. J., Falk, K. 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[...]... recognition AFM tips cleaning, 117 118 crosslinker binding, 119 esterification, 118 ligand binding, 119 ligand density and functionality, 119 – 120 probe surfaces, 120 – 121 applications, 116 dynamic force microscopy bond lifetime, 124 kinetic rates, energies, binding pocket, 125 unbinding force vs loading rate, 124 – 125 unbinding force distribution, 123 force distance cycle, 121 – 123 Ligands, DNA interactions, SFM... changes, 27 2 27 4 native membrane proteins HPI layer changes, 28 0 28 2 OmpF porin, 28 2 28 5 surface structure changes, 27 8 28 0 vascular endothelial cadherin, 126 Contact mode atomic force microscopy cell imaging, 57–58 operation, 35 overview, 26 0 26 1 Contact mode photonic force microscopy, 20 8 20 9 Coverslips, HEC/RL cell culture, 109 Crosslinker binding, AFM tips, 119 Cryo -atomic force microscopy deep etching,... technique, 22 3 22 4 modifications, 22 4 pulling, 22 4 sample preparation, 22 5 typical experiment, 22 5 PFM studies material and size, 20 4 20 5 surface modifications, 20 5 20 6 SFM, DNA, 367–368 Protein adsorption, force spectroscopy, 14–15 Protein engineering, AFM studies, 314–316 Protein folding, force spectroscopy, 15–19 Protein–protein interactions, force spectroscopy, 29 2 Proteins cellular, see Cellular proteins... trough, 23 4 23 5 Langmuir trough and small Teflon wells, 23 5 23 6 small vesicle fusion, 23 2 23 4 surfaces, 21 5 tapping, see Tapping mode atomic force microscopy technique improvements, 28 9 29 0 technology overview, 147–148 tips cleaning, 117 118 crosslinker binding, 119 esterification, 118 ligand binding, 119 ligand density and funtionality, 119 – 120 modifications, 177–178 probe surfaces, 120 – 121 shape, 82 topographs... applications, 188–190 Photonic force microscopy contact recording mode, 20 8 20 9 design principles, 20 0 20 3 experimental considerations, 199 20 0 fast 3D single-particle tracking, 21 0 feedback control, 20 2 20 3 force sensor, calibration, 20 7 instrumentation, 20 24 kinesins basic structure, 24 25 mechanical behavior, 26 mechanical properties, 25 27 local viscosity, 28 29 molecular interactions, 196–199 plasma... see Atomic force microscopy Air bubbles, AFM cantilever, 21 6 Antibody–antigen recognition, force microscopy, 134–137 Antibody labeling, membrane protein AFM imaging, 26 4 26 5 Aqueous fluids, AFM imaging bubble removal from cantilever, 21 6 cantilevers, 21 8 different solutions, 21 8 21 9 modes, 21 7 without O ring, 21 5 21 6 with O ring, 21 6 parameters, 21 7 21 8 when to image, 22 1 when not to image, 22 1 22 3 Atomic. .. imaging, 63 membrane proteins antibody labeling, 26 4 26 5 assembly, 28 5, 29 1 29 2 force- induced conformational changes, 27 2 27 4 oligomerization, 27 0 27 2 polypeptide end removal, 26 7 27 0 polypeptide loop removal, 26 7 polypeptide loop replacement, 26 5 26 7 methodological strategies, 1 42 microscope–AFM interface, 174–176 modes, 338 native membrane protein HPI layer changes, 28 0 28 2 OmpF porin conformation, 28 2 28 5... 21 5 21 6 with O ring, 21 6 parameters, 21 7 21 8 when to image, 22 1 when not to image, 22 1 22 3 bacterial pore, 28 7 28 9 basic setup, 151–153 basic technique, 1 42 bilayers charged lipids, 23 8 24 0 ligand-linked lipids, 23 7 23 8 binding force measurement on intact cells, 178–179 binding map analysis, 179–180 biomechanics, 184 cantilever AFM studies, 21 8 bubble removal, 21 6 JAR cell culture, 109 110 materials,... recognition, 124 Bovine serum albumin, supported bilayer substrate, 23 3 23 4 Brush border membrane vesicles, dynamic force microscopy, 131 BSA, see Bovine serum albumin Buffers, SFM of DNA, 370–371 C Cadherin, VE cells, dynamic force microscopy binding strengh, 128 – 129 Ca2+ dependence, 127 – 128 conformation, 126 overview, 125 – 126 400 Index Cadherin, VE cells, dynamic force microscopy (continued ) thermodynamics... kidney cells, AFM studies, 37–38 Mapping AFM analysis, 179–180 human serum albumin, 133–134 Na+/D-glucose cotransporter, 131–133 recognition imaging, 133–134 MDCK cells, see Madin–Darby kidney cells 404 Index Membrane proteins, AFM studies antibody labeling, 26 4 26 5 assembly, 28 5, 29 1 29 2 force- induced conformational changes, 27 2 27 4 native HPI layer changes, 28 0 28 2 OmpF porin conformation, 28 2 28 5 surface . studies, 20 2 20 3 Finite element modeling, AFM interfacing, 1 82 Fluids advanced handling, AFM studies DNA digestion by DNase, 22 7 future outlook, 22 7 operation principles, 22 6 22 7 overview, 22 5 22 6 aqueous,. 370–371 C Cadherin, VE cells, dynamic force microscopy binding strengh, 128 – 129 Ca 2+ dependence, 127 – 128 conformation, 126 overview, 125 – 126 400 Index Cadherin, VE cells, dynamic force microscopy (continued. functionality, 119 – 120 probe surfaces, 120 – 121 applications, 116 dynamic force microscopy bond lifetime, 124 kinetic rates, energies, binding pocket, 125 unbinding force vs. loading rate, 124 – 125 unbinding