The atomic force microscope (AFM) is proving to be a powerful tool for analysis of biological samples. We provide three examples of the application of AFM to the study of biological questions. First, polytene chromosomes from Drosophila are imaged and manipulated by the AFM. Second, the localization of calcium channels on the release face of a nerve terminal is described. Finally, analyses of a new form of DNA, the G-wire, is presented. These examples illustrate the wide variety of biological questions to which AFM can contribute

Ambrosio, Linda

Haydon, Philip

Henderson, Eric

Jondle, Daniel

Marsh, Thomas

Mosher, Curtis

Stanley, Elis

Vesenka, James

English

The atomic force microscope (AFM) is proving to be a powerful tool for analysis of biological samples. We provide three examples of the application of AFM to the study of biological questions. First, polytene chromosomes from Drosophila are imaged and manipulated by the AFM. Second, the localization of calcium channels on the release face of a nerve terminal is described. Finally, analyses of a new form of DNA, the G-wire, is presented. These examples illustrate the wide variety of biological questions to which AFM can contribute.This is a proceeding from NATO Advanced Research Workshop: "Scanning Probe Microscopies and Molecular Materials"  (1994): 1. </p

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1Analyzing Chromosomes, Ion Channels and Novel Nucleic Acid Structures by AFMEric Henderson, Linda Ambrosio, Curtis Mosher, Daniel Jondle,Elis Stanley*, Philip Haydon, Thomas Marsh, and James VesenkaDepartment of Zoology and Genetics, Iowa State University, Ames, IA 50011*National Institutes of Health, Bethesda, MD 20892.Abstract. The atomic force microscope (AFM) is proving to be a powerful tool for analysis ofbiological samples. We provide three examples of the application of AFM to the study of biologicalquestions. First, polytene chromosomes from Drosophila are imaged and manipulated by the AFM.Second, the localization of calcium channels on the release face of a nerve terminal is described.Finally, analyses of a new form of DNA, the G-wire, is presented. These examples illustrate the widevariety of biological questions to which AFM can contribute.1. IntroductionThe atomic force microscope (AFM) (1) has recently come into vogue as a tool for studying biologicalsamples. Simple sample preparation requirements, generation of three-dimensional data sets, detectionof numerous force interactions and the ability to manipulate as well as image samples have contributedto the popularity of the AFM. In this report we provide an overview of three studies using the AFM toaddress biological questions. The samples under investigation are polytene chromosomes, calciumchannels and a novel DNA structure, the G-wire.     Polytene chromosomes from Drosophila melanogaster have several attributes that make them anexcellent test system for analysis of chromosome and chromatin structure by AFM. They have beenextremely well studied and mapped with light and electron microscopes. The locations of many genesare known with relatively high precision. The chromosomes are very large due to the manyendoduplications of the DNA during larval development. Finally, polytene chromosomes containinterphase chromatin, rather than highly condensed metaphase chromatin, making them an idealsample for study of the relationship between chromatin structure and gene expression.     The movement of calcium in neurons is critically linked to synaptic vesicle release and synaptictransmission. The flow of calcium into the nerve terminal triggers the fusion of secretory vesicles withthe plasma membrane and release of their contents. Calcium enters the nerve terminal through asub-group of membrane proteins, the calcium channels. The organization of these calcium channels inthe nerve terminal is likely to be critical for fast (<l ms) synaptic transmission. However, previousstudies have not identified these proteins, thus, their 3D organization is undefined.     G-rich DNA (and RNA) sequences can form novel structures stabilized by cation coordination anda tetrameric arrangement of guanines, each hydrogen bonded to two neighbors, called the G-quartet (2,3). We have recently discovered conditions under which a simple synthetic oligonucleotide,d(GGGGTTGGGG), can spontaneously assemble into large superstructures stabilized by G-quartets.These structures are termed G-wires (4, 5). G-wires are extraordinarily stable and have unusual kineticproperties with regard to growth. These and other features make them an interesting subject for studyby AFM.     In this paper we will provide an overview of these three projects, all of which have been presentedin detail elsewhere (4, -8).22. Experimental2.1 Polytene Chromosome PreparationPolytene chromosomes of salivary gland cells from Drosophila melanogaster were isolated andsquashed as previously described (9). All of the imaging substrates were glass. Standard microscopeslides (26 X 76 mm2), 12 mm2 round coverslips (Fisher), and #1 glass coverslips; either 22 X 22 mm2or 22 X 44 mm2 (Corning) were used. Some of the coverslips were acid washed. There was noapparent difference between the acid washed and the untreated coverslips.2.2 Chick Giant Synapse PreparationChick ciliary ganglion presynaptic nerve terminals (the calyx) were prepared and the release faceidentified by fluorescence microscopy using the vital dye 4-Di-2-Asp as previously described (10-12).The specimen was kept wet and fixed prior to imaging. The N+ type calcium channel toxinw-conotoxin GVIA (w-CTX), conjugated to biotin, was added to the preparation, allowed to bind tocalcium channels, and the excess removed by rinsing. Streptavidin-gold (30 nm dia.) was added,allowed to bind to the biotinylated w-conotoxin, and the excess rinsed. Samples were then imaged insolution in contact mode as described below.2.3 G-Wire Preparationd(GGGGTTGGGG) was dissolved in the appropriate buffer (typically containing Na+ or K+ ions) aspreviously described (4, 5) at 1-10 mg/ml and incubated at 37°C for 12-24 h. After incubation thesamples were diluted 1: 100 in 10 mM Tris-HCI, pH 7.5 and 1 mM MgCl2.2.4 AFM MethodsAll AFM was performed using a Nanoscope III (Digital Instruments, Inc.) equipped with a J or Dscanner and using either standard silicon nitride tips, oxide sharpened tips or silicon tapping tips. Insome cases a BioScopeTm (combined AFM/optical microscope) was used to facilitate localization ofarea of interest. Both contact and, when possible, TappingModeTm images were obtained. Images in airwere typically collected at relative humidity ≤ 10%.     Polytene chromosomes were imaged either in air, under water or in phosphate buffered saline(PBS) as previously described (7, 8). Chromosome dissection experiments were carried out inphosphate buffered saline (PBS). A typical dissection was achieved by disabling the slow scan axis atthe region to be cut and maximizing both the force and the scan rate (13, 14, 15). By making smalladjustments in the scan angle we were able to increase the size of the cuts. This process can beaccomplished in less than 2 minutes.     Chick ciliary ganglion presynaptic nerve terminal release faces were imaged in solution in contactmode. A statistical analysis of the occurrence of protuberances thought to correspond to gold particlesin control and experimental samples was carried out (6). The relative spacing of the gold particles wasmeasured using NIH Image (version 1.53).     For G-wires, 10µl of the G-wire preparation was deposited onto a freshly cleaved mica substrate.The G-wires were allowed to adsorb for 5 min., washed with 1 ml sterile water, and rapidly dried in astream of N2 gas. For contact mode imaging, the relative humidity was maintained at ≤ 10%.3Figure 1. Images of a polytene chromosome. A,brightfield image of chromosome stained withcommassie blue. B, The same portion of achromosome stained with ethidium bromidehomodimer I and imaged by scanning laserconfocal fluorescence microscopy. C, AFM heightimage of the same chromosome taken in air. D,Higher resolution AFM deflection mode image ofanother portion of the same chromosome, imaged inPBS. Note the details in chromosome substructure.Figure 2. Chromosome dissection by AFM. The panels on the left show a deflection mode image of a polytenechromosome in PBS and a section through the chromosome (from a height mode image) prior to dissection. Inthe right hand panels, the same chromosomes are shown after dissection of a region in the lower chromosome(arrowhead).3. Results and Discussion3.1 Polytene ChromosomesPolytene chromosomes were readily imaged in the AFM (Fig. 1). Their large size facilitated findingthem on the substrate surface, even when present at low surface coverage. This feature, in conjunctionwith use of the combined optical/atomic force microscope (BioScope), greatly enhanced the rate ofprogress of this project.4Bands and interbands, similar to those observed by optical and electron microscopy, were observed.Bands often had bumpy surfaces corresponding to loops at the ends of chromatin strands attached atthe base to the chromosome scaffold (Fig. 1D). Comparing DNA density (using the DNA binding dyeethidium homodimer I) with band height clearly showed that the DNA is packed in the Z dimension,rather than X or Y.The structure of the strands in interband was complex. Numerous particles were apparentlyassociated with the chromatin, most likely including transcription factors and polymerase complexesas well as structural proteins involved in chromosome architecture. A detailed discussion of interbandstructure will appear elsewhere.By increasing the force applied by the AFM tip, and disabling scanning in the slow (Y) direction,it was possible to cut chromosomes (Fig. 2). After cutting, image quality is often compromised,suggesting that some of the excised chromatin had adhered to the scanning tip. Thus, the AFM mayprovide a rapid physical means for obtaining defined regions of chromosomes, which would facilitategene mapping efforts. Details of these studies are presented elsewhere (7, 8).3.2 Calcium Channels at Nerve TerminalsThe general protocol for mapping calcium channels at the nerve terminals was to tag the channel witha toxin, w-conotoxin, conjugated to a gold ball through a streptavidin-biotin bridge (6). Controlexperiments in which various steps in the mapping procedure were blocked clearly showed that theincreased incidence in the appearance of features on the surface of the release face corresponded togold bound to calcium channels through the w-conotoxin-gold conjugate (Fig. 3). Statistical analysisof these data showed that the spacing of calcium channels is 40 nm or larger in increments of 20 nm.Since the gold used in this study was 30 nm in diameter, the 20 nm harmonic indicates that channelsare in fact spaced at 20 nm intervals in the nerve terminal. This high density of calcium channelsfound in the nerve terminal ensures that synaptic vesicles (40-50 nm diameter) are likely to beimmediately adjacent to calcium channels. A detailed description of this study is presented in (6).Figure 3. Tagging calcium channels at the nerve terminal with an w-conotoxin-biotin-streptavidin-colloidal goldconjugate. Panel A shows a calyx surface from a control experiment in which the sample was pre-blocked withnon-biotinylated w-conotoxin. Panel B shows the experimental result. Both images were acquired in deflectionmode. The field size is 1 µm.53.3 G-WiresBiochemical data had strongly suggested that the oligonucleotide d(GGGGTTGGGG) assembled intohigher order structures under the appropriate conditions (5), but the morphological nature of thesestructures was unknown. AFM analyses of G-wires immediately gave significant insight into theirstructural nature (4) (Fig. 4). They are long, linear polymers. Their measured height is approximately25-30 Å, which agrees well with the diameter of a G-quartet determined by NMR and x-raycrystallography (16-18). This is an interesting finding because the height of B-DNA in the AFM istypically 5-7Å, which is less than half its width determined by X-ray crystallography. Thus, it appearsthat G-wires are more robust than B-DNA in the sense that they are not compressed by the scanningtip. An additional possibility is that B-DNA undergoes a severe conformation change when absorbedto a surface, but G-wires do not. However, previous work determining the helical period of B-DNA onmica surfaces suggests that it is not significantly altered upon binding to the mica (19).     The studies briefly described here illustrate the breadth of application of AFM to biologicalquestions. As the interest of biological investigators in AFM methods co-evolves with improved andspecialized AFM instrumentation, it is likely that this technology will make an increasingly significantcontribution to life sciences research.Figure 4. Height mode images of G-wires. All panels show G-wires grown in the presence of sodium ion andspermidine. Panels D-F show co-deposited G-wires and B-form plasmid DNA. Note the height difference (whiteto gray = tall to flat) between the plasmid DNA and the GZLUHV)LHOGVL]HVDUHSDQHOV$%'DQG( Ppanels C and F, 500 nm.4. AcknowledgmentsThe authors thank the sponsor and organizers of the workshop. They also thank their many colleaguesfor providing inspiration and enthusiasm for this area of research.65. References1. Binnig, G., Quate, C.F. and Gerber, C. (1986) Phys. Rev. Lett. 56, 930-933.2. Guschlbauer, W., Chantot, J.-F. and Thiele, D. (1990) J. Biomol. Struct. Dyn. 8, 491-511.3. Williamson, J.R., Raghuraman, M.K. and Cech, T.R. (1989) Cell 59, 871-880.4. Marsh, T.C., Vesenka, J. and Henderson, E. (1994) submitted5. Marsh, T.C. and Henderson, E. (1994) Biochemistry 33, 10718-10724.6. Haydon, P.G., Henderson, E. and Stanley, E.F. (1994) Neuron in press,7. Jondle, D.M., Ambrosio, L., Vesenka, J. and Henderson, E. (1995) Chrom. Res. in press,8. Mosher, C, Jondle, D., Ambrosio, L., Vesenka, J. and Henderson, E. (1995) Scan. Micros. in press.9. Pardue, M.L. (1986) In Roberts, D.B. (ed.), Drosophila a practical approach. IRL Press, Oxford,    Washington DC, pp. 111- 137.10. Stanley, E.F. and Atrakchi, A.H. (1990) Proc. Natl. Acad. Sci. USA 87, 9683-9687.11. Stanley, E.F. (1991) Neuron 7, 585-591.12. Stanley, E.F. and Goping, G.J. (1991) J. Neurosci. 11, 985-993.13. Henderson, E. (1992) Nuc. Acids Res. 20, 445-447.14. Vesenka, J., Hansma, H., Siegerist, C, Siligardi, G., Schabtach, E. and Bustamante, C. (1992) SPIE      1639, 127-137.15. Hansma, H., Vesenka, J., Siegerist, C, Kelderman, G., Morret, H., Sinsheimer, R.L., Elings, V.,      Bustamante, C. and Hansma, P.K. (1992) Science 256, 1180-1184.16. Kang, C.H., Zhang, X., Ratliff, R., Moyzis, R. and Rich, A. (1992) Nature 356, 126-131.17. Laughlan, G., Murchie, A.I.H., Norman, D.G., Moore, M.H., Moody, P.C.E., Lilley, D.M. and      Luisi, B. (1994) Science 265, 520-524.18. Smith, F.W. and Feigon, J. (1992) Nature 356, 164-168.19. Rhodes, D. and Klug, A. (1980) Nature 286, 573-579.

Analyzing Chromosomes, Ion Channels and Novel Nucleic Acid Structures by AFM

Proceedings of the NATO Advanced Research Workshop:  Scanning Probe Microscopies and Molecular Materials , 25.05.1994 - 03.06.1994, Schloss Ringberg, Tegernse

Analyzing Chromosomes, Ion Channels and Novel Nucleic Acid Structures by AFM

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