Atomic Force Microscopy Episode 2 Part 5 docx

A Image of the surface of an A6 renal epithelial cell labeled with colloidal gold particles conjugated to nonimmune IgG showing the microvilli.. B Image of the surface of an A6 cell labe

286 Smith and Benos

Fig 2 Representative images illustrating the localization of epithelial sodium

chan-nels by AFM (A) Image of the surface of an A6 renal epithelial cell labeled with colloidal gold particles conjugated to nonimmune IgG showing the microvilli (B)

Image of the surface of an A6 cell labeled with colloidal gold particles conjugated to

an anti-epithelial sodium channel antibody showing localization of marker to the microvilli Note marked increase in height of microvilli when compared to A

Repro-duced with permission from the American Physiological Society from ref 2.

3 Scan multiple cells/cover slip and multiple cover slips labeled with both antibody and control IgG Also scan cover slips bearing cells that were fixed but not labeled

to obtain dimensions and surface topography of the cells before antibody treat-ment Binding of colloidal gold antibody conjugates to the epitope-tagged ENaC results in a marked difference in height of the cell surface when compared to

controls (see Fig 2; Note 5).

4 Notes

1 cDNAs have been cloned for the α, β, and γ subunits of Xenopus (9), rat (10–13), mouse (14), and human ENaC (15–17) All species can be expressed

heterolo-gously in mammalian cells Typically, the cDNAs are available upon request from the laboratories that cloned them Subcloning of the subunits into a mam-malian expression vector, such as pcDNA3.1, may be required A number of laboratories have produced constructs for ENaC subunits with extracellular

epitope tags that may be available upon request (3,4,18).

2 Although we produce colloidal gold particles following the method of Slot and

Geuze (7), colloidal gold particles produced by this method are available

com-mercially (Sigma; Electron Microscopy Sciences, Fort Washington, PA)

3 HEK 293 cells have been effectively used for the transient expression of ENaC

(19,20) When HEK 293 cells are used with an expression vector that includes

the CMV promoter, such as pcDNA 3.1, high levels of transcription are obtained

COS 7 cells (19) and Fisher rat thyroid cells, which form polarized monolayers

(21), have also proven useful for the transient expression of ENaC Alternatively,

stably transfected cell lines expressing epitope-tagged α, β, and γ ENaC can be

generated (18,20,22) The selection of an appropriate cell line and the use of

tran-sient or stable transfectants depends upon the objectives of the investigation

4 For transient tranfection using liposome-mediated transfection reagents, we sug-gest starting with a plasmid concentration of 0.3 µg for each ENaC subunit This, however, will need to optimized in each lab Follow the manufacturer’s direc-tions for optimization of transfection efficiency and protein expression levels To achieve high efficiency transfections, it is critical that the plasmid DNA used is

of high quality and is free of contaminants

5 For visualization of colloidal gold antibody conjugate on the cell surface, select small areas of interest during scanning and reduce the field to concentrate on these areas In addition, small areas of interest can be selected from stored images, zoomed to full screen and analyzed using the Nanoscope III software To control for bias during both scanning and analysis, it is recommended that a blind study be performed

Acknowledgments

This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grants DK-37206 (Dale J Benos) and DK-56596 (Peter

288 Smith and Benos

R Smith) Peter R Smith is the recipient of an Established Investigator Award from the American Heart Association

References

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Struc-ture and regulation of amiloride-sensitive sodium channels Ann Rev Physiol.

62, 573–594.

2 Smith, P R., Bradford, A L., Schneider, S., Benos, D J., and Geibel, J P (1997) Localization of amiloride-sensitive sodium channels in A6 cells by atomic force

microscopy Am J Physiol 272, C1295–1298.

3 Firsov, D., Schild, L., Gautschi, I Merillat, A M., Schneeberger, E., and Rossier,

B C (1996) Cell surface expression of the epithelial Na channel and a mutant

causing Liddle syndrome: A quantitative approach Proc Natl Acad Sci USA

93, 15370–15375.

4 Konstas, A A., Bielfeld-Ackermann, A., and Korbmacher, C (2001) Sulfony-lurea receptors inhibit the epithelial sodium channel (ENaC) by reducing surface

expression Pflugers Arch 442, 752–761.

5 Ausbel, F M., Brent, R., Kingston, R E., Moore, D D., Seidman, J G., Smith, J A.,

et al (1997) Current Protocols in Molecular Biology, J Wiley and Sons, New York.

6 Putnam, C A.J., deGrooth, B G., Hansma, P K., van Hulst, N F., and Greve, J (1992) Immunogold labels: cell-surface makers in atomic force microscopy

Ultramicroscopy 42, 1549–1552.

7 Slot, W and Geuze, H J (1985) A new method to make gold probes for

multiple-labeling cytochemistry Eur J Cell Biol 38, 87–93.

8 Hartwig, J H (1992) An ultrastructural approach to understanding the

cytoskel-eton, in The Cytoskeleton A Practical Approach (K L Carraway and C A.C.

Carraway, eds), IRL, New York

9 Puoti, A., May, A., Canessa, C M., Horisberger, J-D., Schild, L., and Rossier, B

C (1995) The highly selective low-conductance epithelial Na+ channel of

Xeno-pus laevis A6 kidney cells Am J Physiol 269, C188–C197.

10 Canessa, C M., Horisberger, J.-D., and Rossier, B C (1993) Epithelial sodium

channel related to proteins involved in neurodegeneration Nature 361, 467–470.

11 Canessa, C M., Schild, L Buell, G., Thorens, B., Gautschi, I, Horisberger, J.-D.,

et al (1994) Amiloride sensitive epithelial Na+ channel is made of three

homolo-gous subunits Nature 367, 463–467.

12 Linqueglia, E., Voilley, N., Waldmann, R., Lazdunski, M., and Barbry, P (1993) Expression cloning of an epithelial amiloride-sensitive Na+ channel A new

chan-nel type with homologies to Caenorhabdites elegans degenerins FEBS Lett 318,

95–99

13 Lingueglia, E., Renard, S., Waldmann, R., Voilley, N., Champigny, G., Plass, H.,

et al (1994) Different homologous subunits of the amiloride-sensitive Na+

chan-nel are differently regulated by aldosterone J Biol Chem 269, 13736–13739.

15 McDonald, F J., Snyder, P M., McCray, P B., and Welsh, M J (1994) Cloning, expression, and distribution of a human amiloride-sensitive Na+ channel Am J.

Physiol 266, L728–L734.

16 McDonald, F J., Price, M P., Snyder, P M., and Welsh, M J (1995) Cloning and expression of the β and γ subunits of the human epithelial sodium channel Am J.

Physiol 268, C1157–C1163.

17 Voilley, N., Lingueglia, E., Champigny, G., Mattei, M.-G., Waldmann, R., Lazdunski, M., et al (1994) The lung amiloride-sensitive Na+ channel:

biophysi-cal properties, pharmacology, ontogenesis, and molecular cloning Proc Natl.

Acad Sci USA 91, 247–251.

18 Hanwell, D., Isikawa, T., Saleki, R., and Rotin, D (2002) Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin–Darby

canine kidney cells J Biol Chem 277, 9772–9779.

19 Prince, L S and Welsh, M J (1998) Cell surface expression and biosynthesis of epithelial Na+ channels Biochem J 336,705–710.

20 Staub, O., Gautschi, I., Ishikawa, T., Breitschopf, K., Ciechanover, A., Schild, L.,

et al (1997) Regulation of stability and function of the epithelial Na+ channel

(ENaC) by ubiquitination EMBO J 16, 6325–633.

21 Snyder, P M (2000) Liddle’s syndrome mutations disrupt cAMP-mediated trans-location of the epithelial Na+ channel to the cell surface J Clin Invest 105, 45–53.

22 Shimkets, R A., Lifton, R., and Canessa, C M (1998) In vivo phosphorylation of

the epithelial sodium channel Proc Natl Acad Sci, USA 95, 3301–3305.

21

High-Resolution Imaging of Bacteriorhodopsin

by Atomic Force Microscopy

Dimitrios Fotiadis and Andreas Engel

1 Introduction

In the last years the atomic force microscope (AFM; ref 1) has become a

powerful imaging tool for the biologist The unique features like the possibility

to image biological structures in their native environment (i.e., in buffer solu-tion, at room temperature, and under normal pressure), the high lateral and vertical resolution, and the high signal-to-noise ratio of the topographs acquired

by AFM make this instrument outstanding It has made the observation of dif-ferent single biomolecules at work and the monitoring of biomolecular

interac-tions by time-lapse AFM possible (for recent reviews, see refs 2–4).

This chapter focuses on the application of contact mode AFM to acquire high-resolution structural information of membrane proteins in buffer solu-tion In this scan mode the probing tip touches the surface with a constant force while scanning To minimize possible damage of the biological specimen by the tip, soft cantilevers with spring constants around 0.1 N/m must be used and scanning must be performed at minimal tip force (approx 100–300 pN) Lat-eral resolutions down to 0.41 nm and vertical resolutions down to 0.10 nm

have been achieved on biological membranes in solution (5) Alternative AFM modes to record topographies are the tapping mode (6–8) and the magnetically activated oscillating mode (9,10), better known as MAC mode Both are

simi-lar and frequently used to image the surface topography of weakly immobi-lized biomolecules, that is, single proteins, fibrils, and chromosomes, and have

in common that the AFM tip is oscillated vertically while scanning the sample Thus, frictional forces are reduced by the oscillation of the tip avoiding defor-mation and displacement of the sample However, for high-resolution imaging

of biological membranes contact mode has shown to be the better choice pro-From: Methods in Molecular Biology, vol 242: Atomic Force Microscopy: Biomedical Methods and Applications

Edited by: P C Braga and D Ricci © Humana Press Inc., Totowa, NJ

292 Fotiadis and Engel vided the imaging parameters, for example, force and imaging buffer, are

adjusted correctly (11) To prevent deformation of the structure as a result of

friction, the forces acting between tip and sample should not exceed 300 pN in contact mode However, application of higher forces can sometimes be useful

to perform precise and controlled dissections of biological samples by

manipu-lation with the AFM tip (12).

As biological specimen for the AFM imaging experiment presented here,

we have chosen bacteriorhodopsin (BR) membranes This 26-kDa heptahelical transmembrane protein acts as a light-driven proton pump in the cell

mem-brane of the bacterium Halobacterium salinarum (13,14) Photoisomerization

of the covalently bound chromophore from all-trans to 13-cis retinal initiates

proton translocation across the cell membrane (15,16) This establishes a

pro-ton gradient across the cell membrane for ATP synthesis and other energy requiring processes in the cell BR molecules form highly ordered

two-dimen-sional crystals (17) (trigonal lattice: a = b = 6.2 nm, γ = 60°) in the native

membrane of Halobacterium salinarum, termed purple membrane for its color.

Because of its crystallinity and flatness this sample is very suitable for AFM and cryo-electron microscopy High-resolution three-dimensional structures of

BR (Fig 1) were determined by electron crystallography and X-ray diffraction

(for a recent review, see ref 18) In BR, the retinal (see Fig 1; arrowhead) lies

in the intramembrane cavity formed by the seven transmembrane α-helices generally denoted A to G The main portions of BR that protrude out of the membrane are: The loops connecting the transmembrane α-helices A and B as

well as E and F (AB and EF loops; Fig 1) on the cytoplasmic side and the B-C interhelical loop (BC loop; Fig 1) on the extracellular side The latter forms a

twisted antiparallel β-sheet and is more stable than the wobbly EF loop (19).

2 Materials

2.1 Preparation of Mica Supports for Sample Immobilization

1 Inoxydable and magnetic steel disks of 11 mm in diameter (internal services of the Biozentrum, Basel, Switzerland)

2 Teflon sheets of 0.25-mm thickness (Maag Technic AG, Birsfelden, Switzerland)

3 Mica sheets with a thickness between 0.3–0.6 mm (Mica House, 2A Pretoria Street, Calcutta 700 071, India)

4 “Punch and die” set from Precision Brand Products Inc (Downers Grove, IL)

5 Ethanol (concentration 96% [v/v])

6 Loctite 406 superglue from KVT König, Dietikon, Switzerland

7 Araldit Rapid: Two-component epoxy glue from Ciba-Geigy, Basel, Switzerland

8 Scotch tape

2.2 BR and Buffers (see Note 1)

1 Purple membranes of H salinarum Stock solution: 0.25 mg/mL in double

dis-tilled water containing 0.01% NaN3 Store at 4°C and protect from unnecessary light irradiation

2 Adsorption buffer: 20 mM Tris-HCl, pH 7.8, 150 mM KCl.

3 Imaging buffer for the extracellular side (ES imaging buffer): 20 mM Tris-HCl,

pH 7.8, 150 mM KCl, 25 mM MgCl2

4 Imaging buffer for the cytoplasmic side (CS-imaging buffer): 20 mM Tris-HCl,

pH 7.8, 150 mM KCl.

2.3 AFM and Accessories (see Note 2)

1 A commercial multimode AFM equipped with a 120-µm scanner (j-scanner) and a liquid cell (Digital Instruments, Veeco Metrology Group, Santa Barbara, CA)

2 Oxide-sharpened Si3N4 micro cantilevers of 100 µm in length and a nominal

spring constant of k = 0.08 N/m (Olympus Optical Co., LTD, Tokyo, Japan).

Fig 1 Ribbon diagram of BR The retinal chromophore (arrowhead) is displayed

as a ball-and-stick model This illustration of BR was calculated using the coordinates

of Kimura et al (28) and the three-dimensional visualization program DINO (http://

www.dino3d.org/)

294 Fotiadis and Engel

3 Method

3.1 Preparation of Mica Supports for Sample Immobilization

1 Punch mica disks of 6 mm and Teflon disks of 13 mm diameter using the “punch and die” set and a hammer

2 Clean the Teflon and steel disks with ethanol and paper wipes

3 Glue a Teflon disk on a steel disk using Loctite 406

4 Glue a mica disk on the Teflon surface of the Teflon-steel disk with the two-component epoxy glue

5 Let the supports dry for at least 1 day

3.2 Adsorption of BR to Mica

1 Dilute and mix 3 µL of purple membrane stock solution with 30 µL of adsorption buffer in an Eppendorf tube

2 Cleave mica with Scotch tape

3 Pipet the diluted purple membranes on the freshly cleaved mica support

4 Adsorb BR for 15 to 30 min

5 Wash away the purple membranes that are not firmly attached to the mica by removing approximately two thirds of the fluid volume from the mica surface and readding the same amount of the corresponding imaging buffer Repeat this washing procedure at least three times

6 Transport the support onto the piezo scanner

7 Mount the AFM head containing fluid cell (without o-ring seal) and cantilever on the microscope

8 Fill the space between the mica surface and the fluid cell with the corresponding imaging buffer to avoid drying of the protein

3.3 Operation of the AFM (see Notes 3–5)

After thermal relaxation of the instrument, initial engagement of the tip is performed Specimen deformation and contamination of the tip is minimized during the engagement process by setting the scan size to 0 Prior scanning the surface, the operating point of the instrument is set to forces below 1 nN Dur-ing scannDur-ing the forces are kept as small as possible (<300 pN) and corrected manually to compensate for thermal drift

Two frames of 512 by 512 pixel are simultaneously recorded either showing topography or deflection signal in trace or retrace direction Usually deflection and height signals are recorded at low magnification (frame size >1 µm) whereas height signals were acquired in both, trace and retrace direction at high magnification (frame size <1 µm)

This allows deformation of the sample in the fast scan direction to be detected and to be minimized by lowering the force applied to the stylus Typi-cally, the scan speed is set to 4.7–5.5 Hz (lines per second) At high magnifica-tion the scan range of the z piezo is reduced to avoid limitamagnifica-tion of the axial

3.4 Conclusion

Here we have presented materials and methods to image the native surface

of bacteriorhodopsin at subnanometer resolution with the AFM in buffer

solu-tion (see Note 6) We have demonstrated that forces between stylus and sample

as well as shape and geometry of the AFM tip play an important role for

suc-cessful imaging of the biological sample (see Note 7) Additionally, it was

shown that higher forces may be of advantage to study otherwise hidden

fea-tures of a protein, that is, the AB loop in BR (see Note 5).

4 Notes

1 Buffer conditions for high-resolution AFM imaging: Topographs of native

mem-brane proteins with a lateral resolution of 0.41 nm (5) can reproducibly be

recorded with the AFM provided imaging force and buffer are adjusted correctly

(11) In general, scanning is performed at minimal forces applied to the stylus to

avoid friction and deformation of the biological sample However, often even the smallest force adjustable by the instrument is too high for preventing deforma-tion of the biomolecule

The effective interaction force acting between AFM stylus and specimen is the sum of the force applied to the stylus, the electrostatic repulsion and the van der Waals attraction between the two surfaces By adjusting pH and ion strength of the imaging buffer van der Waals attraction and electrostatic repulsion between tip and sample can be balanced Under these conditions the tip is assumed to surf

on a cushion of electrostatic repulsion minimizing the deformation of the biomolecule The best imaging conditions are determined by recording and ana-lyzing force-distance curves between tip and sample in different buffers Condi-tions that yield force curves with a small repulsive peak are ideal for high-resolution imaging By this screening method the two slightly different

imaging buffers for BR mentioned in Subheading 2.2 (CS and ES imaging

buffer) were found For further reading on this topic, see ref 11.

2 Damping of vibrations: For high-resolution AFM imaging, an acoustic and vibra-tion isolated set-up of the microscope is crucial Antivibravibra-tion and damping tables,

or lead platforms supported by bungees offer excellent vibration damping Acous-tic isolation of the AFM can efficiently be achieved by a vacuum bell jar

3 Morphology of BR crystals Figure 2 shows a typical overview (frame size 25 µm)

of purple membranes adsorbed to freshly cleaved mica The diameter of the BR sheets varies between 0.5–1.5 µm The number of adsorbed membrane patches depends on the adsorption buffer, time and the concentration of the bacteriorhodopsin solution deposited on the mica To avoid contamination of the tip, BR sheets were not adsorbed too densely on the support At higher

magnifi-cation (Fig 3; frame size 3.67 µm) two different types of membranes can be