figure 1 - Intact Red Blood Cell Imaged with the Explorer LifeSciences AFM.

Analysis of Red Blood Cell Cytoskeleton Using the Explorer(tm) LifeSciences(tm) AFM

By Dr. J. M. Mikrut & Dr. R. C. MacDonald, Dept. of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, Il 60208-3500 (708) 491-5062

Synopsis

In this Applications Note we describe how we have successfully used the Explorer LifeSciences AFM to study properties of whole erythrocytes and the cytoskeletal network of the cell membrane.

Introduction

Red Blood Cells (RBCs) have been extensively studied over the years because of their relatively simple structure and ease of isolation. The RBCs are biconcave in shape with an approximate thickness of 2-3 microns and a diameter of around 7.0 microns. An understanding of these cells is important because of their function in transporting oxygen to, and carbon dioxide away from, all the tissues in the body. This is achieved due to the large quantity of the protein hemoglobin that is contained in the cell. It is not understood how the RBCs withstand the large shear forces they experience while passing through the capillaries of the vascular system. Through the use of SDS-PAGE, the integral proteins (e.g. spectrin, actin, and Band 4.1) have been identified. It is believed that these proteins are responsible for the mechanical stability of the cell. While considerable progress has been achieved using the electron microscope to determine the arrangement of the proteins in the cytoskeleton, there remains a large number of dynamic experiments that are precluded by the rigors of electron microscope sample preparation procedures. For example, electron microscopy has yielded information on the disposition of spectrin and actin in stretched RBCs, however its disposition is totally unknown in relaxed conformation. For this reason we turned our attention to using the Atomic Force Microscope (AFM), which combines the advantage of high instrumental resolution with the ability to scan dynamic events under physiological conditions.

Diagram 1. - Tip sample interaction.

Experimental

Fresh RBCs were washed in phosphate buffer and applied to freshly cleaved mica surface which has been treated with poly-l-lysine. The cells were either left intact (Figure 1) or lysed with a low salt buffer and the hemoglobin rinsed away to reveal the cytoskeletal network (Figure 2) All images were acquired with the TopoMetrix Explorer LifeSciences AFM. The Explorer is unique among commercially available instruments in that its scanning mechanism is suspended above the sample. Furthermore, imaging under physiological conditions is enormously simplified by using the liquid scanner. The instrument can be positioned on an inverted optical microscope, so that cells are easily positioned under the scanning tip. Whole cells are easily seen under reflected white light, but in the case of ghosts it is necessary to label them and image them under fluorescence illumination.

Figure 2. - Cytoskeletal network in a lysed RBC sample.

Results and Discussion

The enormous benefit afforded by the AFM for biological studies is the ability to scan a sample, manipulate it, and then scan again. An example of this type of analysis is demonstrated. An image of the intact RBCs are shown in Figure 1. The image is similar to those seen in the SEM, only information from the upper side of the cell is available due to the nature of the tip - sample interaction which is diagramatically depicted in Diagram 1. To expose the cytoskeletal network on the inner surface of the cell membrane the cells were lysed in 5mM sodium phosphate buffer and excess material was washed away to reveal the spectrin scaffold (Figure 2). This experiments represents the first stage of a long term project designed to understand how the cell endures mechanical stresses during circulation through the blood stream. The information gained from this project will have important clinical implications, since many forms of anemia can be directly attributed to cytoskeletal abnormalities.

All images were acquired with the TopoMetrix TMX 2000 Discoverer SPM

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