MICROSCOPY RESEARCH AND TECHNIQUE 69:291–295 (2006)
Dynamic Study of Intramembranous Particles in Human Fresh Erythrocytes Using an ‘‘In Vitro Cryotechnique’’ NOBUO TERADA,* NOBUHIKO OHNO, YASUHISA FUJII, TAKESHI BABA, AND SHINICHI OHNO Department of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi 409-3898, Japan
KEY WORDS
shear stress; freeze-fracture; replica; membrane skeleton
ABSTRACT For analyses of dynamic ultrastructures of erythrocyte intramembranous particles (IMPs) in situ, a quick-freezing method was used to stabilize the flow behavior of erythrocytes embedded in vitreous ice. Fresh human blood was jetted at various pressures through artificial tubes, in which the flowing erythrocytes were elongated from biconcave discoid shapes to elliptical ones, and quickly frozen in liquid isopentane–propane cryogen (1938C). They were freeze-fractured using a scalpel in liquid nitrogen, and routinely prepared for replica membranes. Many IMPs were observed on the protoplasmic freeze-fracture face (P-face) of the erythrocyte membranes. Some control erythrocytes under nonflowing or stationary conditions showed IMPs with their random distribution. However, other jetted erythrocytes under flowing conditions showed variously sized IMPs with much closer distribution. They were also arranged into parallel rows in some parts, and aggregated together. This quick-freezing method enabled for the first time the visualization of time-dependent topology and the molecular alteration of IMPs in dynamically flowing erythrocytes. Microsc. Res. Tech. 69: 291–295, 2006 V 2006 Wiley-Liss, Inc. C
INTRODUCTION Individual erythrocytes are reported to have elongated shapes because of the strength of shear stresses while human blood is subjected to viscometric flow in vivo (Fisher et al., 1978). It is generally accepted that the ‘‘in vivo cryotechnique’’ can stop the time-dependent physiological processes of cells and tissues in vivo (Ohno et al., 1996; Terada et al., 2005). Flowing erythrocytes in blood vessels can easily change their shape because of the surrounding conditions, such as the types of vessels or flow speed, as well as some processes for preparing electron microscopic specimens (Terada et al., 1998a). Using the ‘‘in vivo cryotechnique,’’ we already reported three-dimensional shapes of flowing erythrocytes in sinusoids of living mouse livers (Terada et al., 1998b) and spleens (Xue et al., 2001), large blood vessels, including the aorta and caudal vena cava (Xue et al., 1998), glomerular capillaries of mouse kidneys (Yu et al., 1998), and capillaries of mouse lungs (Takayama et al., 2000). A shear stress from the flow is one of the factors, which affect the erythrocyte shapes (Fisher et al., 1978). To examine morphological changes of human erythrocytes by the shear stress in vitro, we developed one experimental model by a quickfreezing method, designated ‘‘in vitro cryotechnique for erythrocytes’’ (Terada et al., 1998a). Using the ‘‘in vitro cryotechnique,’’ flowing erythrocytes were clearly demonstrated to have elongated shapes because of the high jet-pressures, by freezing immediately after their ejection from artificial tubes (Terada et al., 1998a). In such erythrocytes, the membrane moves around the cell body and cytoplasm is driven into an eddy-like flow (Fisher, 2004). This particular kind of membrane motion has been termed tank-tread motion (Schmid-Schonbein and Wells, C V
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1969). The ‘‘in vitro cryotechnique’’ actually enabled a direct analysis of the morphological changes of human erythrocytes under various flowing conditions using electron microscopy, and not with indirect light microscopic observation as was often performed previously (Fisher et al., 1978). In the present study, the frozen erythrocytes at various jet-pressures were freeze-fractured to expose the integral membrane proteins, corresponding to intramembranous particles (IMPs), after the ‘‘in vitro cryotechnique for erythrocytes.’’ This provided, for the first time, a new image of IMPs’ distribution in human elongated erythrocytes. MATERIALS AND METHODS Samples Venous blood was collected into heparin-coated syringes from healthy volunteers. In the present study, freshly prepared blood, except for the heparin treatment, was used within 30 min to examine the native state of human erythrocytes. ‘‘In Vitro Cryotechnique for Erythrocytes’’ Followed by Freeze-Fracturing For morphological observation of elongated erythrocytes at high jet-pressures, the fresh blood was directly jetted from the tip of a syringe (Fig. 1a, right side), attached to a pump apparatus to control jet-pressures (60–200 mm Hg), into liquid isopentane–propane cryo*Correspondence to: Nobuo Terada, MD, PhD, Department of Anatomy, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Tamaho, Yamanashi 409-3898, Japan. E-mail:
[email protected] Received 19 November 2005; accepted in revised form 16 January 2006 DOI 10.1002/jemt.20315 Published online in Wiley InterScience (www.interscience.wiley.com).
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Fig. 2. Scanning electron micrographs of erythrocytes under dropping condition (a) and ejecting from a syringe at a high jet-pressure (b). Direction of flow is indicated by the arrow (b). Note the elongated erythrocytes, from about 7 lm (a) to 10 lm (b) in length, along the flow direction (large arrow), as previously reported (Terada et al., 1998a). One side of the tip of elongated erythrocytes can be observed (arrows), because another side is stuck into the adjacent erythrocytes. Scale bar ¼ 10 lm.
Fig. 1. Schematic representation of the ‘‘in vitro cryotechnique for erythrocytes’’ (a), following freeze-fracturing (b) and shadowing (c) steps. a: Erythrocytes, dropping (nonflowing erythrocytes) or jetting (flowing erythrocytes) from narrow tubes, are quickly frozen in isopentane–propane cryogen (IP) (1938C). b, c: The frozen specimens are freeze-fractured with a scalpel in liquid nitrogen, and rotationally replicated with platinum (Pt) and carbon (C).
gen (1938C) (Terada et al., 1998a). To obtain a resting state for erythrocytes (i.e., nonflowing erythrocytes), some amount of whole blood was dropped into the cryogen (Fig. 1a, left side). When the blood entered the cryogen, erythrocytes were quickly frozen in situ, as previously reported (Terada et al., 1998a). Superficial layers of the frozen specimens were freeze-fractured using a scalpel in liquid nitrogen for replica membrane preparations, as previously reported (Ohno et al., 1996) (Fig. 1b). Freeze-Substitution for Scanning Electron Microscopy Some frozen blood specimens were submitted to the routine freeze-substitution method to examine the extent of elongation of jetted erythrocytes, as follows. They were transferred into absolute acetone containing 2% osmium tetroxide at 808C and kept for 20 h, at 208C for 2 h, and finally at 48C for 2 h. They were briefly washed in pure acetone at room temperature, and then transferred into t-butyl alcohol for scanning electron microscopy (SEM) preparations. They were routinely frozen and freeze-dried at 58C in ES-2030 apparatus (Hitachi Ltd., Tokyo, Japan). These speci-
mens were then ion-sputtered with platinum/palladium (10–15 nm in thickness), and observed in S-4500 SEM (Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 5 kV. Etching and Shadowing for Replica Membranes Other freeze-fractured specimens were mounted on specimen holders with glycerine in liquid nitrogen, as previously reported (Ohno et al., 1996). They were transferred into an Eiko FD-3AS machine (Eiko Co., Ibaraki, Japan) and etched under vacuum conditions of 1–4 3 107 Torr vacuum at 958C for 5–10 min. Replica membranes of the freeze-fractured erythrocytes were then prepared on a rotation stage by two evaporation steps of platinum at an angle of 258 and subsequently of carbon at an angle of 908 (Fig. 1c). The replica membranes were routinely treated in household bleach and mounted on Formvar-filmed copper grids. They were examined in Hitachi H-600 electron microscope. Electron micrographs were printed from inverted negative films. RESULTS Some clumps of erythrocytes flowing through the tube were immediately frozen in the isopentane–propane cryogen. In the present cryofixation process, surface layers of the jetted whole blood were well frozen, as revealed by SEM (Fig. 2). Native shapes of erythrocytes in the blood could be obtained by this direct cryofixation, and acceptable areas without cryo-damage were restricted to such surface layers. Under the shear stress, flowing erythrocytes were elongated, consistent Microscopy Research and Technique DOI 10.1002/jemt
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Fig. 3. a: As illustrated, P-face (PF), E-surface (ES), and inside of the erythrocyte (IE) are obtained by the freeze-fracturing step. Se: Serum components. b: Low magnified replica electron micrograph, showing the freeze-fractured erythrocyte. An erythrocyte is seen under the flowing condition (arrow), and its elongated shape can be recognized. Scale bar ¼ 1 lm. c: Higher magnification of some parts in the P-face, shown in the square of (b). Note the parallel lining arrangement of IMPs along the long axis direction (arrow) of the elongated erythrocyte. Scale bar ¼ 200 nm. d, e: On higher magnified replica membrane shown in (c), the arrangement of IMPs (arrowheads in (d)) along the direction of flow (arrow in (d)) is obvious, in comparison with those of nonflowing erythrocytes (e). Scale bar ¼ 100 nm.
with the previous studies (Fisher, 2004; Terada et al., 1998a). The nonflowing erythrocytes were observed as typical biconcave discoid shapes (Fig. 2a), whereas the flowing erythrocytes (under 200 mm Hg jet-pressure) were elongated for up to 10 lm in their long axis (Fig. 2b). The actual shear stress was calculated as laminar flows in the tube used in the present study. The length and radius of the tube were 100 and 0.1 cm, respectively. The pressure difference was 200 mm Hg (1 mm Hg ¼ 133 Pa ¼ 1,330 dyn/cm2). Therefore, the shear stress was calculated to be 133 dyn/cm2 by the formula: shear stress ¼ (pressure difference 3 radius of tube)/ (2 3 length of tube). The similar rate of share stress has been well used for the erythrocyte-elongation by the ektacytometer, to simulate the physiological condition (Mazeron et al., 1997). Microscopy Research and Technique DOI 10.1002/jemt
Most upper layers of the erythrocytes were usually freeze-fractured to reveal IMPs. Figure 3 shows replica electron micrographs, prepared by using etching step after the ‘‘in vitro cryotechnique.’’ The frozen clumps were easily freeze-fractured (Fig. 3a), and well-frozen areas were obtained in the surface specimens (Fig. 3b). Appearance and distribution of IMPs could be detected on the protoplasmic-face (P-face; PF in Fig. 3b). Flowing erythrocytes (under 200 mm Hg jet-pressure) showed variously sized IMPs, clustering in some parts to form rows (Figs. 3c and 3d). Moreover, the relation of the erythrocyte elongation axis to IMPs’ arrangement could be assessed using these procedures. The direction of flow was indicated by arrows in Figure 3, judging from the shape of the elongated erythrocyte (arrows in Figs. 3b–3d). IMPs were arranged into rows running in
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the same direction as the long elongation axis (Figs. 3c and 3d). The nonflowing discoid erythrocytes showed variously sized IMPs on the P-face (Fig. 3e), and the IMPs were distributed randomly without aggregation. These results suggest that this simple cryotechnique is useful for visualizing the dynamic movement of IMPs. DISCUSSION This study is the first attempt to visualize IMPs in artificially elongated erythrocytes. The native distribution of IMPs, which were commonly assumed to interact with membrane skeletons under lipid layers, has not been clarified in flowing erythrocytes under any conditions. It is well-known that IMPs in erythrocyte membranes can be directly visualized using the conventional freeze-fracture method (Elgsaeter and Branton, 1974). Thus, a key point of our cryotechnique was to observe the rapidly changed IMPs of variously shaped erythrocytes. This was achieved using the cryofixation step under flowing conditions and the freeze-fracturing step. In the present study, some IMPs were attached to each other and arranged into strand-like structures, as shown in Figure 3, especially in elongated erythrocytes. Some IMPs in erythrocyte membranes have been suggested to consist of many proteins, including ion transporters or ion exchangers. Band 3 and glycophorins are major components of such proteins, as seen on the P-face (Pinder et al., 1995). In normal erythrocytes, some spectrin molecules have been thought to behave like elastic springs, which were capable of resisting mechanical extension stresses (McGough and Josephs, 1990; Terada et al., 1997). The shape changes of membrane skeletons have also been speculated by some simulations (Disher et al., 1998; Picart et al., 2000; Vera et al., 2005). During such extension processes, the spectrin networks probably drag complexes of integral proteins and membrane skeletons along their force direction (Lux and Palek, 1995). Moreover, it was also reported that the moving range of integral proteins might be restricted by underlying membrane skeletons, making ‘‘compartmentation,’’ because of dragging of the erythrocyte membranes (Sako and Kusumi, 1995). By this model, IMPs in erythrocyte membranes, such as band 3, were thought to collide with each other and associated with the membrane skeleton during extension of the membrane (Tomishige et al., 1998). Therefore, it was speculated that IMP distributions would dramatically change, based on the shape of the erythrocyte. This study supports this hypothesis with observations of changes to IMP distribution when the erythrocytes were deformed under a shear stress. One possible explanation for changing IMPs arrangement, observed in this study, is illustrated in Figure 4, although the number of IMPs attaching to the membrane skeletons was nothing more than the speculation at the present time. For example, less than half of band 3, a major intramembranous protein in erythrocytes, was reported to attach to the spectrin-actin membrane skeletons via ankyrin (Casey and Reithmeier, 1991). Although some IMPs are known to associate with the cytoskeleton (Huang et al., 2004), it is difficult to observe this association using the quick-freezing method presented in this study. The next step is to identify each component with the replica immunocytochemistry for the IMPs, which probably changes its or-
Fig. 4. A model of arrangement of IMPs with regard to translocational diffusion. An erythrocyte is in the biconcave discoid shape under the nonflowing condition (a). It changes into an elliptical shape under the flowing condition (c). b, d: The speculated view from the inside of the erythrocyte showing membrane skeletons lying tangent to the lipid bilayer (b: nonflowing condition, d: flowing condition). IMPs undergo free diffusion within each ‘‘compartment.’’ The concept of such a ‘‘compartment’’ by the membrane skeleton is based on the previous study (Tomishige et al., 1998). IMPs, being not attaching to the membrane skeletons, have smaller space to move under the flowing condition (d), than those under the nonflowing one (a).
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