Birefringence measurements of the actin bundle in the acrosomal processes of Limulus sperm.
Actin bundles play an important role in many kinds of cell activities, such as muscle contraction, cytoplasmic streaming, cell migration, and acrosomal reaction. Using our modified polarized light microscope (1), we have measured the birefringence of well-characterized actin bundles to estimate the birefringence of a single actin filament.
The actin bundles in Limulus sperm contain a nearly constant number of actin filaments (80 filaments in the basal region) (2). The actin bundle is discharged from the head of the sperm after an acrosomal reaction and thus is very easy to identify.
Limulus sperm shows two types of discharge of acrosomal process -- true (Fig. 1A) and false (Fig. 1B). True discharge is part of the normal acrosomal reaction, but false discharge is induced by some stress (for example a temperature increase). In both cases, the discharged process consists of an actin bundle and a membrane (2).
To eliminate the effect of cell membrane on the measurements of retardance, we treated the sperm with Triton X-100 (3). Figure 1A shows a Triton-extracted sperm with a true discharge. Birefringence of the actin bundle is larger in the region nearer to the cell body (Fig. 1A). The birefringence, expressed as retardance, was almost constant in the basal region (0.85 [+ or -] 0.11nm, mean [+ or -] SD, n = 5). This observation is consistent with previous findings (2) that the number of actin fibers contained in the basal region is almost constant.
Because electron microscopic observations have shown that the number of the actin filaments in the basal region of the true discharge corresponds to the number in the tip region of the false discharge (2), we compared retardance in the base of the true discharge with that in the tip of the false discharge. The retardance of the tip of the false discharge was 0.72 [+ or -] 0.038 nm (n = 3), which is similar to the retardance measured in the basal region of the true discharge.
To find the retardance of a single actin filament, we divided the measured retardance of the acrosomal process by its number of actin filaments (80). As has been shown previously (4), the retardance of bundled microtubules increases linearly with number of microtubules in the bundle. Thus, the retardance of the single actin fiber ([R.sub.single]) can be calculated as:
[R.sub.single] = R/N
where R is the measured retardance, and N is the number of actin fibers (in this case 80 filaments). [R.sub.single] in artificial seawater is estimated as 0.0107 [+ or -] 0.0013 nm (n = 5). This value is a rough estimate because the actual number of actin filaments in the bundles used in this study was not determined, but the value should be useful for estimating the number of actin filaments contained in cells observed with the new pol-scope.
We are indebted to Dr. L. G. Tilney and S. Inoue for their invaluable suggestions. This work is supported by 1996 Lilli Fellowship awarded to F.O. and NIH grant (RO1 GM49210) to R.O.
1. Oldenbourg, R. 1996 Nature 381:811-812.
2. Tilney L.G. 1975. J. Cell Biol. 64:289-310.
3. Brokaw C.J. 1995. Methods Cell Biol. 47:231-238.
4. Tran P., S. Inoue E.D. Salmon, and R. Oldenbourg. 1995. Biol. Bull. 189:206.
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|Author:||Katoh, Kaoru; Yamada, Katsuyiki; Oosawa, Fumio; Oldenbourg, Rudolf|
|Publication:||The Biological Bulletin|
|Date:||Oct 1, 1996|
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