Identification of dynein as the outer arms of sea urchin sperm axonemes.

Abstract

The location of dynein, the main flagellar ATPase, within the sea urchin sperm axoneme was investigated by the use of immunofluorescence and immunoelectron microscopy, employing an antiserum against a t tic fragment of dynein 1 (Fragment 1A) purified from sea urchin sperm flagella. The axonemes were found to be stained with the antiserum when examined by an indirect immunofluorescence technique. Immunoelectron microscopy with the antiserum and a ferritin-conjugated IgG fraction of goat antiserum to rabbit IgG revealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles. With either the normal serum or antiserum absorbed with Fragment 1A, there were no ferritin particles within the axonemes. When the outer arms were extracted with 0.5 M NaCl, leaving the inner arms intact, again no ferritin dots were detected. Furthermore, it was found that the outer arm on the no. 5 doublet microtubule, which connects with the extra arm projection backward from the no. 6 doublet, had no attached ferritin particles. From these observations, it can be concluded that the outer arm consists of dynein (at least dynein 1) and that Fragment 1A, containing the active site for ATPase activity of dynein 1, is located at the distal end of the outer arms. The significance of the present findings is considered in connection with flagellar movement. Flagellar movement is a particular, rhythmic, propagating bending. The sliding-microtubule model, principally based on the sliding of the arms against the neighboring outer doublet microtubules, has been presented for interpreting the mechanisms that are responsible for producing this bending (1-4). Experimental evidence for such a sliding process was reported by Summers and Gibbons (5) vealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles and that the enzymatically active site is located in the distal part of the outer arm. MATERIALS AND METHODSSpermatozoa of the sea urchin Anthocidaris crassispina were used in this study. Some points were corroborated with other species, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Shedding was induced by introducing a few drops of 0.5 M KCI into the body cavity.The spermatozoan plasma membrane-was removed by adding 20 volumes of 0.025% (vol/vol) Triton X-100 in 0.15 M KCI/4 mM MgSO4/1 mM CaCl2/2 mM EDTA/5 mM 2-mercaptoethanol/2 mM Tris-HCl, pH 8.2. After 1 min at room temperature, the sperm suspension was diluted 6-fold with the above medium without Triton X-100, and then centrifuged at 3000 rpm for 5 min. (Centrifugations were in a Hitachi instrument.) The sedimented spermatozoa were washed three times with the same medium and finally suspended in a small volume of 0.1 M sodium phosphate buffer, pH 7.5. In order to remove the outer arms from the flagellar axoneme, the spermatozoa were first suspended in 0.04% Triton X-100/0.15 M NaCl/4 mM MgSO4/0.5 mM EDTA/1 mM dithiothreitol/2 mM Tris-HCI, pH 8.0, and homogenized to detach the fla...

Immunological dissimilarity in protein component (dynein 1) between outer and inner arms within sea urchin sperm axonemes

“…In the previous experiment (22), no ferritin particles were observed near the inner arms even when the 9 + 2 structures appeared as opened or fragmented arrays. While these axonemes were fragmented after or during the immunocytological processing, axonemes were mechanically disrupted in advance by the standard processing in the present experiment .…”

“…Chemical and functional equivalences have been shown between the outer and inner arms of sea urchin sperm axonemes on the basis of chemical dissection (6,14) . The present work, together with the previous report (22), has tried to demonstrate the immunological dissimilarity in protein component (dynein 1) between the two members of a pair of arms, since a major area of controversy in the eukaryotic flagellar motility at present is the question of whether they are identical in protein component and function . In contrast to the previous ferritin-conjugated IgG, the present peroxidase-conjugated IgG was supposed to exhibit a better permeability with respect to the axonemesbecause of its smaller molecular size .…”

“…6 a-c), the outer arm projecting from the no . 5 doublet hardly received the anti-dynein 1 (present result) and anti-Fragment IA (22) antibodies. The inner arms are also connected with an interdoublet link and do not receive antibodies .…”

“…The former two specimens were obtained as described previously (22) . The last one was prepared by disrupting Tritonated axonemes with repeated suckingblowing in a Pasteur pipet, so that the axonemal peripheral doublet-microtubules appeared as opened or fragmented arrays .…”

“…The intact and salt-extracted axonemes were examined by indirect immunofluorescence microscopy as described in the previous paper (22) .…”