Controls for Kinesin-II Staining
Specific fluorescence was not observed when the primary antibody was replaced by normal mouse IgG, nor was it observed in other controls (Fig. 10). Buy Advair Diskus Online
IC74 Was Enriched in Supernatants From Gelsolin-Treated Spermatid/Junction Complexes
In each of the three experiments, actin filaments, present in controls, were virtually eliminated from plaques attached to spermatid/junction complexes incubated in gelsolin (Fig. 11). Significantly, the intermediate chain of cytoplasmic dy-nein (74 kDa) was obviously and consistently enriched, relative to controls, in supernatants collected from this material after removal of spermatids by slow-speed centrifugation (compare lanes 1 and 2 in each of the dynein immu-noblots in Fig. 11).
The content of kinesin-II (presumably from contaminating tails or protein solubilized from the sperm tails during the protocol) in supernatants was qualitatively much less affected by gelsolin treatment than was dynein (compare lanes 1 and 2 in each of the kinesin-II immunoblots in Fig. 11). Levels of this motor appeared to be noticeably elevated in only one of the three experiments.
FIG. 10. Immunolocalization of kinesin-II in fixed frozen sections of rat testis. The section shown in A was treated first with primary antibody and then with a secondary antibody conjugated to fluorescein. In B and C, the primary antibody was replaced with normal mouse IgG or with buffer alone, respectively, and in C, both the primary and secondary antibodies were replaced by buffer alone. Specific immunoreactivity with the probe occurred in the sperm tails. Also, bright focal signals were emitted from regions near the base of the tails.
FIG. 11. Shown here are the results of three separate experiments in which the actin in Sertoli cell junction plaques was digested with gelsolin and the supernatants from control and digested samples were tested for reactivity, on Western blots, with probes for dynein and kinesin-II. The rationale for using gelsolin was to release the endoplasmic reticulum component of the junction complexes from spermatids. Because motor proteins are thought to be anchored to the endoplasmic reticulum of the junction, the concentration of these motors should be increased in low-speed supernatants collected from digested samples. To verify that filamentous actin was digested by the gelsolin, control (A, C, and E) and experimental (B, D, and F) samples were stained with rhodamine phalloidin. On each of the blots, lane 1 is control supernatant, lane 2 is supernatant from gelsolin digested samples, and lane 3 is gelsolin (which cross-reacted both with the IC74 antibody and with normal mouse IgG—the latter is not shown). Molecular weight markers are in the unlabeled lane (size X 10—3 on right). Dynein reactivity on the blots was noticeably increased in supernatants collected from gelsolin-digested samples in all three experiments. Kinesin-II, localized in the tails of spermatids, was noticeably increased in the second experiment, but much less so in the other two. Results of this series of experiments are consistent with the conclusion that cytoplasmic dynein is a component of the junction plaque, but do not eliminate the possibility that at least some of the dynein could have originated from damaged spermatids themselves.