FIGURE 2. The distribution of Myo1e in frog oocytes as revealed by subcellular fractionation and immunofluorescence microscopy.

A, subcellular fractionation, a homogenate of frog oocytes was subjected to low speed centrifugation (~700 × g for 3 min to generate supernatant 1 (S1) and pellet 1 (P1)) followed by high speed centrifugation (100,000 × g for 1 h to produce S2 and P2), and samples of these fractions equivalent to a single cell were subjected to immunoblot analysis for the presence of Myo1e, β-tubulin (~50 kDa), and Csp. B, immunofluorescence microscopy, indirect immunostaining of oocytes for Myo1e (panels H–K) and csp (panels D–G) was performed as under “Experimental Procedures,” and cortical granule lectin was detected using rhodamine-conjugated D. bifluros lectin (panels A–C). By using epifluorescence microscopy at low magnification (panels A, D, and H), the signal for all three of these proteins is restricted to the cortex of the oocyte. At higher magnification, a narrow band of fluorescence is seen for cortical granule lectin (B). For Csp (panel E), the immunostaining projects deeper into the interior of the oocyte than it does for the lectin, whereas for Myo1e (panel I) a diffuse band of fluorescence is apparent. In en face views obtained using laser scanning confocal microscopy (panels B, F, G, J, and K), the red cortical granules are clearly identified by the binding of the D. bifluros lectin (panels C, G, and K). Concomitant immunostaining for Csp reveals prominent rings of labeling around granules (panels F and G). The Myo1e immunostaining (panel J) also is clearly apparent in the same plane as the cortical granules (panel K), but it is not prominently arrayed around the granules as is the Csp immunostaining.