IMMUNODETECTION OF CYTOSKELETAL STRUCTURES AND THE EG5 MOTOR PROTEIN ON DEEP-ETCH REPLICAS OF XENOPUS EGG CORTICES ISOLATED DURING THE CORTICAL ROTATION

We have developed a new method for immunogold detection on deep-etch r eplicas of isolated Xenopus egg cortices in order to examine the inter actions of different cortical elements in three dimensions at high res olution. We have applied this technique to vegetal cortices isolated d uring the second half of the first cell cycle. The vegetal cortical re gion at this time is the site of cellular machinery responsible for th e 'cortical rotation'. The entire cortex translocates with respect to the inner cytoplasm, relocating dorsalising determinants to the future dorsal side of the egg. The aligned microtubules in the shear zone be tween cytoplasm and cortex, implicated in the cortical rotation, were found to be organised as interweaving loose bundles. Interleaved among st these aligned microtubules were extensive sheets of ER lying in lay ers parallel to the egg surface. Cytokeratin filaments were found to a ssociate closely with the microtubules over short stretches. Putative actin filaments were present in the shear zone and in the cortex. Eg5, an abundant kinesin-related microtubule motor protein, and candidate for a role in generating cortical rotation movement, showed an almost exclusive localisation to microtubules. Immunofluorescence studies of cortices treated with detergent to disrupt ER or cold to depolymerise microtubules confirmed that Eg5 associates primarily with microtubules . We propose revised models for the mechanism of cortical rotation bas ed on these observations and conclude that Eg5 is unlikely to move ER relative to microtubules during the cortical rotation.