Cytokeratin intermediate filament organisation and dynamics in the vegetal cortex of living Xenopus laevis oocytes and eggs.
Cytokeratin intermediate filaments are prominent constituents of developing Xenopus oocytes and eggs, forming radial and cortical networks. In order to investigate the dynamics of the cortical cytokeratin network, we expressed EGFP-tagged Xenopus cytokeratin 1(8) in oocytes and eggs. The EGFP-cytokeratin co-assembled with endogenous partner cytokeratin proteins to form fluorescent filaments. Using time-lapse confocal microscopy, cytokeratin filament assembly was monitored in live Xenopus oocytes at different stages of oogenesis, and in the artificially-activated mature egg during the first cell cycle. In stage III to V oocytes, cytokeratin proteins formed a loose cortical geodesic network, which became more tightly bundled in stage VI oocytes. Maturation of oocytes into metaphase II-arrested eggs induced disassembly of the EGFP-cytokeratin network. Imaging live eggs after artificial activation allowed us to observe the reassembly of cytokeratin filaments in the vegetal cortex. The earliest observable structures were loose foci, which then extended into curly filament bundles. The position and orientation of these bundles altered with time, suggesting that forces were acting upon them. During cortical rotation, the cytokeratin network realigned into a parallel array that translocated in a directed manner at 5 microm/minute, relative to stationary cortex. The cytokeratin filaments are, therefore, moving in association with the bulk cytoplasm of the egg, suggesting that they may provide a structural role at the moving interface between cortex and cytoplasm.
Clarke
Emma J
EJ
School of Biological Sciences, University of Manchester, Manchester, United Kingdom.
Allan
Victoria J
VJ
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000502:physiology; D005260:Female; D005306:Fertilization / Q000502:physiology; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D007382:Intermediate Filaments / Q000502:physiology*; D007633:Keratins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D007700:Kinetics; D007814:Larva / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008845:Microinjections / Q000379:methods; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000276:immunology; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D009866:Oogenesis / Q000502:physiology; D010063:Ovum / Q000166:cytology* / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D014176:Protein Biosynthesis; D021381:Protein Transport / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D012156:Reticulocytes / Q000378:metabolism; D013194:Staining and Labeling; D014162:Transfection; D014982:Xenopus laevis / Q000502:physiology*
D000906:Antibodies; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D007633:Keratins