Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle.
Cajal bodies (CBs) are evolutionarily conserved nuclear organelles that contain many factors involved in the transcription and processing of RNA. It has been suggested that macromolecular complexes preassemble or undergo maturation within CBs before they function elsewhere in the nucleus. Most such models of CB function predict a continuous flow of molecules between CBs and the nucleoplasm, but there are few data that directly support this view. We used fluorescence recovery after photobleaching (FRAP) on isolated Xenopus oocyte nuclei to measure the steady-state exchange rate between the nucleoplasm and CBs of three fluorescently tagged molecules: U7 small nuclear RNA, coilin, and TATA-binding protein (TBP). In the nucleoplasm, the apparent diffusion coefficients for the three molecules ranged from 0.26 to 0.40 microm2 s-1. However, in CBs, fluorescence recovery was markedly slower than in the nucleoplasm, and there were at least three kinetic components. The recovery rate within CBs was independent of bleach spot diameter and could not be attributed to high CB viscosity or density. We propose that binding to other molecules and possibly assembly into larger complexes are the rate-limiting steps for FRAP of U7, coilin, and TBP inside CBs.
Handwerger
Korie E
KE
Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210, USA.
Murphy
Christine
C
Gall
Joseph G
JG
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D020541:Coiled Bodies / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D005260:Female; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D012342:RNA, Small Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000378:metabolism; D014982:Xenopus laevis / Q000033:anatomy & histology*
D009687:Nuclear Proteins; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; C405487:U7 small nuclear RNA; C068921:p80-coilin