Mutational analysis of fibrillarin and its mobility in living human cells.
Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant approximately 0.02 microm(2) s(-1)), and has a significantly larger mobile fraction in CBs than in nucleoli.
Snaar
S
S
Department of Molecular Cell Biology, Sylvius Laboratories, Leiden University Medical Center, 2333 AL Leiden, The Netherlands.
Wiesmeijer
K
K
Jochemsen
A G
AG
Tanke
H J
HJ
Dirks
R W
RW
D021581:Active Transport, Cell Nucleus; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020541:Coiled Bodies / Q000737:chemistry / Q000378:metabolism*; D004058:Diffusion; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D009038:Motion; D009154:Mutation / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002868:Chromosomal Proteins, Non-Histone; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C047458:fibrillarin