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Published 15 March 2004. doi:10.1083/jcb.200311121
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 164, Number 6, 831-842
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Article

 In vivo kinetics of Cajal body components

Miroslav Dundr1, Michael D. Hebert3,4, Tatiana S. Karpova2, David Stanek5, Hongzi Xu3, Karl B. Shpargel4, U. Thomas Meier6, Karla M. Neugebauer5, A. Gregory Matera4, and Tom Misteli1

1 National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
2 Fluorescence Imaging Facility, National Institutes of Health, Bethesda, MD 20892
3 The University of Mississippi Medical Center, Jackson, MS 39216
4 Case Western Reserve University School of Medicine, Cleveland, OH 44106
5 Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
6 Albert Einstein College of Medicine, Bronx, NY 10461

Address correspondence to M. Dundr, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. Tel.: (301) 402-0303. Fax: (301) 402-0055. email: dundrm{at}mail.nih.gov; or A.G. Matera, Case Western Reserve University School of Medicine, Cleveland, OH 44106. Tel.: (216) 368-4922. Fax: (216) 368-1257. email: a.matera{at}case.edu

Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures.

Key Words: Cajal body; gems; coilin; SMN; iFRAP

Abbreviations used in this paper: CB, Cajal body; iFRAP, inverse fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; MEF, mouse embryonic fibroblast; rRNA, ribosomal RNA; SMN, survival of motor neurons; snRNP, small nuclear ribonucleoprotein; snoRNP, small nucleolar ribonucleoprotein; Tgs1, trimethylguanosine synthase 1.


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