High mobility of proteins in the mammalian cell nucleus

Abstract

The mammalian cell nucleus contains numerous sub-compartments, which have been implicated in essential processes such as transcription and splicing. The mechanisms by which nuclear compartments are formed and maintained are unclear. More fundamentally, it is not known how proteins move within the cell nucleus. We have measured the kinetic properties of proteins in the nucleus of living cells using photobleaching techniques. Here we show that proteins involved in diverse nuclear processes move rapidly throughout the entire nucleus. Protein movement is independent of energy, which indicates that proteins may use a passive mechanism of movement. Proteins rapidly associate and dissociate with nuclear compartments. Using kinetic modelling, we determined residence times and steady-state fluxes of molecules in two main nuclear compartments. These data show that many nuclear proteins roam the cell nucleus in vivo and that nuclear compartments are the reflection of the steady-state association/dissociation of its 'residents' with the nucleoplasmic space. Our observations have conceptual implications for understanding nuclear architecture and how nuclear processes are organized in vivo.

Aging by epigenetics—A consequence of chromatin damage?

“…First, essential processes, such as gene transcription, DNA replication and repair, all involve disruption and reassembly of chromatin structure [9,10]. Second, Fluorescence recovery after photobleaching (FRAP) studies have shown that most chromatin-bound proteins are not statically bound, but exhibit relatively high "off" and "on" rates, even in supposedly "closed" heterochromatin [11,12]. Third, remarkably and paradoxically, formation of heterochromatin actually depends on a degree of transcription, which contributes to heterochromatinization through the RNAi pathway [4].…”

Human ribonuclease P: Subunits, function, and intranuclear localization

“…In situ RNA hybridization analysis and microinjection of labeled H1 RNA into human cell lines demonstrate that this RNA transiently enters the nucleolus before it diffuses in the nucleoplasm (Jacobson et al+, 1997; Table 2)+ The endogenous H1 RNA was also detected in the cytoplasm, the nucleoli, and the perinucleolar compartment (see Wolin & Matera, 1999)+ Because H1 RNA is essential for enzyme activity and exists in the nucleoplasm, in contrast to the S. cerevisiae RNase P RNA that is predominantly found in the nucleolus (Bertrand et al+, 1998;Kendall et al+, 2000;Lewis & Tollervey, 2000), human RNase P may function in this compartment+ The nucleolus of human cells seems to serve as an assembly site for RNase P (Pederson & Politz, 2000)+ In contrast to H1 RNA, several protein subunits, including Rpp14, Rpp29, Rpp38, hPop1, and hPop5, are mainly localized in nucleoli of human cells (Lygerou et al+, 1996b;Jarrous et al+, 1999b;Savino et al+, 1999;van Eenennaam et al+, 2001)+ Rpp29 and Rpp38 are also found in Cajal bodies (Table 2; Jarrous et al+, 1999b), nuclear structures in which transcription and processing machineries preassemble (Lamond & Earnshaw, 1998;Gall, 2000Gall, , 2001)+ Unexpectedly, endogenous Rpp21 is mainly found in the nucleoplasm and a small fraction of it exists in nucleoli ( Table 2; )+ In addition, Rpp21 is not concentrated in Cajal bodies, as are Rpp29 and Rpp38+ These observations indicate that the nuclear localization patterns of Rpp21, as well as H1 RNA, are different from those of other Rpp subunits (Table 2)+ Moreover, protein subunits of RNase P may move rapidly from one compartment to another in the nucleus of human cells+ Thus, fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques reveal that Rpp29 exchanges quickly between the nucleolus and nucleoplasm of HeLa cells (Chen & Huang, 2001)+ The high mobility of Rpp29 suggests that the intranuclear pool of this protein is not in association with a stationary complex+ A high mobility in the nucleus has also been demonstrated for RNA and nucleolar proteins required for mRNA processing (Politz et al+, 1998;Phair & Misteli, 2000;Chen & Huang, 2001;Pederson, 2001)+ Moreover, nuclear organelles, such as Cajal bodies, are in continuous movement and contact each other in the nucleus (Platani et al+, 2000)+ Therefore, the localization pattern of Rpp29 in the nucleus does not ...…”

miR-484 regulates mitochondrial network through targeting Fis1

“…Mitochondrial connectivity was analysed by fluorescence recovery after photobleaching as previously described 57,58 . In brief, cardiomyocytes were transfected with mitochondrial matrix-targeted GFP (pAcGFP1-mito, Clontech) using the FuGENE transfection reagent (Roche).…”