Histochem Cell Biol (2008) 129:5–11 sured in living cells (Ritland-Politz et al. 2006; Shav-Tal et al. 2004). These studies show that RNAs synthesized from genes positioned in proximity to the nuclear envelope diVuse away from their site of synthesis in all directions rather than follow a direct path to the nearest nuclear pore. This observation powerfully demonstrates the non-directed, diVusion-based motion of RNAs in the nucleus. TraYcking as a means of targeting One of the conceptually most challenging problems in cell biology is the question of how molecules Wnd their speciWc targets within a cell or within an organelle. This problem is particularly complex in the cell nucleus where transcrip- tional regulators need to Wnd their speciWc target genes amongst the myriad of potential binding sites within the genome. Somewhat counter-intuitively, the non-directional, but rapid, motion of proteins within the cell nucleus pro- vides a means to ensure targeting of proteins to speciWc genome locations (Misteli 2001). The power of targeting by random diVusion within the nucleus is best illustrated when considering how a transcription factor Wnds its target genes but it also applies to targeting of proteins to nuclear com- partments or any other nuclear site. Proteins a priori do not know where their targets are and we do know of any directed transport systems, such as a molecular motor-based mechanism, that would bring a fac- tor to a speciWc gene or a speciWc location within the nucleus. Thus, the only way for a transcription factor to Wnd its target is to scan the genome. The intrinsic ability of pro- teins to rapidly move within the nucleus by diVusion-based mechanisms permits such genome scanning (Misteli 2001). This occurs by a transcription factor freely diVusing within the nucleoplasm until it interacts by chance with chromatin. The molecule will now probe whether the sequence it has encountered is a speciWc binding site such as in the pro- moter of one of its target genes. If it is, the transcription factor will be captured and stably associated with its spe- ciWc target site. If the sequence is not a binding site, the molecule, after a short interaction, will dissociate from chromatin and continue its diVusional journey through the nucleus. It might appear at Wrst glance that such a stop-and-go model for genome scanning would be insuYciently eVec- tive. However, the observed dynamic properties of tran- scription factors are entirely consistent with this model. We know that the residence time of most transcription factors even on speciWc DNA binding sites is in the order of a few seconds and that their interaction with non-speciWc sites is even faster, most likely in the order of tens of milliseconds (Gorski et al. 2006; Houtsmuller et al. 1999; McNally et al. 2000; Phair et al. 2004; Sprague et al. 2006). Assuming 7 these time scales for binding, one can calculate that it takes a single transcription factor molecule only a few minutes to search the entire genome space. Considering that most tran- scription factors exist in several thousand copies and have multiple target genes, their random diVusional motion is entirely suYcient to ensure a steady supply at their target genes. This assumption is further supported by direct mea- surement of transcription factor Xux on an endogenous rRNA promoter demonstrating the collision of several hun- dred molecules per second (Dundr et al. 2002). Since pro- teins similarly move by diVusional motion within the cytoplasm, it stands to reason that the random scanning is also a key mechanism of protein targeting in the cytoplasm and represents a universal mechanism for how proteins Wnd their targets. A key feature, and a requirement, in a genome-scanning model of targeting is that the interactions of proteins with chromatin are transient. This has been conWrmed by photo- bleaching methods on a large number of DNA binding pro- teins (Gorski et al. 2006; Phair et al. 2004; Sprague et al. 2006). The transient nature of protein–chromatin interac- tions is important for three reasons. First, it allows proteins to maintain a high rate of motion and thus allows faster scanning. Were protein–chromatin interactions static, they would get stuck at non-speciWc or incorrect binding sites which would slow down their overall motion. Second, the short-life of protein–chromatin interactions continuously makes available binding sites which can then be scanned by diVusing transcription factors. If proteins interacted for extended periods of time on chromatin, non-speciWc or improper binding would block access of the correct factors. Third, the dynamic dissociation allows for change. Were protein complexes permanently bound to their target sites, changes in transcriptional activity such as in response to physiological stimuli could only occur after active removal of the bound complex, presumably by dedicated and spe- cialized molecular machinery. In contrast, in a dynamic binding model the natural Xux of proteins provides a win- dow of opportunity for association of a distinct regulator each time a bound protein or complex dissociates as part of its normal binding cycle. Protein dynamics as the key for formation of nuclear compartments A hallmark of the mammalian cell nucleus is the presence of distinct subnuclear compartments and domains in which particular functions occur (Handwerger and Gall 2006; Hernandez-Verdun 2006) (Fig. 2). The prototypical nuclear compartment is the nucleolus, a distinct intranuclear com- partment in which ribosomal RNAs are synthesized and partially processed. Other prominent nuclear domains 123