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.

We imaged transcription in living cells using a locus-specific reporter system, which allowed precise, single-cell kinetic measurements of promoter binding, initiation and elongation. Photobleaching of fluorescent RNA polymerase II revealed several kinetically distinct populations of the enzyme interacting with a specific gene. Photobleaching and photoactivation of fluorescent MS2 proteins used to label nascent messenger RNAs provided sensitive elongation measurements. A mechanistic kinetic model that fits our data was validated using specific inhibitors. Polymerases elongated at 4.3 kilobases min −1 , much faster than previously documented, and entered a paused state for unexpectedly long times. Transcription onset was inefficient, with only 1% of polymerase-gene interactions leading to completion of an mRNA. Our systems approach, quantifying both polymerase and mRNA kinetics on a defined DNA template in vivo with high temporal resolution, opens new avenues for studying regulation of transcriptional processes in vivo.Transcription by RNA polymerase II (Pol II) is at the core of gene expression and hence is the basis of all cellular activities. Little information exists about the kinetics of this process in live cells 1 , as understanding of gene expression regulation comes from studies using purified proteins. For instance, the subunits of the elongating Pol II are well known 2 and the crystal structure of this enzyme explains much of its behavior in vitro 3,4 . mRNA transcription can be deconstructed into a succession of steps: promoter assembly, clearanceCorrespondence should be addressed to R.H.S. (rhsinger@aecom.yu.edu). AUTHOR CONTRIBUTIONS All data were initially acquired by X.D. and Y.S.-T. Subsequent data were obtained by V.d.T. (Fig. 4a,b and Fig. 5a) and Y.B. (Fig. 9b). S.M.S. was responsible for the microscopy, built the wide-field microscope for live-cell imaging and wrote analysis software. X.D. performed the kinetic modeling. R.D.P. provided consultation on model formulation and testing, and training in the use of the ProcessDB software. R.H.S. supervised the project. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/nsmb/. HHS Public AccessAuthor manuscript Nat Struct Mol Biol. Author manuscript; available in PMC 2016 July 12. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript and escape 5 , followed by elongation and termination. The process of transcriptional initiation involves several structural changes in the polymerase as the nascent transcript elongates 6 . Early in initiation, the polymerase can produce abortive transcripts 7,8 . These abortive cycles have been observed with a single prokaryote polymerase (RNAP) releasing several transcripts without escaping the promoter 9,10 . The elongation step can be regulated by pausing for various times, as demonstrated using prokaryotic polymerases in vitro 11,12 .For eukaryotic cells, attempts have been mad...

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG–GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG–GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG–GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG–GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments.The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG– GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG–GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG–GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.

Abstract. The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 Њ C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.

Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.Organization of DNA into higher-order chromatin structure serves to accommodate the genome within the spatial confines of the cell nucleus and acts as an important regulatory mechanism (22,36,46,60). Establishment, maintenance, and alterations of global and local chromatin states are modulated by the combined action of a multitude of chromatin-binding proteins. The nucleosome, containing histone proteins, acts as a structural scaffold and as an entry point for regulatory mechanisms (60, 63). Nonhistone proteins, including the HMG proteins, further contribute to the structural maintenance and regulation of chromatin regions (6, 61). In heterochromatin, specific factors such as HP1 convey a transcriptionally repressed state, possibly by influencing higher-order chromatin structure (19,27). Histone-modifying enzymes such as histone acetyl-and methyltransferases are instrumental in generating epigenetic marks on chromatin domains (60). Chromatin remodeling factors act on specific sites to facilitate access to regulatory DNA elements. Once accessible, transcriptional activators bind specific sequences on DNA and recruit the basal transcription machinery (37,44,46). All of these steps involve binding of proteins to chromatin.Due to their functional significance, chromatin-associated proteins have been extensively characterized-mostly by biochemical extraction and in vitro binding assays. Little is known about the dynamics of how chromatin proteins bind to their target sites in native chromatin in living cells. In vivo microscopy techniques are providing novel tools to study chromatin proteins in living cells (32,39,41,50). Qualitative analysis of photobleaching experiments has revealed a wide range of dynamic behavior for chromatin-associated proteins. The bulk of core histones is immobile on DNA, whereas the linker histone H1...

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.

SUMMARY The “CTCF code” hypothesis posits that CTCF pleiotropic functions are driven by recognition of diverse sequences through combinatorial use of its 11 zinc fingers (ZFs). This model, however, is supported by in vitro binding studies of a limited number of sequences. To study CTCF multivalency in vivo, we define ZF binding requirements at ~50,000 genomic sites in primary lymphocytes. We find that CTCF reads sequence diversity through ZF clustering. ZFs 4–7 anchor CTCF to ~80% of targets containing the core motif. Nonconserved flanking sequences are recognized by ZFs 1–2 and ZFs 8–11 clusters, which also stabilize CTCF broadly. Alternatively, ZFs 9–11 associate with a second phylogenetically conserved upstream motif at ~15% of its sites. Individually, ZFs increase overall binding and chromatin residence time. Unexpectedly, we also uncovered a conserved downstream DNA motif that destabilizes CTCF occupancy. Thus, CTCF associates with a wide array of DNA modules via combinatorial clustering of its 11 ZFs.

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.