Fluorescence Loss In Photobleaching Research Articles

FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.

Nucleo-cytoplasmic shuttling is an important feature of proteins involved in nuclear export/import of RNAs, proteins, and also large ribonucleoprotein complexes such as ribosomes. The vast amount of proteomic data available shows that many of these processes are highly dynamic. Therefore, methods are needed to reliably assess whether a protein shuttles between nucleus and cytoplasm, and the kinetics with which it exchanges. Here we describe a combination of the classical heterokaryon assay with fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques, which allows an assessment of the kinetics of protein shuttling in the yeast Saccharomyces cerevisiae.

A hyper-dynamic equilibrium between promoter-bound and nucleoplasmic dimers controls NF-κB-dependent gene activity

Because of its very high affinity for DNA, NF-kappaB is believed to make long-lasting contacts with cognate sites and to be essential for the nucleation of very stable enhanceosomes. However, the kinetic properties of NF-kappaB interaction with cognate sites in vivo are unknown. Here, we show that in living cells NF-kappaB is immobilized onto high-affinity binding sites only transiently, and that complete NF-kappaB turnover on active chromatin occurs in less than 30 s. Therefore, promoter-bound NF-kappaB is in dynamic equilibrium with nucleoplasmic dimers; promoter occupancy and transcriptional activity oscillate synchronously with nucleoplasmic NF-kappaB and independently of promoter occupancy by other sequence-specific transcription factors. These data indicate that changes in the nuclear concentration of NF-kappaB directly impact on promoter function and that promoters sample nucleoplasmic levels of NF-kappaB over a timescale of seconds, thus rapidly re-tuning their activity. We propose a revision of the enhanceosome concept in this dynamic framework.

In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains

The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.

Complex Interactions at the Adherens Junction

Two articles challenge the currently accepted model of interaction between the epithelial adherens junction and the actin cytoskeleton, in which α-catenin forms a stable link between β-catenin (which binds to the cytoplasmic tails of cadherins, junctional cell-cell adhesion proteins) and the actin cytoskeleton (binding to actin or to actin-associated proteins). Yamada et al. found that, in a pelleting assay, α-catenin pelleted with actin but did not promote pelleting with actin of an E-cadherin-β-catenin complex, whereas a chimeric protein in which α-catenin was covalently linked to β-catenin failed to bind actin. Thus, α-catenin bound to either actin or β-catenin but not to both simultaneously. Moreover, actin filaments failed to bind to E-cadherin-β-catenin-α-catenin complexes reconstituted on isolated patches of cadherin-containing epithelial cell membranes. Similarly, the actin-binding proteins α-actinin and vinculin failed to promote association between actin and the cadherin-catenin complex. Fluorescence recovery after photobleaching (FRAP) analysis of MDCK cells stably expressing green fluorescent protein-labeled proteins indicated that E-cadherin, β-catenin, and α-catenin showed similar mobility; in contrast, membrane-associated actin at cell-cell contacts was much more dynamic. Further, disruption of actin organization failed to affect mobility of the cadherin-catenin complex. In the accompanying paper, Drees et al. found that cytoplasmic α-catenin was present as both a monomer and a homodimer. Consistent with the known overlap between the dimerization domain and the β-catenin binding domain, the authors found that the monomer bound more strongly to β-catenin. In contrast, α-catenin preferentially bound to actin as a homodimer. When actin filaments were incubated with cadherin-catenin complexes, some of the α-catenin dissociated from the complex and associated with actin; fluorescence loss in photobleaching (FLIP) analysis indicated that exchange of α-catenin associated with membrane proteins with that in a cytoplasmic pool occurred in living cells. The Arp2/3 complex nucleates actin filaments, a process that is regulated by Wiscott-Aldrich syndrome proteins (WASPs). α-catenin inhibited Arp2/3 binding to actin and suppressed the actin polymerization that occurred in the presence of Arp2/3 and the WASP activation domain, with the dimer suppressing polymerization more effectively. Thus, the authors conclude that the association between the cadherin-catenin complex and the actin cytoskeleton is likely dynamic. In their model, rather than linking the cadherin-β-catenin complex to the actin cytoskeleton, α-catenin--which exists in one form that is bound to β-catenin and another that can bind actin--may act as a molecular switch to regulate organization of the actin cytoskeleton. Gates and Peifer discuss the research in a minireview. S. Yamada, S. Pokutta, F. Drees, W. I. Weis, W. J. Nelson, Deconstructing the cadherin-catenin-actin complex. Cell 123 , 889-901 (2005). [PubMed] F. Drees, S. Pokutta, S. Yamada, W. J. Nelson, W. I. Weis, α-Catenin is a molecular switch that binds E-cadherin-β-catenin and regulates actin-filament assembly. Cell 123 , 903-915 (2005). [PubMed] J. Gates, M. Peifer, Can 1000 reviews be wrong? Actin, α-catenin, and adherens junctions. Cell 123 , 769-772 (2005). [PubMed]

Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells

Recent advances in our understanding of the intracellular trafficking, membrane microenvironment, and subcellular sites of signaling of Ras have been driven by observations of GFP-tagged Ras in living cells. Here, we describe methods to gain further insight into the regulation of these events through the use of quantitative fluorescence microscopy. We focus on three techniques, fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and selective photobleaching. While all of these techniques exploit photobleaching as a tool to monitor protein dynamics, they each provide a unique subset of information. In particular, FRAP provides measurements of protein mobility via lateral diffusion by monitoring recovery of fluorescence into a region following a single photobleaching event. FLIP assesses the level of continuity and communication between subcellular compartments by repetitively photobleaching a region of interest and following concomitant loss of fluorescence from other areas in the cell. Selective photobleaching reveals kinetic information about active and passive transport of proteins into organelles such as the Golgi complex or between areas of protein enrichment such as caveolae. We describe how to implement these techniques using commercially available confocal microscopes and outline methods for data analysis. Finally, we discuss how these approaches are being used to provide new insights into the mechanisms of membrane microdomain localization, vesicular versus non-vesicular transport, and kinetics of exchange of Ras on and off of cell membranes.

Distinct Constrictive Processes, Separated in Time and Space, DivideCaulobacterInner and Outer Membranes

Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.

Inhibition of Nuclear Import of LIMK2 in Endothelial Cells by Protein Kinase C-dependent Phosphorylation at Ser-283

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle.

Highlighting protein kinase CK2 movement in living cells

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (alpha2beta2) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol 23: 975-987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio-temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.

Nuclear Trapping May Help Avoid Aging

In the worm, at least, increased expression of the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase Sir2.1 can prolong life span. Thus, the biochemical effects of such enzymes, known as Sirtuins, are of particular interest. Frescas et al. explored the effects of mammalian Sirtuins on deacetylation of the transcription factor FoxO1. They expressed a FoxO1 molecule modified by fusion with green fluorescent protein (GFP) in cultured cells and monitored its movement to the nucleus and its mobility in FLIP (fluorescence loss in photobleaching) and FRAP (fluorescence recovery after photobleaching) experiments. Their results show that treatment of cells with a Sirtuin activator, resveratrol, or promotion of oxidative stress by treatment of cells with hydrogen peroxide promoted nuclear translocation of FoxO1. The FRAP studies showed that after deacetylation by Sirtuins, FoxO1 remains bound in a particular nuclear compartment. The nuclear sequestration of FoxO1 is correlated with increased expression of FoxO1 target genes. Control of localization is not the whole story, however, as expression of a constitutively nuclear form of FoxO1 was not sufficient to stimulate FoxO1-dependent transcription. Thus, other signals must also modulate FoxO1 activity. Genes regulated by FoxO1 include hepatic enzymes that control gluconeogenesis; better understanding of these regulatory interactions may be useful in achieving therapeutic control of metabolic disorders. D. Frescas, L. Valenti, D. Accili, Nuclear trapping of the Forkhead transcription factor FoxO1 via Sirt-dependent deacetylation promotes expression of glucogenetic genes. J. Biol. Chem. 280 , 20589-20595 (2005). [Abstract] [Full Text]

Dynamic properties of germ line-specific lamin B3: The role of the shortened rod domain

The mammalian lamin B2 gene codes for two proteins, the somatic lamin B2 and the germ line-specific lamin B3. Lamin B3 lacks the N-terminus and a part of the α -helical rod domain present in lamin B2. These domains are substituted by 84 amino acids unique for lamin B3. When ectopically expressed in somatic cells, lamin B3 causes severe deformation of nuclei which adopt a hook-like configuration. Accordingly, it was proposed that lamin B3 provides the germ line cells with a more flexible nuclear periphery that facilitates spermatogenesis-specific nuclear reorganization events. Here we investigated which protein domains of lamin B3 are responsible for nuclear deformation in transfected cells, and how stable is the nuclear periphery of these cells. Expression of wild-type and mutant lamins evidenced that nuclear deformations are due to the shortened rod domain of lamin B3. Cell fractionation experiments revealed that lamin B3 can be solubilized more easily than lamin B2. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analyses of transfected cells showed that lamin B3 has an increased mobility compared to B2. Our results lead to the conclusion that lamin B3 reduces the stability of the nuclear periphery. They are also consistent with the notion that lamin B3 is relevant to specific properties of the nuclear envelope during spermiogenesis.

A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naive MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.

Different populations of RNA polymerase II in living mammalian cells

RNA polymerase II is responsible for transcription of most eukaryotic genes, but, despite exhaustive analysis, little is known about how it transcribes natural templates in vivo. We studied polymerase dynamics in living Chinese hamster ovary cells using an established line that expresses the largest (catalytic) subunit of the polymerase (RPB1) tagged with the green fluorescent protein (GFP). Genetic complementation has shown this tagged polymerase to be fully functional. Fluorescence loss in photobleaching (FLIP) reveals the existence of at least three kinetic populations of tagged polymerase: a large rapidly-exchanging population, a small fraction resistant to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) but sensitive to a different inhibitor of transcription (i.e. heat shock), and a third fraction sensitive to both inhibitors. Quantitative immunoblotting shows the largest fraction to be the inactive hypophosphorylated form of the polymerase (i.e. IIA). Results are consistent with the second (DRB-insensitive but heat-shock-sensitive) fraction being bound but not engaged, while the third (sensitive to both DRB and heat shock) is the elongating hyperphosphorylated form (i.e. IIO).

Intracellular Trafficking and Dynamics of Double Homeodomain Proteins

Double homeodomain (DUX) proteins are encoded by a family of 3.3-kilobase repeated elements dispersed in the human genome. One of these elements named D4Z4 is found in a tandem repeat array on chromosome 4 that is partially deleted in facioscapulohumeral muscular dystrophy. We have evaluated the trafficking and mobility of two DUX proteins, DUX1 and DUX4. We transfected C2C12 myoblasts with cDNA encoding these proteins fused to the green fluorescent protein and studied their intracellular localization and diffusional mobilities using fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We also studied truncated forms of the proteins, containing one or both homeodomains or a region outside the homeodomains. We show that both full-length proteins are actively transported into the nucleus, and that the homeodomains contain the signals required for this localization. DUX1 is more mobile than DUX4 within the nucleus (t(1/2) = 4.8 s for DUX1 and 13.4 s for DUX4), suggesting differences in the way the two proteins interact with nuclear components.

Nucleocytoplasmic shuttling revealed by FRAP and FLIP technologies

Protein mobility within cells is of key importance for many cellular functions. Although immunostaining can reveal protein locations in the steady-state, this might not represent the full picture and provides no information about protein movements. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are two techniques that enable the dynamics of intracellular protein mobility to be studied. These technologies have been successfully used to analyze the nucleocytoplasmic shuttling of STAT1, an intracellular signal transducer and activator of transcription, and can applied to the study of other proteins. Furthermore, FRAP and FLIP approaches have the added advantage of not affecting cell viability and might find application in the imaging of intracellular events in certain tissues and live animals.

Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import

The Cdc25 phosphatases play crucial roles in cell cycle progression by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Cdc25A is an important regulator of the G1/S transition but functions also in the mitotic phase of the human cell cycle. In this paper, we investigate the sub-cellular localisation of exogenously expressed Cdc25A. We show that YFP-Cdc25A is localised both in the nucleus and the cytoplasm of HeLa cells and untransformed fibroblasts. Cell fusion assays and fluorescence loss in photobleaching (FLIP) assays reveal that the localisation is dynamic and the protein shuttles between the nucleus and the cytoplasm. We demonstrate that nuclear export of Cdc25A is partly mediated by an N-terminal nuclear export sequence (NES), in a manner not sensitive to the Exportin 1-inhibitor leptomycin B. A nuclear localisation signal (NLS) is also characterised, mutation of which leads to cytoplasmic localisation of Cdc25A. Our results imply that the Cdc25A phosphatase may interact with substrates and regulators both in the nucleus and the cytoplasm.

Smads on the Move

The general scheme by which transforming growth factor β (TGF-β) receptors signal to the nucleus is one in which activated receptors cause phosphorylation and activation of Smad proteins, which then accumulate in the nucleus. However, closer inspection reveals that the dynamic behavior of Smad proteins is somewhat more complicated. Nicolás et al. made Smad fusion proteins with green fluorescent protein and monitored movement of the proteins in live cells in FLIP (fluorescence loss in photobleaching) experiments. The results show that although most GFPSmad4 is in the cytoplasm of unstimulated cells, the protein does rapidly shuttle between the cytoplasm and the nucleus. Distinctions could be made between the different Smads, and GFPSmad2 was less mobile in the cytoplasm than was GFPSmad4, indicating that interactions with other molecules may tether Smad 2 in the cytoplasm. Using a combination of FLIP and FRAP (fluorescence recovery after photobleaching), the authors found that after stimulation of cells with TGF-β, both Smads showed reduced mobility in the nucleus, which presumably results from interactions that hold the activated proteins in the nucleus. It will now be of interest to determine the precise nature of the interactions that modulate shuttling of Smads between the cytoplasm and the nucleus. F. J. Nicolás, K. De Bosscher, B. Schmierer, C. S. Hill, Analysis of Smad nucleocytoplasmic shuttling in living cells. J. Cell Sci. , 117 , 4113-4125 (2004). [Abstract] [Full Text]

Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-kappaB inducing kinase NIK.

It has been shown previously that the transcription factor NF-kappaB and its inhibitor IkappaBalpha shuttle constitutively between cytosol and nucleus. Moreover, we have recently demonstrated nucleocytoplasmic shuttling of the NF-kappaB-inducing kinase NIK, a component of the NF-kappaB pathway, which is essential for lymph node development and B-cell function. Here we show that nuclear NIK also occurs in nucleoli and that this localization is mediated by a stretch of basic amino acids in the N-terminal part of the protein (R(143)-K-K-R-K-K-K(149)). This motif is necessary and sufficient for nucleolar localization of NIK, as judged by nuclear localization of mutant versions of the full-length protein and the fact that coupling of these seven amino acids to GFP also leads to accumulation in nucleoli. Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) approaches, we demonstrate a dynamic distribution between nucleoli and nucleoplasm and a high mobility of NIK in both compartments. Together with the nuclear export signal in the C-terminal portion of NIK that we have also characterized in detail, the nuclear/nucleolar targeting signals of NIK mediate dynamic circulation of the protein between the cytoplasmic, nucleoplasmic and nucleolar compartments. We demonstrate that nuclear NIK is capable of activating NF-kappaB and that this effect is diminished by nucleolar localization. Thus, subcellular distribution of NIK to different compartments might be a means of regulating the function of this kinase.

Revealing protein dynamics by photobleaching techniques.

Green fluorescent proteins (GFPs) are widely used tools to visualize proteins and study their intracellular distribution. One feature of working with GFP variants, photobleaching, has recently been combined with an older technique known as fluorescence recovery after photobleaching (FRAP) to study protein kinetics in vivo. During photobleaching, fluorochromes get destroyed irreversibly by repeated excitation with an intensive light source. When the photobleaching is applied to a restricted area or structure, recovery of fluorescence will be the result of active or passive diffusion from fluorescent molecules from unbleached surrounding areas. Fluorescence loss in photobleaching (FLIP) is a variant of FRAP where an area is bleached, and loss of fluorescence in surrounding areas is observed. FLIP can be used to study the dynamics of different pools of a protein or can show how a protein diffuses, or is transported through a cell or cellular structure. Here, we discuss these photobleaching fluorescent imaging techniques, illustrated with examples of these techniques applied to proteins of the Saccharomyces cerevisiae pheromone response MAPK pathway.

Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET analyses in living HeLa cells

Barrier-to-autointegration factor (BAF) is a conserved 10 kDa DNA-binding protein. BAF interacts with LEM-domain proteins including emerin, LAP2β, and MAN1 in the inner nuclear membrane. Using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we compared the mobility of BAF to its partners emerin, LAP2β, and MAN1 in living HeLa cells. Like endogenous BAF, GFP–BAF was enriched at the nuclear envelope, and found inside the nucleus and in the cytoplasm during interphase. At every location, FRAP and FLIP analysis showed that GFP–BAF diffused rapidly; the halftimes for recovery in a 0.8 μm square area were 260 ms at the nuclear envelope, and even faster inside the nucleus and in the cytoplasm. GFP-fused emerin, LAP2β, and MAN1 were all relatively immobile, with recovery halftimes of about 1 min, for a 2 μm square area. Thus, BAF is dynamic and mobile during interphase, in stark contrast to its nuclear envelope partners. FLIP results further showed that rapidly diffusing cytoplasmic and nuclear pools of GFP–BAF were distinctly regulated, with nuclear GFP–BAF unable to replenish cytoplasmic BAF. Fluorescence resonance energy transfer (FRET) results showed that CFP–BAF binds directly to YFP–emerin at the inner nuclear membrane of living cells. We propose a “touch-and-go” model in which BAF binds emerin frequently but transiently during interphase. These findings contrast with the slow mobility of both GFP–BAF and GFP–emerin during telophase, when they colocalized at the ‘core’ region of telophase chromosomes at early stages of nuclear assembly.