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• W2113406918 abstract "Article19 March 2013Open Access A fluorescent reporter for mapping cellular protein-protein interactions in time and space Daniel Moreno Daniel Moreno Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Joachim Neller Joachim Neller Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Hans A Kestler Hans A Kestler Research Group for Bioinformatics and Systems Biology, Institute of Neural Information Processing, Ulm University, Ulm, Germany Search for more papers by this author Johann Kraus Johann Kraus Research Group for Bioinformatics and Systems Biology, Institute of Neural Information Processing, Ulm University, Ulm, Germany Search for more papers by this author Alexander Dünkler Alexander Dünkler Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Nils Johnsson Corresponding Author Nils Johnsson Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Daniel Moreno Daniel Moreno Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Joachim Neller Joachim Neller Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Hans A Kestler Hans A Kestler Research Group for Bioinformatics and Systems Biology, Institute of Neural Information Processing, Ulm University, Ulm, Germany Search for more papers by this author Johann Kraus Johann Kraus Research Group for Bioinformatics and Systems Biology, Institute of Neural Information Processing, Ulm University, Ulm, Germany Search for more papers by this author Alexander Dünkler Alexander Dünkler Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Nils Johnsson Corresponding Author Nils Johnsson Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany Search for more papers by this author Author Information Daniel Moreno1, Joachim Neller1, Hans A Kestler2, Johann Kraus2, Alexander Dünkler1 and Nils Johnsson 1 1Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, Ulm, Germany 2Research Group for Bioinformatics and Systems Biology, Institute of Neural Information Processing, Ulm University, Ulm, Germany *Corresponding author. Department of Biology, Institute of Molecular Genetics and Cell Biology, Ulm University, James Franck Ring N27, 89081 Ulm, Germany. Tel.:+49 731 50 36300; Fax:+49 731 50 36302; E-mail: [email protected] Molecular Systems Biology (2013)9:647https://doi.org/10.1038/msb.2013.3 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info We introduce a fluorescent reporter for monitoring protein–protein interactions in living cells. The method is based on the Split-Ubiquitin method and uses the ratio of two auto-fluorescent reporter proteins as signal for interaction (SPLIFF). The mating of two haploid yeast cells initiates the analysis and the interactions are followed online by two-channel time-lapse microscopy of the diploid cells during their first cell cycle. Using this approach we could with high spatio-temporal resolution visualize the differences between the interactions of the microtubule binding protein Stu2p with two of its binding partners, monitor the transient association of a Ran-GTPase with its receptors at the nuclear pore, and distinguish between protein interactions at the polar cortical domain at different phases of polar growth. These examples further demonstrate that protein–protein interactions identified from large-scale screens can be effectively followed up by high-resolution single-cell analysis. Synopsis A method based on a combination of the Split-Ubiquitin system with two spectrally different fluorescent proteins (SPLIFF) is shown to enable measurement of protein interactions in vivo with high spatial and temporal resolution in yeast. SPLIFF visualizes protein interactions with high spatial and temporal resolution. Spc72p and Kar9p interact with the MAP Stu2p at opposite poles of microtubules. Histone chaperone Nap1p and Kcc4 kinase interact preferentially at the bud site. F-BAR protein Hof1p associates with the polarisome during cell fusion and cytokinesis. Introduction A mechanistic understanding of cellular biology requires a comprehensive knowledge about the protein interactions of the cell (Uetz et al, 2000; Gavin et al, 2002; Krogan et al, 2006; Yu et al, 2008; Breitkreutz et al, 2010). Split-protein sensors comprise a family of related techniques that contributed in small- and large-scale experiments to this cumulative endeavour (Miller et al, 2005; Tarassov et al, 2008; Hruby et al, 2011; Lowder et al, 2011; Stynen et al, 2012). The Split-Ubiquitin method (Split-Ub) is the prototype of these techniques (Müller and Johnsson, 2008; Stynen et al, 2012). Here, the N- and C-terminal halves of Ubiquitin (Nub and Cub) are coupled separately to the two proteins under study. Upon interaction of the fusion proteins, Nub and Cub are forced into close proximity and reassemble into a native-like Ubiquitin (Ub). The native-like Ub is recognized by Ub-specific proteases that cleave off a reporter protein that was genetically attached to the C-terminus of Cub (Johnsson and Varshavsky, 1994). The cleavage of the Split-Ub reporter orothic acid decarboxylase (Ura3p, CRU) from Cub leads to a qualitative difference in bulk cell growth (Wittke et al, 1999). This and other proteome-wide interaction techniques produce binary protein–protein interaction maps. The information encoded in these maps could fundamentally transform our understanding of cellular processes. However, to be effectively used in cell biology these networks would need to acquire a spatial as well as temporal dimension in order to place the interactions into a functional and cellular context (Alexander et al, 2009). The reason for the prevalence of binary interaction maps is mainly technical. Currently, robust and easy-to-use approaches for the characterization of cellular protein interactions in space and time are not available. Here, we report on a new method based on Split-Ub and two spectrally different fluorescent proteins (SPLIFF) to monitor the interaction between two proteins during the cell cycle with high spatial and temporal resolution. We further show that SPLIFF can bridge large-scale protein interaction screens with high-resolution single-cell analysis. Results Rational and design of measurements To create a robust and sensitive fluorescent reporter for protein interactions, we designed a Split-Ub module where Cub is sandwiched by two spectrally different fluorescent proteins (Cherry-Cub-GFP, CCG) (Figure 1A). Coupling CCG to the C-terminus of protein Y (Y-CCG) reveals the cellular localization of the fusion protein. Interaction of Y-CCG with a Nub-coupled interaction partner X will result in the cleavage of GFP from Y-CCG to create Y-CC. The liberated GFP is subsequently degraded. As Cherry stays attached to Y, the ratio of red to green fluorescence serves as a ratiometric reporter of protein–protein interactions and constitutes the actual readout of SPLIFF. To improve the spatial and temporal resolution of the assay, we defined the start of each reaction by fusing two yeast cells of opposite mating type, each expressing one half of the Split-Ub sensor (Figure 1B). The conversion of a Cherry/GFP-labelled (Y-CCG) into a Cherry-labelled fusion protein (Y-CC) is then recorded online by two-channel time-lapse fluorescence microscopy. This strategy is borrowed from chemical stopped-flow experiments where the kinetics of chemical reactions is recorded after forcing the reagents into a single chamber. Figure 1.Experimental design of SPLIFF. (A) Protein Y coupled to the Cherry-Cub-GFP (Y-CCG) interacts with the Nub fusion of protein X (Nub-X). Upon reassociation of Nub and Cub, the GFP is cleaved off and Y-CCG is converted to Y-CC. The N-terminally exposed arginine leads to rapid degradation of GFP. (B) Two yeast cells of the a- and α-mating type expressing Y-CCG (connected red and green circles) and Nub-X (yellow ellipsoid), respectively, fuse at t0. The cytosols mix, Y-CCG and Nub-X interact, leading to the progressive conversion of Y-CCG to Y-CC at t1, t2, and t3. Download figure Download PowerPoint In the following experiments, we demonstrate the applicability of SPLIFF by investigating protein interactions that occur at different cellular locations over widely different time periods and by providing examples of successful transitions from large-scale to single-cell analysis. Interactions in the nucleus The interaction between the nucleolar protein Net1p and the NAD-dependent histone deacetylase Sir2p exemplifies the experimental approach (Straight et al, 1999; Figure 2). The CRU or CCG modules were attached in frame behind the ORF of NET1 by homologous recombination. The Nub module was fused 5′ to the SIR2 ORF in α cells. The expression of the Nub fusion was controlled by the PCUP1 promoter and could be adjusted by varying the levels of copper in the medium. The growth on 5-fluoroorotic acid (5-FOA) of cells co-expressing Net1CRU and Nub-Sir2p revealed the interaction in the chosen configuration of Nub and Cub attachment (Figure 2B). Subsequent time-lapse microscopy of the diploid cells originating from the mating of Net1CCG- and Nub-Sir2p expressing a- and α-cell visualized the course of interaction in the nucleus from the a-cells. The reaction was completed within 20 min even before nuclear fusion has occurred (Figure 2C and D; Supplementary Movie 1). Figure 2.SPLIFF analysis of the Net1p/Sir2p interaction. (A) Cartoon of the RENT complex. (B) Interaction between Net1p and Sir2p as measured by the Split-Ub growth assay. Cells expressing an interacting Nub fusion grow on medium containing 5-FOA. (C) Interaction between Net1p and Sir2p as measured by SPLIFF. Selected frames of the time-lapse analysis of the mating of two yeast cells expressing Net1CCG and Nub-Sir2, respectively, (white border) are shown. Nuclei of unfarmed cells belong to haploid a-cells. Note the selective loss of green fluorescence from the a-cell-originated nucleus in the diploid at 10 and 20 min. Time 0 indicates the time point shortly before cell fusion. Scale bar, 5 μm. (D) Quantitative analysis of the experiment shown in (C) (upper panel) or the averages of 6 independent experiments (lower panel). The relative fluorescence of GFP (), Cherry () and the calculated conversion of Net1CCG to Net1CC () are plotted against the time (error bars, standard error (s.e.)). See also Supplementary Figure 1 and Supplementary Movie 1. Source data for this figure is available on the online supplementary information page. Source data for Figure 2D [msb20133-sup-0001-SourceData-S1.xls] Download figure Download PowerPoint To test the workflow for transferring interactions revealed by large-scale interaction experiments into single-cell analysis, we screened Net1CRU against an array of 382 different Nub fusions (Nub array) (Hruby et al, 2011). Among others Ubc9p, Cdc14p, and Fkh1p were identified as further Net1p-binding partners (Table I; Visintin et al, 1999). The interactions of Net1p with all three Nub fusions were subsequently analyzed by SPLIFF. The kinetic profiles of the Net1CCG conversions were very similar to the profile induced by Nub-Sir2p (Supplementary Figure 1). Nub-Cdc14p induced a slightly but significantly slower conversion of Net1CCG than Nub-Sir2p (Supplementary Figure 1; Supplementary Movie 2). Nub-Pea2p, a Nub-fusion that was not identified by the large-scale experiment, did also not interact in the SPLIFF analysis with Net1CCG (Supplementary Figure 1). Table 1. Interaction partners of Net1p, Spa2p, and Stu2p identified by large-scale Split-Ub interaction screens Cub fusions Nub fusions Net1p Scc2p, Smt3p, Swi6p, Irr1p, Net1p, Fkh1p, Cdc14p, Fkh2p, Ubc9p Spa2p Kel1p, Hof1p, Ymr124wp, Spa2p, Bud14p, Sec4p, Pea2p Stu2p Spc24p, Bik1p, Spc72p, Bim1p, Kar3p, Dad1p, Dad3p, Stu2p, Clb4p, Kip2p, Kip3p, Kar9p To identify the rate-limiting steps of these reactions, we compared the accumulation of GFP-Cdc14p in the nuclei of a-cells with the Nub-Cdc14p-induced Net1CCG conversion (Figure 3). In the first 10 min after mating, the kinetic profiles of both reactions were similar. However, whereas the best fit of the Net1CCG conversion is described by a sigmoid curve, the nuclear accumulation of GFP-CDC14p proceeded linear and consequently slower (Figure 3B). We conclude that the conversion of Net1CCG to Net1CC is not limited by the association rate of the fusion proteins and the subsequent Ub assembly and degradation of the attached GFP. The sigmoid shape indicates that the interaction between Cdc14p and Net1p is dynamic. Nub-Cdc14p exchanges binding partners and thereby catalytically converts Net1CCG into Net1CC. Figure 3.Comparison between Nub-Cdc14p-induced Net1CCG conversion and GFP-Cdc14p accumulation. (A) α-Cells expressing Nub-Cdc14p were mated with a-cells expressing Net1CCG and the conversion to Net1CC was recorded after mating (upper panel). Lower panel: α-cells expressing GFP-Cdc14p were mated with wild-type a-cells and the accumulation of the GFP signal was recorded in the a-cell-derived nucleus (white arrowhead) as fraction of total fluorescence measured shortly before cell fusion (t0). The white-framed cells indicate the diploid cells. (B) Pairwise comparison of best fits of independent experiments as shown in (A). Blue curve corresponds to conversion of Net1CCG and black curve to the accumulation of GFP-Cdc14. See also Supplementary Figure 1 and Supplementary Movie 2. Download figure Download PowerPoint Interactions at the nuclear pore: slow exchange and transient interactions The nuclear pore complex of yeast consists of >30 different proteins that are organized into three different layers. Integral membrane proteins (POMs) anchor a ring of coat nucleoporins followed by adaptor nucleoporins. The adaptors position the channel nucleoporins to regulate the transfer of cargo between the nucleus and the cytoplasm (Figure 4A; Aitchison and Rout, 2012). Nup49p and Nic96p together with Nsp1p and Nup57p form the Nic96p sub-complex (Figure 4A). Nic96p is a member of the adaptor nucleoporins whereas Nup49p, Nsp1p, and Nup57p belong to the FG repeat-bearing channel nucleoporins. FG repeats interact transiently with importins and exportins that are bound to cargo and the Ran-GTPase Gsp1p during their shuttle across the pore (Figure 4A; Aitchison and Rout, 2012). After mating, the nuclear membranes of the two yeast nuclei fuse and the nuclear pore complexes from both cells mix (Bucci and Wente, 1997). According to the growth rate of the co-expressing diploids Nup49CRU interacted strongly with Nub-Nup57p, -Nic96p, -Nsp1p, and only very weakly with Nub-Gsp1p (Supplementary Figure 2). We measured the kinetic profiles of these interactions with Nup49p as CCG fusion. The SPLIFF analysis of Nup49CCG visualized for the first time the interaction between a Ran-GTPase and a FG-repeat protein in a living cell (Figure 4B, C and F; Supplementary Movie 3). Nub-Gsp1p converted Nup49CCG much faster than the Nub fusions of Nic96p or Nsp1p (Figure 4F; Supplementary Figure 2; Supplementary Movie 4). The nuclear resident Nub-Cdc14p did not interact with Nup49CCG (Figure 4D and E). Contrary to the transport factors, constitutive members like Nsp1p or Nic96p are known to be stably incorporated into the nuclear pore complex (Rabut et al, 2004). We therefore surmise that the slow exchange of the unlabelled proteins against the corresponding Nub fusions of Nsp1p or Nic96p is rate limiting for the interaction with Nup49CCG. Notably, the interaction between Nup49CCG and its Nub-labelled interaction partners started only after nuclear fusion was completed (Supplementary Movie 4). Figure 4.Protein interactions at the nuclear pore. (A) Overview of the structure of the nuclear pore, the Nic96p sub-complex, and the nucleo-cytoplasmic traffic in yeast. (B) Selected frames of the time-lapse analysis of a cell (white frame) expressing Nup49CCG and Nub-Gsp1p after mating. The haploid cells are not framed. (C) Time-dependent relative fluorescence intensity of Cherry (), GFP (), and the calculated fraction of converted Nup49CC () from the experiment shown in (B). Time 0 indicates the time point shortly before the fusion of the two nuclei. (D) As in (B) but showing a diploid cell expressing Nup49CCG and Nub-Cdc14p after mating. (E) Analysis as in (C) but of the experiment shown in (D). The nuclear protein Nub-Cdc14p does not interact. (F) Time-dependent change of the fractions of converted Nup49CC through interaction with Nub-Gsp1p (), Nub-Nic96p (), Nub-Nsp1p (), and Nub-Cdc14p (). The averages of n=8 independent matings (error bars, s.e.) are shown. The expressions of the Nub fusions were induced by 100 μM copper. Scale bar, 5 μm. See also Supplementary Figure 2 and Supplementary Movies 3 and 4. Source data for this figure is available on the online supplementary information page. Source data for Figure 4C [msb20133-sup-0002-SourceData-S2.xlsx] Source data for Figure 4E [msb20133-sup-0003-SourceData-S3.xls] Source data for Figure 4F [msb20133-sup-0004-SourceData-S4.xls] Download figure Download PowerPoint Interactions between components of the polar cortical domain: tracking interactions during the cell cycle The proteins of the polar cortical domain (PCD) form a protein network below the plasma membrane. The composition of the PCD in yeast is not fully defined and highly dynamic (Gao et al, 2011). The PCD is located first at the mating projection (PCDI), later at the site of bud growth (PCDII), and finally at the site of cytokinesis (PCDIII). During these transitions, the components of the PCDs are dissolved before they are recruited to their new site (Figure 5A). Measuring the interactions between members of the PCD thus requires tracking of the CCG fusion in their different locations during the cell cycle. Spa2p is member of the polarisome complex that organizes the actin cytoskeleton at the PCD (Sheu et al, 1998). We first identified the pool of Nub-labelled interaction partners by mating a Spa2CRU expressing strain against the Nub array and selected a subset of those for single-cell analysis (Table I). Among the found binding partners was a further member of the polarisome Nub-Pea2p (Sheu et al, 1998). Time-lapse microscopy of the mated Spa2CCG and Nub-Pea2p expressing cells revealed for the first time that both proteins interact throughout the cell cycle (Figure 5A, B; Supplementary Movie 5). The specificity of the measurements was confirmed by co-expressing Spa2CCG and Nub-Ptc1p, a Nub fusion that, according to the large-scale analysis, does not bind to Spa2CRU (Figure 5C–E). Figure 5.SPLIFF analysis of the interactions between components of the polar cortical domain. (A) Selected frames of the time-lapse analysis of a cell (white frame) expressing Spa2CCG and Nub-Pea2p after mating. The haploid cells are not framed. PCDI indicates the stained region below the membrane of the shmoo tip, PCDII the region below the membrane of the growing bud, and PCDIII the region of cell separation. (B) Time-dependent relative fluorescence intensity (RFI) of Cherry (), GFP (), and the calculated fraction of converted Spa2CC () of the experiment shown in (A). Time 0 indicates the time point shortly before the fusion of the cells. (C, D) As (A) and (B) but with diploid cells expressing Spa2CCG and Nub-Ptc1p after mating. The cytosolic protein Nub-Ptc1p does not interact with Spa2CCG. (E) Time-dependent change of converted Spa2CC through interaction with Nub-Spa2p (), Nub-Pea2p (), and Nub-Pea2p in cells lacking the native Pea2p (), Nub-Hof1p (), Nub-Hof198–669 (), and Nub-Ptc1p (). Dashed lines separate the analyses of the subsequent PCDs. The averages of n=6 independent matings (error bars, s.e.) are shown. Scale bar, 5 μm. See also Supplementary Figure 3; Supplementary Movies 5–8. Source data for this figure is available on the online supplementary information page. Source data for Figure 5B [msb20133-sup-0005-SourceData-S5.xls] Source data for Figure 5D [msb20133-sup-0006-SourceData-S6.xls] Source data for Figure 5E [msb20133-sup-0007-SourceData-S7.xlsx] Download figure Download PowerPoint Spa2p forms homo-oligomers (Table I). Our SPLIFF analysis revealed that this reaction occurred much faster than the Spa2p-Pea2p heteromerization (Figure 5E; Supplementary Figure 3; Supplementary Movie 6). Why is the Spa2p-Pea2p interaction slower? We presumed that the continuous presence of the unlabelled Pea2p in the polarisome might effectively hinder Nub-Pea2p from binding and converting Spa2CCG. Consequently, cells lacking unlabelled Pea2p should display a faster interaction. Consistent with our hypothesis, we observed a 5.3-fold increase in the rate of Nub-Pea2p induced Spa2CCG conversion upon deleting the chromosomal PEA2 in the Spa2CCG expressing a-cells (Figure 5E; Supplementary Figure 3; Supplementary Movie 7). The ratio of the initial rates of conversion RCiPEA2 to RCiΔpea2 (Δf=5.3) provides a quantitative measure (Supplementary Figure 3). By using simplifying assumptions, we calculated the fraction of bound Spa2p (Fb) by Fb=(Δf−1)/Δf. Based on this calculation, we estimate that only 19% of the Spa2CCG molecules in wild-type cells are free to react with Nub-Pea2p. The remaining 81% (Fb) of the binding sites are occupied by endogenous Pea2p. We identified Hof1p, a member of the cytokinesis machinery, as a new ligand of Spa2p (Table I) (Lippincott and Li, 1998; Meitinger et al, 2011). The SPLIFF analysis revealed that Hof1p already interacted with Spa2p at the PCDI during cell fusion. Furthermore, the kinetic profile of the interaction between Spa2CCG and Nub-Hof1p differed strikingly from the profile of the Spa2p/Pea2p interaction in tracing interactions during fusion and cytokinesis but not during bud growth (Figure 5E; Supplementary Movie 8). An allele of HOF1 lacking the N-terminal FCH domain (Nub-Hof198–669) added an interesting mechanistic detail to the understanding of this protein interaction. In contrast to the full-length protein, Nub-Hof198–669 converted Spa2CCG only during cytokinesis but not during cell fusion (Figure 5E). The results thus allude to different modes and degrees of interaction between Hof1p and Spa2p during the three phases of polar growth. Mapping the cellular space of protein–protein interactions Many proteins simultaneously occupy different locations in the cell. Each of these locations might reflect an altering set of binding partners. We examined the cellular distributions of four different protein interactions to demonstrate the spatial resolution of SPLIFF. The Nub fusion of Kel1p was found as interaction partner of Spa2CRU (Table I). Kel1p is involved in cell fusion during mating (Philips and Herskowitz, 1998). Kel1CCG is spread across the entire bud during its growth (Figure 6A). To specify the regions of Kel1p–Spa2p interaction, we mated Kel1CCG- with Nub-Spa2p expressing cells. Time-lapse analysis of the diploids revealed that the interaction occurs not only during cell fusion but also during bud growth (Figure 6A and B). We analyzed the spatial distribution of the red and green fluorescence in medium-sized buds (Figure 6C–E). Both intensities matched closely at the base of the bud yet segregated at its tip (Figure 6C–E). The reconstituted differential interaction maps describe a gradient in the ratio of red to green fluorescence that peaks at the bud tip and trails off toward the mother cell (Figure 6D and E). This distribution very probably corresponds with a gradient of interactions caused by the unequal distribution of Spa2p and Kel1p across the bud (Figures 5A and 6A). Figure 6.Map of the cellular location of the Spa2p/Kel1p interaction. (A) Left panel: Selected frames of the time-lapse analysis of a diploid cell (white frame) expressing Kel1CCG and Nub-Spa2p after mating. The distribution of Kel1CCG and Kel1CC is shown. Right panel: Blow-ups of the purple-framed buds showing the distribution of GFP (Kel1CCG) and Cherry (Kel1CCG+Kel1CC) at the indicated times. (B) Time-dependent change of the relative fluorescence intensity (RFI) of Cherry (), GFP (), and the calculated fraction of converted Kel1CC () in the experiment shown in (A). Time 0 indicates the time point shortly before cell fusion. (C) Colour-coded maps of the RFIs of cherry (Kel1CCG+Kel1CC) and GFP (Kel1CCG) obtained from the area within the white squares of (A), right panel. White arrows point to identical regions of the bud in the two channels and mark regions of relative depletion of GFP (Kel1CCG). (D) Left panels: 3D reconstructions of confocal images from GFP, Cherry and merged channels of yeast cells expressing Kel1CCG, or Kel1CCG and Nub-Spa2p after mating (white frame). Right panels show the profiles of the relative GFP and Cherry intensities from the yellow- (diploid) and white-framed (haploid) buds obtained by summing over all fluorescence intensities encountered orthogonal to the direction of the respective arrows. Arrow *: across the tip of the bud of haploid cells. Arrow **: from the tip to the base of the bud of diploid cells. Arrow ***: across the tip of the bud of diploid cells. (E) Analysis as in (D) but of cells expressing Kel1CCG and a Nub fusion not binding to Kel1p. Scale bar, 5 μm. Source data for this figure is available on the online supplementary information page. Source data for Figure 6B [msb20133-sup-0008-SourceData-S8.xls] Source data for Figure 6D [msb20133-sup-0009-SourceData-S9.xls] Source data for Figure 6E [msb20133-sup-0010-SourceData-S10.xls] Download figure Download PowerPoint Nap1p is a multifunctional protein that is involved in the transport and assembly of histones and the formation of the septin ring at the bud neck of the cells (Mortensen et al, 2002; Ohkuni et al, 2003). Nap1p is localized in the cytosol, the nucleus, and the bud neck. We recently confirmed Nub-Kcc4p as binding partner of Nap1CRU (Hruby et al, 2011). Kcc4p is a septin-localized protein kinase (Barral et al, 1999; Okuzaki and Nojima, 2001). To find out exactly when and where the interaction between Nap1p and Kcc4p occurs, we monitored the Nub-Kcc4p-induced conversion of Nap1CCG in the cytosol as well as in the nucleus. The interaction started immediately after cell fusion (Figure 7A and B). The slower decrease in nuclear versus cytosolic green fluorescence indicated that Kcc4p interacts with Nap1p primarily in the cytosol (Figure 7B; Supplementary Figure 4). Continuous time-lapse analysis of Nap1CCG identified at a later time point during early bud formation a red fluorescent zone devoid of green fluorescence beneath the incipient bud site (Figure 7A and C; Supplementary Movie 9). This region of preferred complex formation contrasted with the nucleus as well as with the cytosol as two compartments of comparatively weak Nap1p-Kcc4p interaction activity (Figure 7C). Fifty minutes after mating a decrease in the nuclear Cherry signal might indicate a trapping of Nap1CCG in the cytosol through the continuous co-expression of Nub-Kcc4p (Figure 7B). Nub-Pea2p is also concentrated at the incipient bud site but does not interact with Nap1CRU. Consequently, Nub-Pea2p did not induce a similar dissociation of red from green fluorescence in Nap1CCG-expressing cells (Figure 7D and E). Figure 7.Dynamic map of the Nap1p/Kcc4p interaction. (A) Selected frames of the time-lapse analysis of a diploid cell (white frame) expressing Nap1CCG and Nub-Kcc4p after mating. White arrowheads point the region of high interaction activity at the site of a newly emerging bud. (B) Time-dependent change of the relative fluorescence intensity (RFI) of Cherry (), and GFP (), as well as the calculated fraction of converted Nap1CC () in the experiment shown in (A). The analysis distinguishes between the cytoplasmic (upper panel) and the nuclear-localized Nap1CCG (lower panel). The cytoplasmic fluorescence is defined as the difference between total cellular and nuclear fluorescence. Note that the reaction occurs faster in the cytosol than in the nucleus (see also Supplementary Figure 4). The white arrowhead indicates the time of the first appearance of the region of high interaction activity in the emerging bud. (C) Colour-coded map of the RFI of Cherry (Nap1CCG+Nap1CC) and GFP (Nap1CCG) in the white-boxed cell (left panel) in the experiment shown in (A) at 35 min. White arrows point to the emerging bud. (D, E) Analysis exactly as in" @default.
• W2113406918 created "2016-06-24" @default.
• W2113406918 creator A5000554347 @default.
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• W2113406918 date "2013-01-01" @default.
• W2113406918 modified "2023-10-15" @default.
• W2113406918 title "A fluorescent reporter for mapping cellular protein‐protein interactions in time and space" @default.
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