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Chromobodies to Quantify Changes of Endogenous Protein Concentration in Living Cells*

Open AccessPublished:September 18, 2018DOI:https://doi.org/10.1074/mcp.TIR118.000914
      Understanding cellular processes requires the determination of dynamic changes in the concentration of genetically nonmodified, endogenous proteins, which, to date, is commonly accomplished by end-point assays in vitro. Molecular probes such as fluorescently labeled nanobodies (chromobodies, CBs) are powerful tools to visualize the dynamic subcellular localization of endogenous proteins in living cells. Here, we employed the dependence of intracellular levels of chromobodies on the amount of their endogenous antigens, a phenomenon, which we termed antigen-mediated CB stabilization (AMCBS), for simultaneous monitoring of time-resolved changes in the concentration and localization of native proteins. To improve the dynamic range of AMCBS we generated turnover-accelerated CBs and demonstrated their application in visualization and quantification of fast reversible changes in antigen concentration upon compound treatment by quantitative live-cell imaging. We expect that this broadly applicable strategy will enable unprecedented insights into the dynamic regulation of proteins, e.g. during cellular signaling, cell differentiation, or upon drug action.

      Graphical Abstract

      Several methods are available to detect changes in the concentration of specific proteins in biological samples. The rise of mass spectrometry (MS)-based analysis has enabled relative and absolute quantification of proteins with unprecedented sensitivity and accuracy (
      • Li H.
      • Han J.
      • Pan J.
      • Liu T.
      • Parker C.E.
      • Borchers C.H.
      Current trends in quantitative proteomics - an update.
      ,
      • Lindemann C.
      • Thomanek N.
      • Hundt F.
      • Lerari T.
      • Meyer H.E.
      • Wolters D.
      • Marcus K.
      Strategies in relative and absolute quantitative mass spectrometry based proteomics.
      ). Considering that MS-based quantification requires expensive equipment and trained personnel, this technology is mostly applied for large-scale proteomic analyses. Because of the ever-growing availability of specific antibodies, antibody-based techniques such as enzyme-linked immunosorbent assay (ELISA) or immunoblotting are commonly used to analyze relative concentration changes of single proteins of interest (POIs)
      The abbreviations used are:
      POI
      protein of interest
      AMCBS
      antigen-mediated chromobody stabilization
      BIO
      6-bromoindirubin-3′-oxime
      CA
      capsid
      CB
      chromobody
      CCC
      cell cycle chromobody
      CHX
      cycloheximide
      CMV
      cytomegalovirus
      CTNNB1
      beta-catenin
      DMEM
      Dulbecco's Modified Eagle Medium
      EDTA
      ethylenediaminetetraacetic acid
      ELISA
      enzyme-linked immunosorbent assay
      FP
      fluorescent protein
      GSK3-β
      glycogen synthase kinase 3 beta
      HEK293T
      human embryonic kidney 293T
      HIV
      human immunodeficiency virus
      IF
      immunofluorescence
      KRAB
      krüppel associated box
      MS
      mass spectrometry
      NB
      nanobody
      PCNA
      proliferating cell nuclear antigen
      PEI
      polyethylenimine
      PEST
      sequence rich in proline, glutamate, serine, threonine
      PFA
      paraformaldehyde
      PMSF
      phenylmethylsulfonyl fluoride
      RNAi
      RNA interference
      S.D.
      standard deviation
      S.E.M.
      standard error of the mean
      siRNA
      small interfering RNA
      Ub
      ubiquitin
      VHH
      variable domain of heavy-chain antibodies
      VIM
      Vimentin.
      1The abbreviations used are:POI
      protein of interest
      AMCBS
      antigen-mediated chromobody stabilization
      BIO
      6-bromoindirubin-3′-oxime
      CA
      capsid
      CB
      chromobody
      CCC
      cell cycle chromobody
      CHX
      cycloheximide
      CMV
      cytomegalovirus
      CTNNB1
      beta-catenin
      DMEM
      Dulbecco's Modified Eagle Medium
      EDTA
      ethylenediaminetetraacetic acid
      ELISA
      enzyme-linked immunosorbent assay
      FP
      fluorescent protein
      GSK3-β
      glycogen synthase kinase 3 beta
      HEK293T
      human embryonic kidney 293T
      HIV
      human immunodeficiency virus
      IF
      immunofluorescence
      KRAB
      krüppel associated box
      MS
      mass spectrometry
      NB
      nanobody
      PCNA
      proliferating cell nuclear antigen
      PEI
      polyethylenimine
      PEST
      sequence rich in proline, glutamate, serine, threonine
      PFA
      paraformaldehyde
      PMSF
      phenylmethylsulfonyl fluoride
      RNAi
      RNA interference
      S.D.
      standard deviation
      S.E.M.
      standard error of the mean
      siRNA
      small interfering RNA
      Ub
      ubiquitin
      VHH
      variable domain of heavy-chain antibodies
      VIM
      Vimentin.
      . However, the informative value of these methods is limited because only average protein amounts are determined and no intercellular resolution is provided. In addition, tracing changes in protein concentration over time is very laborious and time-consuming. Alternatively, immunofluorescence (IF) can be used to assess the subcellular localization and the relative concentration of POIs on single-cell level. Like any immunodetection method, this can suffer from inaccuracies based on batch-to-batch antibody variability, epitope inaccessibility and cross reactivity (
      • Walker J.M.
      ). In addition, because of cell fixation and permeabilization procedures, IF allows no direct analysis of dynamic changes (
      • Schnell U.
      • Dijk F.
      • Sjollema K.A.
      • Giepmans B.N.
      Immunolabeling artifacts and the need for live-cell imaging.
      ). Considering that most cellular processes are dynamic in nature and rely on the spatiotemporal orchestration under native conditions, the assessment of time-dependent changes of endogenous protein levels within the physiological environment of living cells is preferable.
      With the rise of genome editing techniques, fluorescent protein (FP) tagging of endogenous proteins provides a straightforward approach to optically monitor the relative amount of a POI (
      • Leonetti M.D.
      • Sekine S.
      • Kamiyama D.
      • Weissman J.S.
      • Huang B.
      A scalable strategy for high-throughput GFP tagging of endogenous human proteins.
      ). However, as repeatedly described, FP tagging can interfere with crucial protein parameters such as turnover, subcellular localization, and participation in multi-protein complexes (
      • Snapp E.L.
      Fluorescent proteins: a cell biologist's user guide.
      ,
      • Stadler C.
      • Rexhepaj E.
      • Singan V.R.
      • Murphy R.F.
      • Pepperkok R.
      • Uhlen M.
      • Simpson J.C.
      • Lundberg E.
      Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells.
      ,
      • Virant D.
      • Traenkle B.
      • Maier J.
      • Kaiser P.D.
      • Bodenhofer M.
      • Schmees C.
      • Vojnovic I.
      • Pisak-Lukats B.
      • Endesfelder U.
      • Rothbauer U.
      A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging.
      ). During the last decade, intrabodies have emerged as beneficial tools to study the dynamic behavior of endogenous proteins in various cellular models. Because of their compact structure, small size, high stability and solubility, single-domain antibody fragments from camelids (VHH, nanobodies) possess many advantageous properties to be employed within living cells (
      • Kaiser P.D.
      • Maier J.
      • Traenkle B.
      • Emele F.
      • Rothbauer U.
      Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells.
      ,
      • Helma J.
      • Cardoso M.C.
      • Muyldermans S.
      • Leonhardt H.
      Nanobodies and recombinant binders in cell biology.
      ,
      • Ingram J.R.
      • Schmidt F.I.
      • Ploegh H.L.
      Exploiting Nanobodies' Singular Traits.
      ). Acknowledging the potential of these binding molecules, numerous protocols and synthetic nanobody libraries for targeted selection of intracellularly functional nanobodies have been developed (
      • Pellis M.
      • Pardon E.
      • Zolghadr K.
      • Rothbauer U.
      • Vincke C.
      • Kinne J.
      • Dierynck I.
      • Hertogs K.
      • Leonhardt H.
      • Messens J.
      • Muyldermans S.
      • Conrath K.
      A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.
      ,
      • Moutel S.
      • Nizak C.
      • Perez F.
      Selection and use of intracellular antibodies (intrabodies).
      ,
      • Traenkle B.
      • Rothbauer U.
      Under the Microscope: single-domain antibodies for live-cell imaging and super-resolution microscopy.
      ). By fusing nanobodies (NBs) to fluorescent proteins, so-called chromobodies (CBs) are generated. Upon cellular expression, they allow optical detection of endogenous proteins in live cells. With regard to imaging purposes transiently binding CBs addressing functionally inert epitopes are preferable to avoid unwanted effects on antigen mobility or by displacing natural interaction partners. To date, multiple target-specific CBs have been applied to visualize cellular processes in cultured cells and entire organisms without functional interference (
      • Rothbauer U.
      • Zolghadr K.
      • Tillib S.
      • Nowak D.
      • Schermelleh L.
      • Gahl A.
      • Backmann N.
      • Conrath K.
      • Muyldermans S.
      • Cardoso M.C.
      • Leonhardt H.
      Targeting and tracing antigens in live cells with fluorescent nanobodies.
      ,
      • Helma J.
      • Schmidthals K.
      • Lux V.
      • Nuske S.
      • Scholz A.M.
      • Krausslich H.G.
      • Rothbauer U.
      • Leonhardt H.
      Direct and dynamic detection of HIV-1 in living cells.
      ,
      • Irannejad R.
      • Tomshine J.C.
      • Tomshine J.R.
      • Chevalier M.
      • Mahoney J.P.
      • Steyaert J.
      • Rasmussen S.G.
      • Sunahara R.K.
      • El-Samad H.
      • Huang B.
      • von Zastrow M.
      Conformational biosensors reveal GPCR signalling from endosomes.
      ,
      • Panza P.
      • Maier J.
      • Schmees C.
      • Rothbauer U.
      • Sollner C.
      Live imaging of endogenous protein dynamics in zebrafish using chromobodies.
      ,
      • Maier J.
      • Traenkle B.
      • Rothbauer U.
      Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies.
      ,
      • Schorpp K.
      • Rothenaigner I.
      • Maier J.
      • Traenkle B.
      • Rothbauer U.
      • Hadian K.
      A multiplexed high-content screening approach using the chromobody technology to identify cell cycle modulators in living cells.
      ).
      Recently, we have generated a CB (BC1-TagGFP2), which specifically targets the soluble, nonmembrane-associated fraction of endogenous β-catenin (CTNNB1) without affecting its transcriptional activity. By live-cell imaging of cells stably expressing BC1-TagGFP2 we observed an increased CB signal along with an elevation of intracellular CTNNB1 level upon compound-mediated induction of the WNT/β-catenin pathway (
      • Traenkle B.
      • Emele F.
      • Anton R.
      • Poetz O.
      • Haeussler R.S.
      • Maier J.
      • Kaiser P.D.
      • Scholz A.M.
      • Nueske S.
      • Buchfellner A.
      • Romer T.
      • Rothbauer U.
      Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.
      ). Notably, this was not because of altered transcription of the CB but attributed to a yet unexplained mechanism of antigen-mediated stabilization on protein level, which is in accordance to previous findings describing higher levels of bacterially injected NBs within the cytoplasm of mammalian cells in the presence of their cognate antigen (
      • Blanco-Toribio A.
      • Muyldermans S.
      • Frankel G.
      • Fernandez L.A.
      Direct injection of functional single-domain antibodies from E. coli into human cells.
      ).
      Here, we demonstrate that antigen-mediated CB stabilization (AMCBS) is applicable for numerous CBs by showing this phenomenon for four different CBs targeting unrelated endogenous and nonendogenous antigens. To adapt CBs for monitoring changes in antigen concentration more precisely, we screened for N-terminal amino acids, which induce accelerated CB turnover. Based on our findings, we generated highly antigen-responsive CBs. As exemplarily shown for CTNNB1-specific CBs, stable chromobody cell lines allow visualization of rapid and reversible changes in the concentration of endogenous proteins upon compound treatment by quantitative live-cell imaging.

      DISCUSSION

      Chromobodies (CBs) comprising nanobody (NB)-derived binding moieties genetically linked to fluorescent proteins have become valuable tools to visualize endogenous antigens within living cells (
      • Helma J.
      • Cardoso M.C.
      • Muyldermans S.
      • Leonhardt H.
      Nanobodies and recombinant binders in cell biology.
      ,
      • Traenkle B.
      • Rothbauer U.
      Under the Microscope: single-domain antibodies for live-cell imaging and super-resolution microscopy.
      ). Recently, we and others observed that NBs are stabilized in the presence of their antigen (
      • Traenkle B.
      • Emele F.
      • Anton R.
      • Poetz O.
      • Haeussler R.S.
      • Maier J.
      • Kaiser P.D.
      • Scholz A.M.
      • Nueske S.
      • Buchfellner A.
      • Romer T.
      • Rothbauer U.
      Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.
      ,
      • Blanco-Toribio A.
      • Muyldermans S.
      • Frankel G.
      • Fernandez L.A.
      Direct injection of functional single-domain antibodies from E. coli into human cells.
      ,
      • Tang J.C.
      • Drokhlyansky E.
      • Etemad B.
      • Rudolph S.
      • Guo B.
      • Wang S.
      • Ellis E.G.
      • Li J.Z.
      • Cepko C.L.
      Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies.
      ). Originally observed for a CB targeting hypo-phosphorylated CTNNB1, here we show antigen-mediated stabilization (AMCBS) for four different CBs targeting soluble (cytoplasmic/nuclear CTNNB1), structural (vimentin), nuclear (PCNA) and virus-derived (p24-CA) antigens. Considering similar observations reported by others, it is conceivable that AMCBS is a general phenomenon common to numerous CBs. Notably, by monitoring CB signals in different stable cell systems, we not only visualized elevation but also, for the first time, depletion of cellular antigens over time. Although substantial changes in protein levels over longer periods can be sufficiently visualized using CBs in their original format, it has to be considered that specific signals of bound CBs in response to smaller and/or more rapid changes in protein levels can be obscured by the diffuse signal of nonbound CBs. To cope with this issue, recently, several approaches have been described to modify such intracellular nanoprobes accordingly.
      To repress the expression of nontarget-bound intrabodies a DNA-binding KRAB domain was fused to an intrabody, thereby establishing a negative transcriptional feed-back mechanism (
      • Gross G.G.
      • Junge J.A.
      • Mora R.J.
      • Kwon H.B.
      • Olson C.A.
      • Takahashi T.T.
      • Liman E.R.
      • Ellis-Davies G.C.
      • McGee A.W.
      • Sabatini B.L.
      • Roberts R.W.
      • Arnold D.B.
      Recombinant probes for visualizing endogenous synaptic proteins in living neurons.
      ). However, like any regulatory circuit involving transcription, this approach reacts sluggishly to rapid changes of cellular POI levels. Moreover, because of DNA-binding, KRAB domain-containing intrabodies accumulate in the nucleus even in the absence of the target protein (
      • Gross G.G.
      • Junge J.A.
      • Mora R.J.
      • Kwon H.B.
      • Olson C.A.
      • Takahashi T.T.
      • Liman E.R.
      • Ellis-Davies G.C.
      • McGee A.W.
      • Sabatini B.L.
      • Roberts R.W.
      • Arnold D.B.
      Recombinant probes for visualizing endogenous synaptic proteins in living neurons.
      ). When we added the KRAB domain to our VIM- and CTNNB1-specific CBs, we observed a strong enrichment of both CBs in the nucleus, which impedes target concentration analysis by AMCBS (data not shown). Consequently, this system is not suitable to monitor nuclear proteins and rapid changes in protein levels. Another approach to lower the concentration of unbound intrabodies was reported for constructs comprising a PEST domain, which promotes rapid ubiquitin-independent proteasomal degradation (
      • Sibler A.P.
      • Courtete J.
      • Muller C.D.
      • Zeder-Lutz G.
      • Weiss E.
      Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells.
      ). Upon introduction of PEST-modified CB expression constructs in live cells, we observed a substantial decrease in CB fluorescence. However, this was accompanied by a rapid onset of cell death irrespective of the addressed antigen (data not shown).
      Recently distinct point mutations within the framework regions were described to destabilize, and accordingly lower the amount of intracellular NBs. Such modified NBs were shown to be re-stabilized in the presence of overexpressed antigen and thus are functional to detect recombinant or viral antigens e.g. by flow cytometry (
      • Tang J.C.
      • Drokhlyansky E.
      • Etemad B.
      • Rudolph S.
      • Guo B.
      • Wang S.
      • Ellis E.G.
      • Li J.Z.
      • Cepko C.L.
      Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies.
      ). Although it was stated that only NBs comprising framework mutations are stabilized in the presence of the antigen, our analysis revealed substantial antigen responsiveness even for nonmodified CBs. This indicates that antigen-mediated stabilization is inherent to CBs per se and does not depend on mutational destabilization. Moreover, as shown for the BC1C92Y-TagGFP2 version, the introduction of mutations within the framework regions bears the risk to lose functional binding molecules. Notably, this is in line with previous reports of multiple NBs that show a participation of the framework regions in antigen binding (
      • Kirchhofer A.
      • Helma J.
      • Schmidthals K.
      • Frauer C.
      • Cui S.
      • Karcher A.
      • Pellis M.
      • Muyldermans S.
      • Casas-Delucchi C.S.
      • Cardoso M.C.
      • Leonhardt H.
      • Hopfner K.P.
      • Rothbauer U.
      Modulation of protein properties in living cells using nanobodies.
      ,
      • Schmidt F.I.
      • Hanke L.
      • Morin B.
      • Brewer R.
      • Brusic V.
      • Whelan S.P.
      • Ploegh H.L.
      Phenotypic lentivirus screens to identify functional single domain antibodies.
      ,
      • Fanning S.W.
      • Horn J.R.
      An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop.
      ,
      • Noel F.
      • Malpertuy A.
      • de Brevern A.G.
      Global analysis of VHHs framework regions with a structural alphabet.
      ).
      Here, we conceived a strategy to reduce base levels of nonbound CBs, which do not affect antigen binding. We focused on the N terminus of CBs, which has never been reported to participate in antigen binding and employed the rather old concept of the N-end rule (
      • Bachmair A.
      • Finley D.
      • Varshavsky A.
      In vivo half-life of a protein is a function of its amino-terminal residue.
      ,
      • Gonda D.K.
      • Bachmair A.
      • Wunning I.
      • Tobias J.W.
      • Lane W.S.
      • Varshavsky A.
      Universality and structure of the N-end rule.
      ). With either Met or Ala at the N terminus, CBs are subjected to the Ac/N-end rule pathway that involves N-terminal acetylation, which presumably results in a long half-life of the protein (
      • Bachmair A.
      • Finley D.
      • Varshavsky A.
      In vivo half-life of a protein is a function of its amino-terminal residue.
      ). The other major degradation pathway of the ubiquitin proteasome system is the Arg/N-end rule pathway. To screen for CB turnover accelerating N-terminal amino acid residues we implemented the ubiquitin fusion technique (
      • Varshavsky A.
      Ubiquitin fusion technique and related methods.
      ) and identified Arg and Phe, which, when exposed at the N terminus of all tested constructs, mediates the fastest CB turnover. The identification of these representatives of basic or bulky hydrophobic residues is in accordance with short half-lives described for other proteins displaying those residues at their N termini (
      • Varshavsky A.
      The N-end rule pathway and regulation by proteolysis.
      ). Additionally, we observed significant differences in the degradation velocities of CBs comprising different fluorescent proteins. We assume that variances in position and number of lysine residues accessible for ubiquitinylation within the fluorescent moiety have an impact on turnover rates of the corresponding CBs.
      Although our findings provide strong evidence that CBs are degraded via the ubiquitin proteasomal system, the precise molecular and structural mechanisms, which are responsible for the stabilization of CBs upon antigen binding within living cells remain to be elucidated. E3 ubiquitin ligases recognize the N-terminal amino acid and initiate ubiquitinylation of accessible nearby lysine residues (
      • Varshavsky A.
      The N-end rule pathway and regulation by proteolysis.
      ). Notably, such ubiquitinylated proteins are stable within living cells unless they also expose an unstructured region, which is needed to initiate degradation (
      • Prakash S.
      • Tian L.
      • Ratliff K.S.
      • Lehotzky R.E.
      • Matouschek A.
      An unstructured initiation site is required for efficient proteasome-mediated degradation.
      ). CBs have two tightly folded domains: the binding moiety, which shows a typical immunoglobulin fold and the cylindrical FP structure. Consequently, unstructured regions are restricted to the complementarity determining regions (CDRs) of the NB, the interconnecting linker, and to the short alpha helices forming the caps on the ends of the FP beta-barrel. Forming the paratope, the CDRs are in close contact with the antigen and thus it can be speculated that antigen binding masks these potential initiation sites and thus prevents degradation of the CB. Additionally, it is conceivable that CB molecules become partially immobilized in the cell upon antigen binding and are therefore less likely to interact with proteasomes compared with freely diffusible CBs. The possible reduced mobility of the bound CBs is in agreement with findings showing that larger protein complexes have a limited and/or reduced diffusion coefficient (
      • Verkman A.S.
      Diffusion in cells measured by fluorescence recovery after photobleaching.
      ). Finally, competition of antigen and ubiquitin ligases for CBs, which would also facilitate the escape of antigen-bound CBs from ubiquitinylation could also contribute to the observed phenomenon of AMCBS.
      For monitoring protein levels optically, FP tagging of endogenous proteins is a straightforward approach. However, FP tagging can interfere with crucial protein parameters such as turnover, subcellular localization, and participation in multi-protein complexes (
      • Snapp E.L.
      Fluorescent proteins: a cell biologist's user guide.
      ,
      • Stadler C.
      • Rexhepaj E.
      • Singan V.R.
      • Murphy R.F.
      • Pepperkok R.
      • Uhlen M.
      • Simpson J.C.
      • Lundberg E.
      Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells.
      ,
      • Virant D.
      • Traenkle B.
      • Maier J.
      • Kaiser P.D.
      • Bodenhofer M.
      • Schmees C.
      • Vojnovic I.
      • Pisak-Lukats B.
      • Endesfelder U.
      • Rothbauer U.
      A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging.
      ). To avoid a permanent FP fusion, recently a technique was described, which relies on the co-translational separation of the POI and the fused FP reporter translated from a bicistronic expression constructs (
      • Lo C.A.
      • Kays I.
      • Emran F.
      • Lin T.J.
      • Cvetkovska V.
      • Chen B.E.
      Quantification of protein levels in single living cells.
      ). Although monitoring FP fluorescence indicative for the expression of the POI provides a simple and efficient read-out, endogenous proteins can be only addressed upon genome editing bearing the risk, that only one allele is modified. Moreover, as the half-life of the POI might differ from that of the FP, this method is suited to measure relative amounts at steady state but is likely insufficient to measure rapid dynamic changes in POI concentration (
      • Lo C.A.
      • Kays I.
      • Emran F.
      • Lin T.J.
      • Cvetkovska V.
      • Chen B.E.
      Quantification of protein levels in single living cells.
      ). For proteins such as CTNNB1, whose concentration is not primarily regulated by transcription but degradation, this approach is not applicable.
      The herein described turnover-accelerated chromobodies substantially expand the possibilities of these multifunctional nanoprobes. AMCBS with highly antigen-responsive CBs combines for the first time visualization of subcellular localization and redistribution of endogenous proteins with monitoring and quantification of rapid changes of protein levels by quantitative live-cell imaging. Like for any molecular probe applied in quantitative live-cell imaging, a potential influence of CB binding on antigen levels has to be carefully evaluated. Here, we demonstrated that the level and the dynamics of endogenous CTNNB1 is not affected by the presence of CTNNB1-specific CBs. Notably, similar observations were made for the PCNA-CB (CCC-TagRFP) and VIM-CB (VB6-eGFP) as reported previously (
      • Panza P.
      • Maier J.
      • Schmees C.
      • Rothbauer U.
      • Sollner C.
      Live imaging of endogenous protein dynamics in zebrafish using chromobodies.
      ,
      • Maier J.
      • Traenkle B.
      • Rothbauer U.
      Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies.
      ). Moreover, the generation of organisms stably expressing CBs also strongly indicates that CB binding in trans does not affect the levels of tightly regulated antigens (
      • Panza P.
      • Maier J.
      • Schmees C.
      • Rothbauer U.
      • Sollner C.
      Live imaging of endogenous protein dynamics in zebrafish using chromobodies.
      ,
      • Jullien D.
      • Vignard J.
      • Fedor Y.
      • Bery N.
      • Olichon A.
      • Crozatier M.
      • Erard M.
      • Cassard H.
      • Ducommun B.
      • Salles B.
      • Mirey G.
      Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells.
      ). From a technical perspective, this AMCBS approach is readily applicable, as fluorescence microscopy instrumentation is widely available in cell biology laboratories. Because of continuous improvements of nano-/chromobody screening protocols (
      • McMahon C.
      • Baier A.S.
      • Pascolutti R.
      • Wegrecki M.
      • Zheng S.
      • Ong J.X.
      • Erlandson S.C.
      • Hilger D.
      • Rasmussen S.G.F.
      • Ring A.M.
      • Manglik A.
      • Kruse A.C.
      Yeast surface display platform for rapid discovery of conformationally selective nanobodies.
      ,
      • Rothbauer U.
      Speed up to find the right ones: rapid discovery of functional nanobodies.
      ), the number of available chromobodies is constantly growing (
      • Traenkle B.
      • Rothbauer U.
      Under the Microscope: single-domain antibodies for live-cell imaging and super-resolution microscopy.
      ) and will enable time-resolved quantification of many further proteins of interest in the near future. AMCBS in combination with target-specific chromobodies could be further adapted to detect post-translational modifications or the presence and abundance of specific splice variants, which cannot be detected with conventional FP fusions.

      Acknowledgments

      We thank Marion Jung (ChromoTek GmbH) for providing reagents and the HeLa_CCC-TagRFP cell line.

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