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Visualizing ubiquitination in mammalian cells

2019; Springer Nature; Volume: 20; Issue: 2 Linguagem: Inglês

10.15252/embr.201846520

1469-3178

Sjoerd J. L. van Wijk, Simone Fulda, Ivan Đikić, Mike Heilemann,

Autophagy in Disease and Therapy

Review21 January 2019free access Visualizing ubiquitination in mammalian cells Sjoerd JL van Wijk Corresponding Author [email protected] orcid.org/0000-0001-6532-7651 Institute for Experimental Cancer Research in Paediatrics, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Simone Fulda Institute for Experimental Cancer Research in Paediatrics, Goethe University, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Heidelberg, Germany German Cancer Research Centre (DKFZ), Heidelberg, Germany Search for more papers by this author Ivan Dikic orcid.org/0000-0001-8156-9511 Institute of Biochemistry II, Goethe University – Medical Faculty, University Hospital Frankfurt, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University, Frankfurt am Main, Germany Search for more papers by this author Mike Heilemann Corresponding Author [email protected] orcid.org/0000-0002-9821-3578 Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Sjoerd JL van Wijk Corresponding Author [email protected] orcid.org/0000-0001-6532-7651 Institute for Experimental Cancer Research in Paediatrics, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Simone Fulda Institute for Experimental Cancer Research in Paediatrics, Goethe University, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Heidelberg, Germany German Cancer Research Centre (DKFZ), Heidelberg, Germany Search for more papers by this author Ivan Dikic orcid.org/0000-0001-8156-9511 Institute of Biochemistry II, Goethe University – Medical Faculty, University Hospital Frankfurt, Frankfurt am Main, Germany Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University, Frankfurt am Main, Germany Search for more papers by this author Mike Heilemann Corresponding Author [email protected] orcid.org/0000-0002-9821-3578 Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Author Information Sjoerd JL van Wijk *,1, Simone Fulda1,2,3, Ivan Dikic4,5 and Mike Heilemann *,6 1Institute for Experimental Cancer Research in Paediatrics, Goethe University, Frankfurt am Main, Germany 2German Cancer Consortium (DKTK), Heidelberg, Germany 3German Cancer Research Centre (DKFZ), Heidelberg, Germany 4Institute of Biochemistry II, Goethe University – Medical Faculty, University Hospital Frankfurt, Frankfurt am Main, Germany 5Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University, Frankfurt am Main, Germany 6Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany *Corresponding author. Tel: +49 69 6786 6574; E-mail: [email protected] *Corresponding author. Tel: +49 69 7982 9736; E-mail: [email protected] EMBO Rep (2019)20:e46520https://doi.org/10.15252/embr.201846520 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Covalent modification of proteins with ubiquitin is essential for the majority of biological processes in mammalian cells. Numerous proteins are conjugated with single or multiple ubiquitin molecules or chains in a dynamic fashion, often determining protein half-lives, localization or function. Experimental approaches to study ubiquitination have been dominated by genetic and biochemical analysis of enzyme structure–function relationships, reaction mechanisms and physiological relevance. Here, we provide an overview of recent developments in microscopy-based imaging of ubiquitination, available reagents and technologies. We discuss the progress in direct and indirect imaging of differentially linked ubiquitin chains in fixed and living cells using confocal fluorescence microscopy and super-resolution microscopy, illustrated by the role of ubiquitin in antibacterial autophagy and pro-inflammatory signalling. Finally, we speculate on future developments and forecast a transition from qualitative to quantitative super-resolution approaches to understand fundamental aspects of ubiquitination and the formation and distribution of functional E3 ligase protein complexes in their native environment. Glossary ACTL8 actin-like protein 8 ARIH1 E3 ubiquitin-protein ligase protein ariadne-1 homolog BiFC bimolecular fluorescence complementation CALCOCO2/NDP52 calcium-binding and coiled-coil domain-containing protein 2/Nuclear domain 10 protein NDP52 CCCP carbonylcyanid-m-chlorphenylhydrazon Cdt1 DNA replication factor Cdt1 CHIP caryl terminus of Hsp70-interacting protein CLSM confocal laser scanning microscopy CRISPR/Cas9 clustered regularly interspaced short palindromic repeats CRL3KLHL21 cullin RING E3 ligase with the KLHL21 adaptor protein Cy5 cyanine dye 5 deGradFP proteasomal degradation of GFP fusions Dha dehydroalanine diGly lys-ϵ-Gly-Gly dSTORM direct stochastic optical reconstruction microscopy DUB deubiquitinating enzyme E1 ubiquitin-activating enzyme E2 ubiquitin-conjugating enzyme E3 ubiquitin protein ligase E6AP human papillomavirus E6-associated protein FC(C)S fluorescence (cross) correlation spectroscopy FCCP carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone FK1 anti-ubiquitin antibody FK1 FK2 anti-ubiquitin antibody FK2 FRAP fluorescence recovery after photobleaching FRET Förster resonance energy transfer FUCCI fluorescence ubiquitination cell cycle indicator GFP green fluorescent protein GFPu 16-residue CL1 degron fused to GFP HECT homologous to the E6-AP carboxyl terminus HOIL1 RanBP-type and C3HC4-type zinc finger-containing protein 1 HUWE1 HECT, UBA and WWE domain-containing protein 1 IĸBα NF-kappa-B inhibitor alpha IKK inhibitor of nuclear factor kappa-B kinase INT-Ub.7KR lysine-less, internally tagged ubiquitin ISG15 interferon stimulated gene 16 KG Kusabira-Green K lysine LRSAM1 E3 ubiquitin-protein ligase leucine-rich repeat and sterile alpha motif-containing protein 1 LUBAC linear ubiquitin chain assembly complex M(et) methionine M1-SUB Met1-linkage-specific Ub-binder mKO2 monomeric Kusabira-Orange 2 NEDD8 neural precursor cell expressed, developmentally down-regulated 8 NEMO NF-kappa-B essential modulator NF-ĸB nuclear factor NF-kappa-B nm nanometre NSlmb-vhhGFP4 F-box-anti-GFP nanobody fusion protein N-terminal amino-terminal NZF Npl4 zinc finger OPTN optineurin OTULIN OTU domain-containing deubiquitinase with linear linkage specificity OUT orthogonal Ub transfer (OUT) method p65/RelA nuclear factor NF-kappa-B p65 subunit PAGFP photoactivatable GFP PAINT point accumulation for imaging in nanoscale topography PALM photoactivation localization microscopy PARKIN E3 ubiquitin-protein ligase parkin PINK1 serine/threonine-protein kinase PINK1, mitochondrial PML promyelocytic leukaemia protein PolyUb-FC polyubiquitin-mediated fluorescence complementation PROTACs proteolysis-targeting chimeric molecules PSF point-spread function Pup prokaryotic ubiquitin-like protein RBR RING-in-between-RING RFP red fluorescent protein RING really interesting new gene SCF SKP1-CUL1-F-Box SCV salmonella-containing vacuole SIM structured illumination microscopy SLBP histone RNA hairpin-binding protein SMLM single-molecule localization microscopy Smurf2 E3 ubiquitin-protein ligase SMAD ubiquitination regulatory factor 2 SQSTM1/p62 sequestosome-1 SRM super-resolution microscopy STED stimulated emission depletion SUMO small ubiquitin-like modifier TAB 2 TGF-beta-activated kinase 1 and MAP3K7-binding protein 2 TNFR1 tumour necrosis factor receptor superfamily member 1A TOM20 mitochondrial import receptor subunit TOM20 homolog TUBE tandem ubiquitin-binding entity UBact ubiquitin bacterial UBAIT ubiquitin-activated interaction trap UBAN ubiquitin-binding in ABIN and NEMO UbDha Ub-dehydroalanine UBD ubiquitin-binding domain UBE1 ubiquitin-activating enzyme 1 UBE2J2 ubiquitin-conjugating enzyme J2 UblA-MS ubiquitin interactor affinity enrichment-mass spectrometry UBL ubiquitin-like Ub-ProT ubiquitin chain protection from trypsinization Ub ubiquitin UCHL3 ubiquitin carboxyl-terminal hydrolase isozyme L3 UiFC ubiquitination-induced fluorescence complementation UIM ubiquitin-interacting motif UPS ubiquitin proteasome system USP30 ubiquitin carboxyl-terminal hydrolase 30 VHH single-domain antibody fragments VPS27 vacuolar protein sorting-associated protein 27 xE1, xE2 and xE3 engineered E1, E2 and E3 OUT enzymes YFP yellow fluorescent protein Ubiquitination Major parts of eukaryotic proteomes are controlled and regulated by post-translational modifications and among the most prominent is the covalent modification with the strictly conserved protein ubiquitin (Ub) (ubiquitination or ubiquitylation). Originally identified as a trigger for protein degradation by the 26S proteasome, ubiquitination serves many more proteasome-independent functions, including signal transduction and selective autophagy 1-3. Ubiquitination thus influences and controls the majority of cellular processes and is implicated in a wide variety of pathophysiological states and diseases, ranging from cancer to infections and hereditary disorders 4. Ubiquitin is a small, 76-residue regulatory protein that is universally expressed in eukaryotic organisms 2, 3. Ubiquitin is a member of the ubiquitin-like (UBL) protein family and shares sequence and structural homology with proteins like Small Ubiquitin-like Modifier (SUMO) 5, 6, Interferon Stimulated Gene 15 (ISG15) 7 and Neural precursor cell Expressed, Developmentally Down-regulated 8 (NEDD8) 8. Although for many years believed to be strictly expressed in eukaryotes, ubiquitin-like proteins with some similarities in structure and conjugation systems have now been identified in Mycobacterium tuberculosis (Pup: prokaryotic ubiquitin-like protein) 9 and in some Gram-negative bacteria (UBact: Ubiquitin Bacterial) 10. Ubiquitination is mediated by the sequential action of an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin protein ligase (E3) (Fig 1A and B) 3, 11-14. The substrate can be modified with a single ubiquitin (mono-ubiquitination) or with polymeric Ub chains. Depending on which internal lysine (K6, K11, K27, K29, K33, K48, K63) or whether the N-terminal methionine residue (M1, linear or head-to-tail chains) of Ub is used for linkage to the distal Ub different chain types can be generated (Fig 1C and D; Box 1) 3, 15, 16. To add complexity, the differential use of Ub lysine residues can generate homotypic chains (linked through one type of residues) or heterotypic or branched chains, such as K63-linear and K48-K11 hybrid polymers, respectively 17, 18. Importantly, the type of ubiquitin signal determines the biological effects of these modifications; for example, K48 and heterotypic K11/K48 chains generally target substrates for degradation by the 26S proteasome. In contrast, chains linked through other residues, like K6, K27, K33, K63 and linear ubiquitin chains, are often involved in non-degradative purposes, like selective autophagy, DNA damage repair and innate immunity 3. This information is decoded by proteins containing ubiquitin-binding domains (UBDs) that recognize chain-specific residues exposed on proximal and distal ubiquitin molecules and within the linker regions connecting two ubiquitin molecules (Fig 1B) 19-22. Deubiquitinating enzymes (DUBs) counterbalance chain-growing capacities by removing ubiquitin modifications (Fig 1B) 23, 24. The concerted interplay of chain/linkage formation, recognition by UBDs and Ub hydrolysis creates dynamic networks that control the distribution of different ubiquitin signals, which in turn regulate a plethora of biological processes within the cell. Figure 1. The complexity of ubiquitin conjugation(A) Schematic representation of the abundance and interactions of human ubiquitin-activating enzyme (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin protein ligases (E3s) involved in ubiquitination. (B) E3 ubiquitin-protein ligases (like for example RING E3s) recruit ubiquitin-loaded E2 enzymes and substrates and mediate the formation of ubiquitin chains. These chains can be recognized by ubiquitin-binding domain (UBD) proteins and/or degraded by deubiquitinating enzymes in a chain-selective manner. (C) The repertoire of ubiquitin chains, linked through methionine (M) 1 (linear/head-to-tail) or through the internal lysine (K) residues 6, 11, 27, 29, 33, 48 and 63 with a short description of their cellular function. (D) Overview of several modes of substrate ubiquitination including different forms of mono- and polyubiquitination and the post-translational modification of ubiquitin itself by acetylation (Ac) and phosphorylation (P). Download figure Download PowerPoint Box 1: Ubiquitin mutants and derivatives for microscopic analysis of cellular ubiquitination Schematic representation of the ubiquitin molecule. (A) Depicted are the N- and C-termini, the initiator methionine (M1) for linear ubiquitination, the seven internal lysine residues and the C-terminal glycine-76. (B) Two exemplary ubiquitin-green fluorescent protein (GFP) fusion protein reporters, used to image ubiquitin/proteasome-dependent proteolysis and the degradative functions of ubiquitin. DUB-mediated cleavage of ubiquitin-(R)-GFP or ubiquitin-(L)-GFP give rise to GFP molecules with arginine or leucine at the N-terminus that determine the half-lives of the GFP molecules by the N-end rule pathway (upper). The deubiquitinating enzyme (DUB)-resistant ubiquitin G76V mutant becomes modified with ubiquitin chains that mediate subsequent proteasomal degradation of the reporter, leading to a decrease in GFP signals (lower). (C) Ubiquitin derivatives with dehydroalanine (Dha) at position 76 can be used as cascade probes to investigate the cellular paths of ubiquitin, including the E1, E2 and HECT E3 ligase (see text for info). Methods to study ubiquitination Ever since its discovery, biochemistry- and imaging-based approaches to study ubiquitination are continuously evolving, improving and adapting. In recent years, ubiquitin biochemistry has profited from the identification of novel key enzymes, new heterotypic/branched chain types and novel pathways relying on ubiquitin. Imaging-based approaches are now more and more complementing biochemical methods due to novel developments and applications in reagents to visualize ubiquitin chains with confocal and super-resolution microscopy. Biochemical approaches to study ubiquitination Ubiquitination is classically studied by resolving ubiquitin chains and/or ubiquitinated substrates on Western blot, and biochemical experiments are the method of choice for substrate identification. Antibody-/affinity reagent-based substrate/chain enrichment allows mass spectrometry to further discover novel aspects of ubiquitination. Since these methods have been extensively reviewed elsewhere (see, for example, the reviews of 25 and 26), here we only want to highlight some of the most useful tools that were developed in recent years. In particular, the antibody-based enrichment of Ub Gly-Gly-Lysine substrate peptides upon trypsinization ((diGly) ubiquitin remnant proteomics) has enabled powerful and versatile substrate identification in complex biological specimens 26. Furthermore, the development of tandem ubiquitin-binding entities (TUBEs), in which one or multiple UBDs are fused, has proven to be powerful for chain enrichment and substrate identification 27-29. Since then, application-specific TUBE adjustments have been introduced, such as ubiquitin chain protection from trypsinization (Ub-ProT) to determine ubiquitin chain length 30 and sensor-based chain-specific TUBEs, like the linear ubiquitin-specific M1-specific ubiquitin binder (SUB) 31. Interestingly, TUBE-like chain-binding sensors are used in cellular imaging-based experiments as well (discussed in more detail later). Genetic trapping approaches like ubiquitin ligase trapping 32, 33 and ubiquitin-activated interaction traps (UBAITs) further facilitated substrate identification 34. Additionally, orthogonal Ub transfer (OUT) relies on the expression of engineered E1, E2 and E3 enzymes (xE1, xE2 and xE3) that possess reactivity towards an affinity tagged ubiquitin mutant (xUb), but not to endogenous Ub, leading to the identification of selective substrates 35, 36. The applicability of this elegant approach has been demonstrated successfully by the identification of novel E6AP and CHIP E3 ligase substrates 37, 38. Although these approaches have not been adapted to microscopy-based settings yet, it should theoretically be possible to image substrate ubiquitination with known, tagged E3 ligase and substrate pairs and labelled ubiquitin. Finally, chemical biology, combined with structural information, has yielded a wealth of activity-based probes that can be used to manipulate and study key enzymes in ubiquitin research (see for an overview for example 39-41). Chemical and semi-chemical probes have been developed that target E1, E2 and E3 enzymes and E3-substrate interactions 42-44, DUBs 45-48, UBDs 49 or can be applied for the induction of protein degradation, such as proteolysis-targeting chimeras (PROTACs) 50-54. In conclusion, biochemistry-based methods are ideally suited for substrate identification, the verification of chain specificity, the differentiation between mono- and polyubiquitination and can be applied on a wide range of biological materials, ranging from in vitro ubiquitination reactions, cellular lysates to whole tissues and organisms. However, biochemical measurements often occur post-lysis and can potentially increase the incidence of artefacts. Moreover, protein interactions might be too weak to be detected by immunoprecipitation and Western blotting. Furthermore, restriction of Ub reactions to specific cellular compartments or subsets of targets often require cell fractionation to enrich specific substrates or chain types. Scaling-up to high-throughput or high-content settings is also difficult to achieve and provides limited spatial-temporal resolution (Table 1). Table 1. Comparative advantages and disadvantages of biochemistry- and imaging-based approaches to study cellular aspects of ubiquitination Advantages Disadvantages Biochemistry-based Method of choice for substrate identification Chain-specific Differentiation between mono- and polyubiquitination Qualitative/quantitative applications Applicable on tissues and intact organisms Cell lysis required Cell fractionation often required Quantitative (near) single-molecule experiments are difficult High-throughput/high-content analysis difficult Often time-consuming Limited spatial resolution Limited temporal resolution Often specialized (and expensive) measurement/analysis set-ups needed Imaging-based Real-time live-cell imaging possible Chain-specific Native environment No cell fractionation required, use of organelle markers Quantitative single-molecule imaging possible Qualitative/quantitative applications High-throughput/high-content analysis possible Applicable on tissues and intact organisms High spatial resolution High temporal resolution Not well suitable for substrate identification Potential fixation and permeabilization artefacts Limited differentiation between mono- and polyubiquitination Often specialized (and expensive) imaging set-ups needed Strategies to monitor ubiquitination in mammalian cells using microscopy Complementing the abovementioned approaches, recent developments in optical microscopy have opened the door to image, visualize and trace ubiquitin-related processes directly in native and live-cell settings. In particular, the development of microscopy techniques that achieve a spatial resolution approaching the size of single proteins allows functional studies on how proteins organize and interact at the molecular level in their physiological environment. In the following paragraphs, we highlight three important strategies to image cellular ubiquitination and discuss specific advantages and disadvantages. The first approach utilizes fluorescently labelled reporter and model substrates to indirectly image the degradative functions of ubiquitination and the UPS. The second approach applies tagged ubiquitin reagents, chain-specific antibodies and chain-specific sensors to directly image ubiquitination in cellular compartments and biological processes. Finally, the application of super-resolution microscopy allows to image ubiquitin signals in mammalian cells with unprecedented spatial resolution. Indirect imaging of ubiquitination in protein degradation The role of ubiquitination in proteasomal degradation has been extensively studied by imaging the stability and degradation of artificial reporter proteins and physiological model substrates. Certain ubiquitin signals like ubiquitin chains linked through K11 and K48 serve as recognition signals for degradation by the 26S proteasome 2, 17, 55. Fluorescently labelled, degradation-sensitive reporters have been developed, often based on green fluorescent protein (GFP) or derivatives, which are stabilized or degraded when expressed in isolated cells or intact organisms (Fig 2A) 56, 57. Monitoring changes in fluorescence intensity serves as indirect read-out for ubiquitination and proteasome function and has for example facilitated the development and evaluation of proteasome inhibiting compounds 56, 57. Figure 2. Indirect imaging of the degradative functions of ubiquitinApproaches to image the proteasome-related functions of Ub in the control of protein stability and breakdown. Darker and lighter green colours indicate the accumulation and breakdown of proteins, respectively. (A) GFP-labelled Ub or model substrates are modified with degradative Ub signals and degraded by the 26S proteasome (blue barrel). Proteasome inhibition induces stabilization and accumulation of these GFP reporters as ubiquitinated forms. (B) Upon IKK activation, GFP-tagged IκBα becomes modified with K48-linked polyubiquitin chains and degraded by the 26S proteasome. This releases mCherry-RelA/p65 that subsequently translocates in the nucleus to control NF-κB-dependent gene expression. IĸBα is among these NF-κB target genes and shuttles back into the cytosol, creating dynamic NF-κB degradation/translocation loops. Grey ovals: additional NF-κB transcription factors (C) FUCCI: co-expression of the UPS substrates GFP-Geminin and RFP-Cdt1 allows microscopic analysis of cell cycle phases by phase-dependent Ub-dependent degradation of GFP-Geminin and RFP-Cdt1. Download figure Download PowerPoint One of the first GFP-based UPS reporter (GFPu) was generated by fusing GFP to the 16-residue CL1 degron that becomes degraded by the 26S proteasome in an ubiquitin-dependent manner 58, 59. This GFP-based reporter has been applied to indirectly study UPS function in isolated cells and intact organisms and can be used to investigate protein degradation in specific cellular compartments, like cytosol and the nucleus 60. Another reporter that probes the activity of DUBs consists of a direct fusion of ubiquitin to GFP. DUB-dependent cleavage of the ubiquitin molecule generates free GFP with different N-terminal residues that serve as indicators for N-end rule pathways degradation since the N-terminal residue determines the protein half-live 61, 62. In addition, the non-cleavable UbG76V-GFP and –Dendra2 fusion reporters become modified with polyubiquitin chains that subsequently target the complete fusion protein for 26S proteasomal degradation (Fig 2A) and (Box 1) 61, 63, 64. These reporters have been applied in isolated cells and transgenic UbG76V-GFP reporter mice to study proteasome activity 64. The above listed reporters either employ artificial substrates or indirectly monitor DUB and proteasome activity. An alternative approach is the direct fluorescent labelling of the physiological ubiquitin substrate itself. This strategy has been applied to NF-κB signalling (Fig 2B). Under basal conditions, the transcription factor p65/RelA is retained in the cytoplasm by its interaction with IκBα. Pathway activation induces IKK-dependent IκBα phosphorylation and its ubiquitin-dependent degradation leading to the liberation and nuclear translocation of p65 65. The co-imaging of the nucleo-cytoplasmic shuttling of GFP-labelled p65 with the oscillatory accumulation/degradation of mCherry-tagged IĸBα protein has allowed to draw conclusions on NF-κB kinetics, dynamics and oscillations 66-68. Another prominent example is the cell cycle indicator FUCCI (fluorescence ubiquitination cell cycle indicator). Here, two proteins known to be degraded in specific phases of the cell cycle are tagged with two different fluorescent reporters: RFP-Cdt1 and GFP-Geminin (Fig 2C) 69. During S, G2 and M phases of the cell cycle, RFP-Cdt is degraded by the UPS, giving rise to GFP-positive nuclei, whereas GFP-tagged geminin is degraded in G1 with the red signal remaining. The G1/S transition phase shows yellow fluorescent nuclei, due to the green and red overlay, since Cdt1 levels are decreasing and geminin levels increase 69. A variant of FUCCI, called FUCCI4, combines four different fluorescent substrates (mKO2-Cdt (30–120), mTurquoise2-SLBP (18–126), Clover-Geminin (1–110) and H1.0-Maroon) to allow visualization of each cell cycle phase 70. In addition, Fly-FUCCI has been developed to monitor proliferation in tissues, based on the FUCCI principle 71. The FUCCI principle allows thus a dynamic, indirect imaging of the degradative functions of ubiquitination during cell cycle progression and division. An interesting development is the application of nanobodies. These are single-chain VHH antibody regions specifically designed to recognize a specific epitope. These small, monomeric and stable reagents, derived from immunized Camelidae sp., can be labelled with fluorophores and expressed in cells 72, 73. Expression of these so-called chromobodies can be achieved by conventional transient expression methods or through stable integration using viral transduction in a constitutive or inducible manner, depending on experimental constraints. Chromobodies against a wide variety of endogenous epitopes 72, 73 including GFP are available. GFP nanobodies can have GFP-quenching or GFP-stimulating properties 74 and can be genetically tagged to be used as biochemical matrices to enrich GFP-tagged proteins 74. In an elegant approach, GFP nanobodies have been employed to achieve the selective (ubiquitin-dependent) degradation of any GFP-tagged protein of interest 75. The authors fused the Drosophila F-box protein Slmb to GFP nanobodies (NSlmb-vhhGFP4). Since F-box proteins are the substrate specifying determinants of large multimeric E3 ligase complexes, called SKP1-CUL1-F-Box (SCF) complexes 76, recognition of GFP-tagged proteins by NSlmb-vhhGFP4 induces their degradation by the SCF complex. The authors demonstrated the applicability of deGradFP (degrade green fluorescent protein) in isolated cells and intact organisms 75. It would be interesting to apply this GFP knockout technique on the degradation of GFP-tagged ubiquitin chain-specific sensors proteins, like GFP-UBAN, that selectively bind linear Ub chains. Theoretically, one would expect proteasomal degradation of the GFP chain sensors and perhaps of the endogenous linear Ub chains as well. This would imply novel modes of manipulation of ubiquitin signalling that can easily be combined with imaging-based experiments. In conclusion, several reporters, methods and reagents have been developed and applied that are useful for studying Ub and protein breakdown by the 26S proteasome in imaging-based set-ups. Although these techniques do not allow direct imaging of the degradative Ub signals, the focus is on imaging the functional consequences of these signals on reporter stability. For this reason, this approach is only suitable for imaging degradative functions of Ub in well-characterized biological scenarios that rely on known and well-studied substrates, E3 ligases and molecular mechanisms. These reporter-based read-outs are less suitable for monitoring proteasome-independent functions of ubiquitin and do not answer questions about the specific type of ubiquitin modification. Direct imaging of ubiquitination in fixed and living cells Microscopy-based imaging of ubiquitination in cells is mostly focused on the visualization of local accumulations of different ubiquitin signals. In contrast to diffuse ubiquitination reactions, that might take place freely in certain organelles, accumulated ubiquitinated structures, like aggregates, foci or puncta, provide assemblies that can be imaged easily. These structures are in most cases ensembles of mixed types of ubiquitin chains, likely combined with (multiple) mono-ubiquitination and/or branched chains. Two main approaches are currently in use to image ubiquitin, a direct one in which genetically labelled ubiquitin is used and an indirect one using reagents that recognize certain types of ubiquitin signals (Table 2). Table 2. Useful reagents for monitoring cellular ubiquitination using