A Genetically Encoded Toolkit Of Functionalized Nanobodies .
Prole and Taylor BMC Biology(2019) ODOLOGY ARTICLEOpen AccessA genetically encoded toolkit offunctionalized nanobodies againstfluorescent proteins for visualizing andmanipulating intracellular signallingDavid L. Prole* and Colin W. Taylor*AbstractBackground: Intrabodies enable targeting of proteins in live cells, but generating specific intrabodies against thethousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescentlylabelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodiestargeting fluorescent protein tags.Results: We generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFPand GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2 , H andATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteinsand identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescentdyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartmentsof the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody,and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We showvarious applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2 to the outer membraneof only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts withthe plasma membrane.Conclusions: This toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studyingand manipulating cell signalling in live cells.Keywords: Cell signalling, Endoplasmic reticulum, Fluorescence microscopy, Fluorescent protein, GFP, Intrabody,Membrane contact site, Mitochondria, Nanobody, RFPBackgroundVisualizing the location of specific proteins within cellsand manipulating their function is crucial for understanding cell biology. Antibodies can define protein locations in fixed and permeabilized cells, but antibodies arelarge protein complexes that are difficult to introduceinto live cells [1]. This limits their ability to interrogatethe dynamics or affect the function of proteins in livecells. Small protein-based binders, including nanobodiesderived from the variable region of the heavy chains* Correspondence: dp350@cam.ac.uk; cwt1000@cam.ac.ukDepartment of Pharmacology, University of Cambridge, Tennis Court Road,Cambridge CB2 1PD, UK(VHH) of camelid antibodies, offer a promising alternative [2]. Nanobodies can be encoded by plasmids andexpressed in live cells. However, generating nanobodiesagainst thousands of protein variants is daunting, andeven for single targets, it can be time-consuming, costlyand not always successful. A solution to this bottleneckis provided by fluorescently tagged proteins, which arewidely used in cell biology [3, 4] after heterologousexpression of proteins or gene editing of endogenousproteins [5–7]. The most common application of fluorescent protein (FP) tags is to visualize protein locations,but they have additional potential as generic affinity tags The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication o/1.0/) applies to the data made available in this article, unless otherwise stated.
Prole and Taylor BMC Biology(2019) 17:41to manipulate and visualize protein functions in livecells. These opportunities are under-developed.Green fluorescent protein (GFP) has undergone numerous cycles of optimization as a reporter and nonperturbing tag [3, 8]. Most GFP-tagged proteins therefore retain their endogenous localization and function [9].Large libraries of plasmids encoding GFP-tagged proteinsare now available [10]. Proteome-scale expression ofGFP-tagged proteins or genome-scale tagging of geneproducts with GFP has been reported for Drosophila [11],fungi [12–14], plants [15, 16] and bacteria [17].Proteins tagged with red fluorescent proteins (RFPs)such as DsRed, mRFP and mCherry (mCh) are also popular. Extensive optimization has made them attractive tags[3, 18], and libraries of RFP-tagged proteins have beendeveloped in mouse stem cells [19] and yeast [14].Nanobodies that bind to RFP [20, 21] or GFP [21, 22] aremost commonly used in their purified forms for immunoprecipitation and immunocytochemistry. However, they alsooffer a generic means of targeting in live cells the hugevariety of available tagged proteins and the many emergingexamples of endogenous proteins tagged with FPs by geneediting. GFP-targeting nanobodies have been used for applications such as targeted proteasomal degradation [23, 24]and relocation of proteins in cells [25], but these and otherapplications are less developed for RFP-targeting nanobodies.Here we develop a plasmid-encoded toolkit of nanobodies that bind common FP tags, including RFPs,CFP, GFP and YFP, fused to functional modules forvisualization and manipulation of cell signalling (Fig. 1).We fused the nanobodies to a variety of functionalmodules: fluorescent sensors for Ca2 , H and ATP/ADP; optimized SNAP tags for labelling with bright andphotostable dyes [26]; and hetero-dimerizing partnersthat allow inducible recruitment or sequestration of proteins and visualization of membrane contact sites (MCS)between organelles. We developed two methods to allowcrosslinking of RFP-tagged and GFP-tagged proteins: adimeric nanobody, and co-expression of RFP-targetingand GFP-targeting nanobodies fused to complementaryhetero-dimerizing domains. We also describe functionalized nanobodies directed to lumenal sub-compartmentsof the secretory pathway. We demonstrate the utility ofnanobody fusions by visualizing local Ca2 dynamics atthe surface of mitochondria, by manipulating the locationsof proteins and organelles within cells, by characterizingMCS between mitochondria and the plasma membrane(PM) and by targeting lumenal Ca2 sensors to asub-compartment of the endoplasmic reticulum (ER).This versatile toolkit of genetically encoded, functionalized nanobodies greatly expands the utility of RFP- andGFP-targeting nanobodies. It will provide a valuableresource for studying protein function and cell signalling inlive cells. We illustrate some applications and demonstrate,Page 2 of 24for example, that IP3 receptors deliver Ca2 to the outermembrane of only some mitochondria and that MCSbetween mitochondria and the plasma membrane occur atonly one or two sites on each mitochondrion.ResultsTargeting RFP and GFP variants with genetically encodednanobody fusions in live cellsThe RFP nanobody (RNb) and GFP nanobody (GNb)used are the previously described llama variants LaM4and LaG16, respectively [21]. They were chosen for theirfavourable combinations of high affinity (Kd values of0.18 nM and 0.69 nM, respectively) and the ability tobind a variety of RFP or GFP variants [21]. The latterattribute maximizes their potential for targeting a widevariety of FPs [3, 4]. LaM4 binds both mCh and DsRedvariants, but not GFPs [21]. In addition to binding GFP,LaG16 binds cyan, blue and yellow FPs (CFP, BFP andYFP), but not RFPs [21]. In contrast, the widely usedVhhGFP4 nanobody binds GFP, but not CFP [22].In HeLa cells with organelles (ER, mitochondria, nucleusand lysosomes) labelled with mCh or mRFP markers,expression of RNb-GFP (Fig. 2a) specifically identified thelabelled organelle (Fig. 2b). Similar results were obtainedwith GNb-mCh (Fig. 2c) and organelles (ER, mitochondriaand nucleus) labelled with GFP or mTurquoise (Fig. 2d).These results demonstrate that plasmid-encoded RNb andGNb allow specific labelling of a variety of RFP and GFPvariants in live cells.Targeting sensors to RFP and GFPThe effects of intracellular messengers such as Ca2 [27],H [28] and ATP/ADP [29] can be highly localized withincells. To enable visualization of these intracellular messengers in microdomains around RFP-tagged and GFP-taggedproteins, we fused RNb and GNb to fluorescent sensorsfor Ca2 [30], H [31, 32] or ATP/ADP [33].RNb was fused to the green fluorescent Ca2 sensorG-GECO1.2 (Fig. 3), and GNb was fused to the redfluorescent Ca2 sensors, R-GECO1.2 or LAR-GECO1.2[30] (Fig. 4). The affinities of these sensors for Ca2 (KCaDof 1.2 μM for G-GECO1.2 and R-GECO1.2, and 10 μM forLAR-GECO1.2) are low relative to global changes in thecytosolic free Ca2 concentration ([Ca2 ]c) after receptorstimulation (typically 300 nM) [34]. This facilitatesselective detection of the large, local rises in [Ca2 ] thatare important for intracellular signalling, at the contactsbetween active inositol 1,4,5-trisphosphate receptors(IP3Rs) and mitochondria, for example [27]. To allowtargeted measurement of relatively low resting [Ca2 ] withincellular microdomains, we also fused RNb to the ratiometricCa2 -sensor, GEMGECO1 ðKCaD 300 nM) [30], to giveRNb-GEMGECO1 (Additional file 1: Figure S1).
Prole and Taylor BMC Biology(2019) 17:41Page 3 of 24Fig. 1 Nanobody fusions for visualizing and manipulating intracellular signalling. Plasmids were generated that encode nanobodies specific forGFP variants (GNb) or RFP variants (RNb), fused to functional modules. Nanobody fusions with an N-terminal signal sequence to target them tothe secretory pathway are also shown (ssGNb and ssRNb)In HeLa cells expressing TOM20-mCh or TOM20GFP to identify the outer mitochondrial membrane(OMM), the RNb-Ca2 sensors (Fig. 3 and Additional file 1:Figure S1) and GNb-Ca2 sensors (Fig. 4) were targeted tothe OMM. Both families of targeted sensor reported an increase in [Ca2 ] after treatment with the Ca2 ionophore,ionomycin (Figs. 3 and 4 and Additional file 1: Figure S1).This confirms the ability of the sensors to report [Ca2 ]changes when targeted to the OMM microdomain.In some cells, the targeted Nb-Ca2 sensors revealedlocal changes in [Ca2 ]c after receptor stimulation withhistamine, which stimulates IP3 formation and Ca2
Prole and Taylor BMC Biology(2019) 17:41Page 4 of 24ACBDFig. 2 RNb and GNb fusion proteins bind to their respective tagged proteins in live cells. a Schematic of the RNb-GFP fusion binding to RFP. b HeLacells expressing RNb-GFP with RFP-tagged markers for the ER surface (mCh-Sec61β), the mitochondrial surface (TOM20-mCh), the nucleus (H2B-mCh),or the surface of lysosomes (TPC2-mRFP). Cells were imaged in HBS using epifluorescence microscopy (cells expressing H2B-mCh) or TIRFM(other cells). Yellow boxes indicate regions enlarged in the subsequent panels. Colocalization values (Pearson’s coefficient, r) were mCh-Sec61β (r 0.93 0.09, n 10 cells), TOM20-mCh (r 0.94 0.09, n 10 cells), H2B-mCh (r 0.97 0.06, n 10 cells), and TPC2-mRFP (r 0.78 0.09, n 5 cells). c Schematicof the GNb-mCh fusion binding to GFP. d HeLa cells co-expressing GNb-mCh with GFP-tagged markers for the ER surface (GFP-ERcyt), the mitochondrialsurface (TOM20-GFP), and the nucleus (H2B-GFP), or an mTurquoise2-tagged ER surface marker (mTurq-ERcyt). Cells were imaged using epifluorescencemicroscopy (cells expressing H2B-GFP) or TIRFM (other cells). Yellow boxes indicate regions enlarged in the subsequent panels. Colocalization values wereGFP-ERcyt (r 0.92 0.08, n 8 cells), TOM20-GFP (r 0.87 0.05, n 7 cells), H2B-GFP (r 0.94 0.07, n 6 cells), and mTurq-ERcyt (r 0.97 0.03, n 7cells). Scale bars 10 μm (main images) or 2.5 μm (enlargements)release from the ER in HeLa cells [34]. Imperfect targeting of the RNb-GGECO1.2 to the OMM allowed Ca2 signals at the surface of individual mitochondria to bedistinguished from those in nearby cytosol in some cells(Fig. 3d–f and Additional file 2: Video 1). In the exampleshown, RNb-GGECO1.2 at both the OMM and nearbycytosol responded to the large, global increases in [Ca2 ]evoked by ionomycin. However, cytosolic RNb-GGECO1.2did not respond to histamine, while the sensor at theOMM responded with repetitive spiking (Fig. 3d–f andAdditional file 2: Video 1). The GNb-LARGECO1.2 sensor,which has the lowest affinity for Ca2 of the sensors used,revealed changes in [Ca2 ]c at the surface of some mitochondria, but not others in the same cell (Fig. 4d–f, Fig. 4hand Additional file 3: Video 2). In the example shown,GNb-LARGECO1.2 at the OMM in all mitochondriawithin the cell responded to the large, global increases in[Ca2 ] evoked by ionomycin. However, in response to histamine, mitochondria in the perinuclear region responded,but not those in peripheral regions (Fig. 4d–f, Fig. 4h andAdditional file 3: Video 2). Ca2 uptake by mitochondriaaffects many cellular responses, including mitochondrialmetabolism, ATP production and apoptosis [35]; andCa2 at the cytosolic face of the OMM regulatesmitochondrial motility [36]. The subcellular heterogeneityof mitochondrial exposure to increased [Ca2 ] suggeststhat these responses may be very localized in cells.These observations align with previous reports showingthat Ca2 -mobilizing receptors evoke both oscillatory [Ca2 ]changes within the mitochondrial matrix [37], and largelocal increases in [Ca2 ] at the cytosolic face of the OMM[38]. Our results establish that nanobody-Ca2 -sensorfusions are functional and appropriately targeted and can beused to detect physiological changes in [Ca2 ] within cellularmicrodomains such as the OMM.For targeted measurements of intracellular pH, RNbwas fused to the green fluorescent pH sensor super-ecliptic pHluorin (SEpHluorin) [31], and GNb was fused to thered fluorescent pH sensor pHuji [32]. Both Nb-pH sensorswere targeted to the OMM by the appropriate fluorescenttags, where they responded to changes in intracellular pHimposed by altering extracellular pH in the presence ofthe H /K ionophore nigericin (Fig. 5).For targeted measurements of ATP/ADP, RNb wasfused to the excitation-ratiometric ATP/ADP sensorPerceval-HR [33]. RNb-Perceval-HR was targeted to thesurface of mitochondria and responded to inhibition ofglycolysis and oxidative phosphorylation (Fig. 6).The results demonstrate that nanobodies can beused to direct sensors for Ca2 , H or ATP/ADP to
Prole and Taylor BMC Biology(2019) 17:41Page 5 of 24ABCDEFFig. 3 Targeting RNb-Ca2 sensors to RFP-tagged proteins. a Schematic of RNb-GGECO fusion binding to RFP. b–d HeLa cells expressing RNbGGECO1.2 and TOM20-mCh, before and after addition of histamine (100 μM) and then ionomycin (5 μM). Cells were imaged in HBS using TIRFM.The TOM20-mCh image is shown after the histamine and ionomycin additions. The merged images are shown using images of RNb-GGECO1.2after ionomycin (b, c) or histamine (d). The yellow and cyan-boxed regions in panel b are shown enlarged in panels c and d, respectively. Scalebars are 10 μm (b) or 1.25 μm (c, d). e Timecourse of the effects of histamine (100 μM) and ionomycin (5 μM) on the fluorescence of RNb-GGECO1.2 (F/F0,where F and F0 are fluorescence recorded at t and t 0). The traces are from regions coinciding with a single mitochondrion or cytosol (regions identifiedin panel d), indicating changes in [Ca2 ] at the OMM. f Enlarged region (70–180 s) of the graph is shown in e. Results are representative of cells from 13independent experimentsspecific subcellular compartments tagged with variantsof RFP or GFP.Targeting SNAPf tags to RFP and GFP in live cellsSNAP, and related tags, are versatile because a range ofSNAP substrates, including some that are membranepermeant, can be used to attach different fluorophores orcargoes to the tag [39]. Purified GFP-targeting nanobodiesfused to a SNAP-tag have been used to label fixed cells foroptically demanding applications [40]. We extended thisstrategy to live cells using RNb and GNb fused to theoptimized SNAPf tag [41] (Fig. 7a, b). In cells expressingthe mitochondrial marker TOM20-mCh, RNb-SNAPfenabled labelling of mitochondria with the cell-permeablesubstrate SNAP-Cell 647-SiR and imaging at far-redwavelengths (Fig. 7c). In cells expressing lysosomalLAMP1-mCh and RNb-SNAPf, SNAP-Cell 647-SiRinstead labelled lysosomes (Fig. 7d), demonstrating thatSNAP-Cell 647-SiR specifically labelled the organellestargeted by RNb-SNAPf. Similar targeting of SNAP-Cell647-SiR to mitochondria (Fig. 7e) and lysosomes(Fig. 7f ) was achieved by GNb-SNAPf co-expressedwith the appropriate GFP-tagged organelle markers.Chromophore-assisted light inactivation (CALI) caninactivate proteins or organelles by exciting fluorophoresattached to them that locally generate damaging reactivesuperoxide. Historically, antibodies were used to direct aphotosensitizer to its target, but the fusion of fluorescentproteins or SNAP-tags to proteins of interest is nowwidely used [42]. RNb-SNAPf and GNb-SNAPf make
Prole and Taylor BMC Biology(2019) 17:41Page 6 of 24ABCDEFGHFig. 4 Targeted GNb-Ca2 sensors detect changes in [Ca2 ] at the surface of mitochondria. a Schematic of GNb-RGECO fusion binding to GFP.b, c Representative HeLa cells co-expressing TOM20-GFP and GNb-RGECO1.2 imaged in HBS using TIRFM before and after addition of histamine(100 μM) and then ionomycin (5 μM). The TOM20-GFP images are shown after the histamine and ionomycin additions. Histamine and ionomycin evokedchanges in fluorescence of GNb-RGECO1.2 at the OMM. The yellow boxed region in panel B is shown enlarged in panel c. d–f Similar analyses of HeLacells co-expressing TOM20-GFP and GNb-LAR-GECO1.2 (GNb-LARG1.2). Histamine (100 μM) evoked changes in fluorescence of GNb-LARG1.2 at the OMMof mitochondria in the perinuclear region (region of interest 1 (ROI 1) in e), but not in a peripheral region (ROI 2 in f). All mitochondria responded toionomycin (5 μM), indicating that histamine evoked local changes in [Ca2 ] at the OMM. The cyan and yellow boxed regions in d are shown enlarged ine and f, respectively. Scale bars 10 μm (b, d) or 2.5 μm (c, e and f). g Timecourse of the changes in fluorescence of GNb-RGECO1.2 at the OMMevoked by histamine and ionomycin for the entire cell shown in B. h Fluorescence changes recorded from ROI 1 and ROI 2 in panels e and f. Resultsare representative of cells from 4 independent experiments
Prole and Taylor BMC Biology(2019) 17:41APage 7 of 24BCDEFGHFig. 5 Targeting H sensors to RFP-tagged and GFP-tagged proteins. a Schematic of RNb fused to the pH sensor super-ecliptic pHluorin (RNbSEpH) and bound to RFP. b Schematic of GNb-pHuji binding to RFP. c, d HeLa cells co-expressing RNb-SEpH and TOM20-mCh were imaged inmodified HBS (MHBS) using epifluorescence microscopy and exposed to extracellular pH 6.5 (c) or pH 8 (d) in the presence of nigericin (10 μM).Scale bars 10 μm. e, f HeLa cells co-expressing GNb-pHuji and TOM20-GFP were exposed to extracellular pH 6.5 (e) or pH 8 (f) in the presence ofnigericin. Scale bars 10 μm. g, h Timecourse from single cells of the fluorescence changes (F/F0) of mitochondrially targeted RNb-SEpH or GNbpHuji evoked by the indicated manipulations of extracellular pH. Results shown are representative of 3 independent experimentsthe SNAP strategy more broadly applicable to CALIapplications. We demonstrate this by targeting CALI tothe outer surface of lysosomes. We anticipated thatCALI in this microdomain might, amongst other effects,disrupt the motility of lysosomes, which depends on theirassociation with molecular motors [43]. RNb-SNAPfenabled labelling of lysosomes with the CALI probefluorescein, using the cell-permeable substrate, SNAP-Cellfluorescein
GNb allow specific labelling of a variety of RFP and GFP variants in live cells. Targeting sensors to RFP and GFP The effects of intracellular messengers such as Ca2 [27], H [28]andATP/ADP[29] can be highly localized within cells. To enable visualization of these intracellular messen-gers in microdomains around RFP-tagged and GFP-tagged
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