Figure 6 - uploaded by Jonathan Tang
Content may be subject to copyright.
Retrofitting a Transgenic GFP Mouse Line for GFP-Dependent Manipulation of Gene Expression and Neural Circuit Activities (A) Tg(GUS8.4GFP) expresses GFP in type 7 cone bipolar and rod bipolar cell types (green fill) of the retina. Adopted schematic (Ghosh et al., 2004). (B) Cryosection of electroporated Tg(GUS8.4GFP) retina expressing Gal4-GBP2 p65-GBP7 and UAStdT. Scale bar, 20 mm. (C) Type 7 (left) and rod bipolar (right) cell types labeled by UAS-tdT. Anti-Calretinin (left) or antiCalbindin (right) staining identify specific layers of the IPL. Scale bar, 10 mm. GFP was immunostained in (B and C). (D) Schematic of ChR2 experiment. Electroporated Tg(GUS8.4-GFP) retinas expressing 103 UAS-ChR2/H134R-mCherry and 53UAS-tdT were analyzed for ChR2-mediated responses in random GCL cells. (E) Cumulative plot of ON responses in GCL cells. Number of spikes counted during the first 300 ms after stimulus onset, normalized to control (minus APB). APB blocks ON responses originating from photoreceptors. Plots are mean ± SEM (n = 4 per condition). (F) Spiking response of a GCL cell. Gray bar, duration of light stimulus. Response to normal light stimuli under control condition (top) or in the presence of APB (middle). Light stimuli focused on INL activate ChR2/H134R in the presence of APB (lower). (G and H) Top and side views of a neurobiotin-filled (green) ganglion cell identified by light stimulation of ChR2. Magenta lines indicate level of anti-Chat bands (not shown). Scale bar, 20 mm.
Source publication
Fluorescent proteins are commonly used to label cells across organisms, but the unmodified forms cannot control biological activities. Using GFP-binding proteins derived from Camelid antibodies, we co-opted GFP as a scaffold for inducing formation of biologically active complexes, developing a library of hybrid transcription factors that control ge...
Contexts in source publication
Context 1
... found that excitability and passive membrane properties were similar for the three groups of neurons (Figures 5C-5D and Table S7) and were consistent with intrinsic cellular properties previously reported for cortical neurons of this age (Oswald and Reyes, 2008). Moreover, transducing T-DDOGs did not impact mor- phological features such as dendritic spine density and length ( Figure S6 and Table S7). Thus, T-DDOGs are compatible with electrophysiological assays and do not induce functional and structural alterations in the developing brain, within the tested time frame. ...
Context 2
... almost none of the cone bipolar types can be singly isolated for genetic manipulation, but multiple GFP lines do label subsets of bipolar types (Siegert et al., 2009;Wä ssle et al., 2009). The a-gustducin-GFP trans- genic line, Tg(GUS8.4-GFP) ( Huang et al., 2003), expresses GFP in type 7 cone bipolar cells and in rod bipolar cells (Fig- ure 6A). Both cell types respond to light increments and are called ON bipolar cells. ...
Context 3
... of T-DDOGs and UAS-tdT into Tg(GUS8.4-GFP) retinas resulted in tdT induction selectively in these two cell types; identification was based on morphology and axonal stratification in the inner plexiform layer (IPL), aligned to the IPL markers Calbindin or Calretinin ( Ghosh et al., 2004) ( Figures 6A-6C). Importantly, 98.9% of tdT+ cells were positive for GFP expression (n = 91 cells, sampled from three retinas). ...
Context 4
... We asked whether light-driven ChR2 activation in GFP-labeled bipolar cells could trigger downstream spiking responses in cells of the ganglion cell layer (GCL) ( Figure 6D). Electroporated retinas were presented with two different light stimuli, and recordings were performed on GCL cells. ...
Context 5
... first stimulus had low light intensity and could evoke photoreceptor-mediated responses in GCL cells but was not bright enough to activate ChR2. We used this stimulus to select GCL cells that responded to both light incre- ments and decrements (ON/OFF cells) ( Figures 6E and 6F). We next blocked synaptic communication between photoreceptors and ON bipolar cells with 2-amino-4-phosphonobutyrate (APB) (Slaughter and Miller, 1981) and presented the retina with a brighter light stimulus that could activate ChR2. ...
Context 6
... ON/ OFF GCL cells receive excitatory input from ON bipolar cells, some of these cells should be connected via excitatory synapses (directly or indirectly) to ChR2-expressing ON bipolar cells. Indeed, the brighter stimulus elicited ON responses in some recorded GCL cells in the presence of APB (Figures 6G and 6H). In contrast, recordings made from ON and ON/OFF GCL cells in nonelectroporated regions of multiple retinas did not reveal any response after the onset of the brighter stimulus in the presence of APB (data not shown). ...
Similar publications
The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD) and modulation of risk by common variation in PD has been well established through GWAS. Anticipating that a fraction of PD-associated genetic variation mediates their effects within this neuronal population, we acquired open chromat...
Citations
... FPs not only illuminate cells and biological processes but also make excellent scaffolds due to their apparent lack of linkage to numerous host protein networks. Using GBPs and GFP as a scaffold to drive biologically active complex formation, Tang et al. created a library of hybrid transcription factors that exclusively control gene expression in the presence of GFP and its derivatives [77]. The production of GFP controls the expression of cell-specific genes ( Figure 5E) and promotes the dysfunction of the mouse retina and brain. ...
Since the discovery of fluorescent proteins (FPs), their rich fluorescence spectra and photochemical properties have promoted widespread biological research applications. FPs can be classified into green fluorescent protein (GFP) and its derivates, red fluorescent protein (RFP) and its derivates, and near-infrared FPs. With the continuous development of FPs, antibodies targeting FPs have emerged. The antibody, a class of immunoglobulin, is the main component of humoral immunity that explicitly recognizes and binds antigens. Monoclonal antibody, originating from a single B cell, has been widely applied in immunoassay, in vitro diagnostics, and drug development. The nanobody is a new type of antibody entirely composed of the variable domain of a heavy-chain antibody. Compared with conventional antibodies, these small and stable nanobodies can be expressed and functional in living cells. In addition, they can easily access grooves, seams, or hidden antigenic epitopes on the surface of the target. This review provides an overview of various FPs, the research progress of their antibodies, particularly nanobodies, and advanced applications of nanobodies targeting FPs. This review will be helpful for further research on nanobodies targeting FPs, making FPs more valuable in biological research.
... As many research and therapeutic applications of nanobody technology will require expression in vivo, we wanted to test whether improved nanobody intracellular stability established in cell lines would apply to in vivo settings. Electroporation of the retina in newborn mice is a means to deliver nanobody expression vectors to multiple cell types (Tang et al., 2013). Plasmids encoding wild-type or partial consensus mutant 3V0A-TagBFP were injected into the subretinal space of postnatal day 2 (P2) mice, along with a dsRed control plasmid, and electroporation was carried out. ...
Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli . These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.
... In this set-up, GFP acts as a scaffold that aggregates modular transcription domains and assembles a hybrid transcription complex to activate the target gene, where GFP recognition is mediated by paired anti-GFP Nbs (36). Using similar ideas, the team also developed a GFP-dependent transcription system (37), termed flippase dependent on GFP (FLP-Dog) (38), and other gene manipulation methods, providing reliable tools for photogenetics and other technologies. ...
Nanobodies are antibody fragments derived from camelids, naturally endowed with properties like low molecular weight, high affinity and low immunogenicity, which contribute to their effective use as research tools, but also as diagnostic and therapeutic agents in a wide range of diseases, including brain diseases. Also, with the success of Caplacizumab, the first approved nanobody drug which was established as a first-in-class medication to treat acquired thrombotic thrombocytopenic purpura, nanobody-based therapy has received increasing attention. In the current review, we first briefly introduce the characterization and manufacturing of nanobodies. Then, we discuss the issue of crossing of the brain-blood-barrier (BBB) by nanobodies, making use of natural methods of BBB penetration, including passive diffusion, active efflux carriers (ATP-binding cassette transporters), carrier-mediated influx via solute carriers and transcytosis (including receptor-mediated transport, and adsorptive mediated transport) as well as various physical and chemical methods or even more complicated methods such as genetic methods via viral vectors to deliver nanobodies to the brain. Next, we give an extensive overview of research, diagnostic and therapeutic applications of nanobodies in brain-related diseases, with emphasis on Alzheimer’s disease, Parkinson’s disease, and brain tumors. Thanks to the advance of nanobody engineering and modification technologies, nanobodies can be linked to toxins or conjugated with radionuclides, photosensitizers and nanoparticles, according to different requirements. Finally, we provide several perspectives that may facilitate future studies and whereby the versatile nanobodies offer promising perspectives for advancing our knowledge about brain disorders, as well as hopefully yielding diagnostic and therapeutic solutions.
... Specific recognition of target proteins by nanobodies can be utilized for not only visualization of target proteins [22][23][24] but also protein degradation 25,26 , signal inhibition 27 and property manipulation 28 . Recently, GFP-dependent gene regulation methods using GFP-specific nanobodies have been reported 29,30 . These techniques enable selective gene expression in GFP-expressing cells by utilizing nanobodies as specific binding modules to recognize GFPs. ...
Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (Esr2)-mRFP1 mice and gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.
... This plasmid encodes a fusion protein that has a 107-amino acid N-terminal GBP fused to a 355-amino acid C-terminal Lac I domain separated by a 7-amino acid linker. The experimental approach for using the LacI-GFP nanobody (GBP) to recruit GFP-tagged proteins to the LacO locus is well characterized (Herce et al, 2013;Tang et al, 2013). ...
The BTB domain is an oligomerization domain found in over 300 proteins encoded in the human genome. In the family of BTB domain and zinc finger–containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site, whereas the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study, we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed cell-based binding assays with fluorescent protein–BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo- and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family.
... Among antibody fragments that can be used inside the cell, nanobodies have emerged as promising molecular tools (5,6). For instance, nanobodies from immunized animals or from synthetic scaffolds were efficiently engineered to specifically recognise not only an ectopic protein such as the Green Fluorescent Protein (GFP) (7,8), or several linear epitope tags (9,10), but also endogenous proteins (11,12). In this regard, intracellular antibodies (also called intrabodies) present the advantage to disrupt endogenous protein functions either by functionalization through targeted protein degradation (13), ERrerouting (14), or by competing with endogenous protein partners, thus offering an alternative strategy to small molecule inhibitors. ...
Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.
... [22] Their small recognition epitopes even facilitate tools wherein two nanobodies that bind different regions of GFP can control transcription by using GFP as a bridge to bring together split proteins, such as split transcription factors and split Cre recombinase. [23,24] Nanobodies have been employed as tools in a wide range of model systems, including mammalian cells, mice, zebrafish, and Caenorhabditis elegans. [2] A limitation of native nanobodies is that their binding cannot be modulated after expression. ...
... We set out to photocage nanobodies for use in C. elegans, initially focussing on a well-established anti-GFP nanobody, the "enhancer" nanobody (eNB), [39] as it is utilised for many in vivo tools [13,14,[22][23][24] and has successfully been expressed and shown Uncaging of NPY follows the same mechanism. C. Schematic for photoinducible protein-protein interactions using photocaged amino acids. ...
... Using our approach we were able to generate photo-controllable variants of the anti-GFP enhancer nanobody (eNB), which is used as the basis of a wide range of in vivo tools. [2,13,14,[22][23][24] These variants may thus immediately add the ability to use light for the precise spatiotemporal control of these tools, with the photocaged tyrosine that is most amenable to in vivo systems, NPY. ...
Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo‐activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo‐activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti‐GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein‐protein interactions.
... We analyzed the endosomal and PMresiding SNAREs VAMP2, VAMP3, VAMP7, STX2, STX4 and SNAP23, which have been shown to have roles in ECM degradation either from our screen (Fig. 1A) or as reported previously (Miyagawa et al., 2019). A modified nanobody-based pulldown approach was employed (Rothbauer et al., 2006(Rothbauer et al., , 2008Tang et al., 2013). GFP-SNAP23, GFP-VAMP2 and GFP-VAMP3 were able to pull down STX7 from cell lysates ( Fig. 5A; Fig. S4A,B). ...
Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia-associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion.
This article has an associated First Person interview with the first author of the paper.
... pUAS-Cre was a gift from Connie Cepko (Addgene plasmid #50797; http://n2t.net/addgene:50797; RRID: Addgene_50797) [9], and pSpCas9 (BB)-2A-miRFP670 was a gift from Ralf Kuehn (Addgene plasmid #91854; http://n2t.net/addgene:91854 ; RRID: Ad-dgene_91854). pUAS-Cre was digested with FseI (New england biolabs, ipswich, Ma, usa) and hpai (Takara, San Jose, CA, USA) in CutSmart buffer (New England Biolabs), followed by bacterial alkaline phosphatase (Takara) treatment and Klenow fragment (Takara) treatment. ...
Cell labeling technologies, including the Cre/loxP system, are powerful tools in developmental biology. Although the conventional Cre/loxP system has been extensively used to label the expression of specific genes, it is less frequently used for labeling protein-protein interactions owing to technical difficulties. In the present study, we generated a new Gal4-dependent transgenic reporter mouse line that expressed Cre recombinase and a near-infrared fluorescent protein, miRFP670. To examine whether this newly generated transgenic mouse line is applicable in labeling of protein-protein interaction, we used a previously reported transgenic mouse lines that express Notch1 receptor with its intracellular domain replaced with a yeast transcription factor, Gal4. Upon the binding of this artificial Notch1 receptor and endogenous Notch1 ligands, Gal4 would be cleaved from the cell membrane to induce expression of Cre recombinase and miRFP670. Indeed, we observed miRFP670 signal in the mouse embryos (embryonic day 14.5). In addition, we examined whether our Cre recombinase was functional by using another transgenic mouse line that express dsRed after Cre-mediated recombination. We observed dsRed signal in small intestine epithelial cells where Notch1 signal was suggested to be involved in the crypt stem cell maintenance, suggesting that our Cre recombinase was functional. As our newly generated mouse line required only the functioning of Gal4, it could be useful for labeling several types of molecular activities in vivo.
... This example of intracellular expression of Nanobody genetic fusions was rapidly followed and expanded to express Nanobodies linked to the F-box, the von Hippel-Lindau protein, to transcription activation or DNA binding domains. These innovative tools were employed to degrade specifically particular cytoplasmic proteins or to activate reporter or killing genes at particular timings during cell development [61][62][63][64]. ...
The presence of unique heavy chain-only antibodies (HCAbs) in camelids was discovered at Vrije Universiteit Brussel (VUB, Brussels, Belgium) at a time when many researchers were exploring the cloning and expression of smaller antigen-binding fragments (Fv and Fab) from hybridoma-derived antibodies. The potential importance of this discovery was anticipated, and efforts were immediately undertaken to understand the emergence and ontogeny of these HCAbs as well as to investigate the applications of the single-domain antigen-binding variable domains of HCAbs (nanobodies). Nanobodies were demonstrated to possess multiple biochemical and biophysical advantages over other antigen-binding antibody fragments and alternative scaffolds. Today, nanobodies have a significant and growing impact on research, biotechnology, and medicine.
