Mara Ceridono

Sylvette Chasserot-Golaz

Nicolas Vitale

Stéphane Gasman

Stéphane Ory
email: ory@inci-cnrs.unistra.fr

Measurements of compensatory endocytosis by antibody internalization and quantification of endocytic vesicle distribution in adrenal chromaffin cells

Keywords: compensatory endocytosis, large dense core vesicle, neuroendocrine cells, chromaffin cells, internalization, dopamine--hydroxylase

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1.

Introduction

A long standing question is how cell plasma membrane homeostasis is maintained especially in cells with high exchange rate between pools of intracellular vesicles and the plasma membrane. Neurons and neuroendocrine cells are heavily challenged to maintain cell to cell communication by releasing neurotransmitters and hormones through regulated exocytosis.

To release their content upon cell stimulation, synaptic vesicles and secretory granules fuse with the plasma membrane resulting in a massive supply of vesicular membrane. Efficient compensatory endocytosis has to take place to maintain plasma membrane homeostasis and restore vesicular pools (1, 2). Several accurate and sensitive methods are widely used to study exocytosis including electrophysiological and electrochemical approaches that measure changes in electrical properties of the plasma membrane and detect the released molecules respectively (3-5). Live cell imaging with the use of pH-sensitive fluorescent proteins which shine when pH increases upon fusion pore opening represents an alternative approach (6, 7).

Since plasma membrane recovers basal properties and fluorescent reporters are quenched upon acidification in endocytic compartments, such methods are however less suitable to follow the fate of proteins once the endocytic vesicle has formed. Therefore, the main challenge to study compensatory endocytosis is to specifically identify and track proteins that had been transiently exposed to the plasma membrane. Biochemical methods can give an overview of protein fate but often require large amounts of proteins that are not necessarily easily obtained, especially in model system with limited amount of cells such as mouse chromaffin cells for example. Adrenal medulla chromaffin cells have been an instrumental model to study exocytosis and compensatory endocytosis. In contrast to synaptic vesicles which size range is below the resolution limits of conventional light microscopy, secretory granules of chromaffin cells allows for single-vesicle exocytosis and endocytosis analysis with conventional microscopy. By the use of specific antibodies that recognize luminal domain of secretory granule proteins, exocytic sites can be stained. Since vesicular trafficking and especially endocytosis requires energy to proceed, chilling cells at 4° after exocytic stimulation temporarily blocks endocytosis, providing time to specifically label proteins of interest with antibodies. By replacing cells at physiological temperature, compensatory endocytosis gets started and antibodies bound to vesicular proteins are uptaken. We are providing here protocols for time course experiments and immunofluorescence analysis of endocytic vesicle containing antibodies directed against the luminal domain of the dopamine--hydroxylase (DBH), a secretory granule resident enzyme converting dopamine into noradrenaline. These protocols give access to spatio-temporal measurement of compensatory endocytosis in bovine chromaffin cells.

Materials

Equipment

1. 12 mm coverslips cleaned and sterilized (see Note 1). Centrifuge for 15 min at 13000 g, aliquot and store at 4°C for several months.

Methods

Anti-DBH antibodies internalization assay

1. Plate freshly isolated chromaffin cells on fibronectin coated coverslips at 1250 cells/mm 2 .

Assay can be performed from 48 hours to 10 days after plating (see Note 7). 4. DBH staining is then segmented using wavelet transform decomposition of original image to efficiently detect DBH spots (8). This is integrated in Spot detector plugin of Icy (Fig. 1).

Place cells on heated

Parameters have to be empirically defined and binary image compared to original image (see

Note 11).

5. Since detections are exported as ROI on the original pictures, basic measurements such as mean spot intensity, spot number and size using ROI measurements plugin can be easily obtained. Such parameters are, for example, useful to compare exocytosis efficiency between two conditions (9, 10) 6. To measure the efficiency of endocytosis, the position of endocytosed DBH antibodies at different incubation time at 37°C has to be defined. To do so, ROI corresponding to cell area is converted into Euclidean distance map (EDM) which gives a gray intensity to each pixel as the function of their distance to the edge of the ROI (Fig. 2A). Consequently, pixel intensity increases as it separates from the ROI edge. Then, ROI corresponding to DBH staining are transferred onto EDM picture (Fig. 2A). Mean intensity of each ROI are measured and exported into Excel files. By compiling all files, distribution of spots intensity can be obtained. An example of DBH-positive spot distribution for a single cell is shown (Fig. 2B andC). A distance threshold can be applied to estimate the percentage of stained vesicles remaining at a specific distance from the cell edge (9-11). Biotinylated proteins are revealed by incubating cells with fluorescently-labelled streptavidin.

11. Typically, at 90 nm pixel resolution, sensitivity can be set to scale 2 and threshold between 30 and 70 to obtain good results. To get the best accuracy between number of spots before and after segmentation, ImageJ watershed is applied to separate spots that are too close to be resolved by spot detector.

5.

7 . 8 .

 78 Since cells tend to spread out and to adopt an elongated and flattened morphology with time in culture, we recommend performing experiments within the first 5 days of cell culture to get accurate and reproducible measurements of compensatory endocytosis. Chromaffin cells in culture are highly sensitive to mechanical stress. Manipulation and changes in medium must be gentle to avoid mechanical cell stimulation and limit DBH staining in resting state.

9 .

 9 After 2 to 3 min incubation at 37°C, most DBH staining remains close to the plasma membrane. It is therefore difficult to assess whether DBH is internalized or still at the cell surface. Acidic buffer might be used to strip off cell surface antibodies. Conversely, at 5 min, most DBH staining is seen inside the cell at distance from the plasma membrane. 10. For accurate cell delineation, plasma membrane proteins can be biotinylated by amine reactive biotin (Sulfo NHS-SS-Biotin) and then revealed using fluorescent streptavidin.Plasma membrane protein biotinylation can be done before or after cell fixation but cell contour are better define when cells are biotinylated before fixation. Following anti-DBH endocytosis assay, cells are incubated an additional 30 min on ice in PBS containing 120 g/ml Sulfo NHS-SS-Biotin. Cells are rinsed twice in ice cold PBS and fixed.

  Locke's solution: 140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM EDTA, 11 mM glucose, 0.57 mM ascorbic acid, and 15 mM HEPES, pH=7.5.2. Nicotine solution:1 M nicotine stock is prepared in Locke's solution and conserved on ice protected from light. 10 M nicotine in Locke's solution is freshly prepared and warmed up to 37°C just before use. Coverslips mounting medium: dissolve 1 g of Mowiol 4-88 in 4 ml PBS by gentle steering for 16 h at room temperature. Add 2 ml anhydrous glycerol and steer for additional 16 h.

	2. Tissue culture well plates with a diameter able to accept coverlips (typically 24 well plates
	or 4 well plates with diameter of 15 mm)
	9. Paraformaldehyde (PFA) solution: 16 % PFA stock solution is diluted at 4% in PBS to fix
	cells.
	10. Permeabilization solution: 0.1% (v/v) Triton X-100 diluted in PBS.

3. Ice bucket to transfer plates or maintain coverslips in plates at 4°C to stop vesicular trafficking during antibodies incubation. 4. Hot plate is used to maintain cells at 37°C during cell stimulation and endocytosis timecourse experiments. 5. Forceps to transfer coverlips between plates. 6. Confocal microscope 7. Open source Image analysis software (see Note 2).

8. Primary chromaffin cells cultured on coverslips (see Note 3).

2.2 Solution and reagents

1.

3. High potassium

Locke's solution: 85,7 mM NaCl, 59 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM EDTA, 11 mM glucose, 0.57 mM ascorbic acid, and 15 mM HEPES, pH=7.5. 4. Phosphate buffer saline: 1 mM KH 2 PO 4 , 155 mM NaCl, 3 mM KH 2 PO 4 , pH 7.4 5. Fibronectin from bovine plasma for coverslip coating: cleaned coverslips are incubated at least 2 hours at 37°C in 50 g/ml fibronectin diluted in water. 6. Cell culture medium: bovine chromaffin cells are maintained in DMEM supplemented with 10% fetal bovine serum previously heat-inactivated for 30 min at 56°C, 1% glutamine, 1% cytosine arabinoside and 0.1% fluorodeoxyuridine solutions to prevent fibroblasts proliferation. 7. Bovine serum albumin (BSA): 0.2 % (w/v) BSA diluted in Locke's solution (see Note 4). 8. Anti-dopamine -hydroxylase (DBH) antibodies (see Note 5).

11. Fluorophore-conjugated secondary antibodies (see Note 6).

12.


Guy Zuber
email: zuber@unistra.fr

Nadja Groysbeck

Mariel Donzeau

Audrey Stoessel

Anne-Marie Haeberle

Stéphane Ory

Danièle Spehner

Patrick Schultz

Ovidiu Ersen

Mounib Bahri

Dris Ihiawakrim

Gold labelling of Green Fluorescent Protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolatecoated gold nanoparticles

Object: Submission of a revision of the manuscript "Gold immunolabeling of Green Fluorescent Protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles" for

nanobody can be synthesized in a reproducible fashion and has a specific known sequence, the nanobody-gold particle is very small, and using silver enhancement methods the size of the particle can be increased to allow simple electron microscopy (EM) or for detection by light microscopy. One possible drawback is the slightly variable stoichiometry but this is well-characterized in the manuscript and, as the authors point out, the multivalency can increase the avidity. One technical concern is that the only characterization of the clumping/crosslinking of the multivalent probe is by STEM (Figure 4) and no quantitation is provided. This issue should be examined more thoroughly.

Point to be addressed:

1. Characterization of the clumping/crosslinking and quantification issue (Figure 4) Answer: The Figure 4 was modified to include a histogram reporting the distribution of sizes of the probe (e.g. the gold particle domain). The image is provided in Figure S7. The image displays 180 particles. Only 4 clusters were observed. Other fields (not shown) display the absence of clusters. To summarize these data, we added the following paragraph in the main text: "The High-Angle annular dark field Scanning Transmission Electron Microscopy (HAADF-STEM) observation of (Nb-E3)2:(K3)2AuNP assemblies revealed gold particles of roughly spherical shape (Fig. 4 B). Analysis of a TEM image containing 180 gold particles (The sizes of the AuNP were distributed mostly between 2 and 2.6 nm TEM image containing 180 gold particles indicates that the diameters were mostly within the 2.0 to 2.6 nm range (average diameter: 2.31 nm; standard deviation: 0.31 nm) (Supporting information, Figure S7). Moreover, the image was almost devoid of clustered particles (less than 2%) indicating that the used synthetic condition mainly favours coordination of the two thiols of the bidentate K3 dimer onto gold atoms of one single AuNP, as drawn in the illustration (Figure 1)."

2. The main use of the probe is for pre-embedding labelling for EM, as shown in the manuscript. The examples presented use a protocol that is not compatible with good resolution EM (initial paraformaldehyde fixation and Triton X100 permeabilization). It would have been good to see how effective the probes can be with a protocol that minimizes cellular damage, for example an initial glutaraldehyde fixation and milder permeabilization protocol, as this would provide a practical example of its use in EM and the effective size of the particles in a real application where cellular preservation is optimal.

Answer: We did immunolabelling of GFP in 2%PFA, 0.02% glutaraldehyde fixed specimen. These data are now reported in the supporting information (Figures S9 andS10) and demonstrated the ability of the probe to detect GFP as well in glutaraldehyde fixed cells. We tried also 0.1% glutaraldehyde but we experienced silver staining of the control as well (eg the cell that was not incubated with gold particle). Weak coloration of the control was somehow also observed with the 2%PFA 0.02% glutaraldehyde fixed cells but never with 4%PFA (see Figures S9 and10). We therefore prefer to use 4% PFA as fixative on a first base. The following sentence and the reference 39 was added in the main text:

The ability of the (Nb-E3)2:(K3)2AuNP to detect GFP in cellular specimen fixed with low percentage of glutaraldehyde 39 was preserved although with a lost in sharpness (Supporting information, Figures S9 andS10).

3. Have the authors tested the probes for on-section labelling for EM (eg. on thawed frozen sections or Lowicryl sections)? This would be another useful application as it would provide an alternative to commercial GFP antibodies.

Answer: no, we did not test these probes on thawed frozen section or lowicryl section due to time constraints. We feel confident that these probes will do the job as well for these applications since there are usefull for immunolabelling of glutarldehyde fixed specimen. 4. In terms of the practicalities of the methods and their adoption by other groups, some details of the stability of the probes should be provided. Can the authors provide guidelines on storage of the final gold-nanobody particles or should the individual components be prepared and combined just before use?

Answer: we provided data on the stability of Nb-E3 (Figure S4) and guideline for storage. We include as well data showing that the full probe remain active after 7 days storage in the fridge. The (K3)2AuNP was stored in the fridge and was still active 4 months after synthesis. We added this sentence in the material and method section: "To note, the (K3)2AuNP was stored at 4°C and was seen to remain stable for at least 4 months."

Referee: 2

Comments to the Author Molecular contrast in electron microscopy --allowing localization of specific molecules in cellular electron microscopy --is a major challenge. This manuscript addresses this long-standing major challenge. Described is an approach for gold labeling nanobodies that bind to Green Fluorescent Protein. The rationale (although not specifically stated) appears to be to enable Correlative Light -Electron Microscopy (CLEM)--by imparting fluorescent contrast to tagged proteins for optical imaging and gold-cluster contrast to GFP for electron microscopy imaging. Much of what is reported is incremental. Gold cluster labeled nanobodies, labeled through Murray Place Exchange reactions were described in 2006 (reference 24, and a few other papers from Kornberg's lab --e.g., Bioconjugate chemistry 21 (2), 214-218). It's really unclear why the dimerization domain from p53 is used--why not just link directly to the nano body (as previously described in the Kornberg work).

Answer: we would have loved to be able to directly link the Nb to the AuNP using S-Au bond. However, we run in issues relative to lost of binding affinity and lost of integrity of protein. Passivation of the gold nanoparticle was also mandatory to minimize unspecific binding. The noncovalent method used in here provided a straightforward solution. We added the two sentences in the main text:

To work out a Nb-AuNP conjugate without loss of binding affinity of the Nb for its target and loss of protein integrity, we could have played around by comprehensive modification of the peptide sequence at the C-terminus. Instead of undergoing this classical approach, we chose to investigate a non-covalent functionalization approach using the properties of 2 peptides derived from the p53 protein to specifically form a stable heterotetramer composed of two homodimers (Figure 1).

We also added the following sentence in the main text as an other advantage of the non-covalent approach:

If the (K3)2AuNP is preferably mixed with the (Nb-E3)2: right before use, the (K3)2AuNP complex was seen to remain active 7 days after storage at 4°C (Supporting Information, Figures S15). The selectivity of E3 dimer for the K3 dimer could also be exploited by detecting the specimen-bound (GFP Nb-E3)2 with (K3)2AuNP through sequential incubations (Supporting Information, Figure S15).

It is very true for cellular work that smaller nanoparticle/antibody conjugates are desirable, since they will diffuse into fixed and permeabilized cells more effectively. This is also given as a motivation for the work. However, to this end, it would be really useful to compare the new construct presented in this work to FAB-NP or IgG-NP conjugates--to show that the assertion is correct.

Answer:

We linked the TAB, TNB AuNB to a monoclonal mouse anti-GFP antibody and evaluate its ability to label GFP-protein with gold in H2B HeLa cells (Figures S11 to S14). The following sentence was added in the main text to summarize results of experiments:

For comparison of labelling activity, a mouse anti-GFP antibody-gold particle conjugate made from the same TAB-, TNB-coated gold particle and a monoclonal 150 kDa IgG was prepared (Supporting Information, Figure S11). Its gold labelling ability was similar to the one of (Nb-E3)2:(K3)2AuNP (Supporting Information, Figures S12-S14). A tendency of the anti-GFP IgG-AuNP conjugate to preferentially label the nuclear periphery of the H2B-GFP HeLa was noticed (Figures S12 andS14).

Ultimately the authors have to silver-enhance the 2.4nm gold particles in order to see them in the EM of cells. It'd be interesting for the authors to compare this to Leapman's prior work in imaging gold nanoparticles in cells, where smaller particles than the ones used here could be viewed in HAADF-STEM mode. Small 8 (14), 2277-2286 Answer: We do not wish to compare our work to Leapman's prior work as it is not the main focus of our manuscript. We were actually able to see 2.4 nm particles inside the cell nucleus using HAADF-STEM in a work analogous to Leapman's study. We permeabilize living HeLa cells by electroporation to allow the entry of 2.4 nm particle inside cells. We were able to detect the particle inside the cell after silver enhancement but also without silver enhancement using HAADF-STEM. It was challenging but occurrence of aggregated AuNPs as seen in Leapman's images) help us to zoom in the "good" area/spot. In the immunolabelling case, most particles (if not all) are individual. It does indicate probe of excellent quality: however this behavior does not facilitate detection of the "good" spot containing the cells. We will keep on trying but we also have to manage with access to facility and cost of use.

The manuscript does show correlative imaging of HeLa cell sections where β-Gal-GFP), Mitochondria-Targeting Signal tagged GFP (MTS-GFP), GFP tagged-PCNA-Interacting Peptide (GFP-PIP), and H2B-GFP are imaged. This is on the one hand definitely showing things that haven't been seen before (great!), and on the other hand, showing that with silver enhancement, you don't really get better resolution than you can get in optical microscopy --therefore why do the electron microscopy work at all (or --tell the reader what additional information is gained in the EM micrographs compared to the light microscope micrographs --which are much easier to acquire.)

Overall --the paper should be improved before publication in Nanoscale Advances. The improvement could be in one of the following areas:

1. Data that compares the efficacy of the nano body constructs to more commonly used FAB or IgG constructs --testing the hypothesis that smaller is better in this case.

Answer:

We linked the TAB, TNB AuNP to a monoclonal mouse anti-GFP antibody and evaluate its ability to label GFP-protein with gold in H2B HeLa cells (Figures S11 to S14). As seen the synthetic procedure is straightforward as well but implies a sequential procedure in where the Ab the initial "substrate". The following sentence was added in the main text to summarize results of experiments:

"For comparison of labelling activity, a mouse anti-GFP antibody-gold particle conjugate made from the same TAB-, TNB-coated gold particle and a monoclonal 150 kDa IgG was prepared (Supporting Information, Figure S11). Its gold labelling ability was similar to the one of (Nb-E3)2:(K3)2AuNP (Supporting Information, Figures S12-S14). A tendency of the anti-GFP IgG-AuNP conjugate to preferentially label the nuclear periphery of the H2B-GFP HeLa was noticed (Figures S12 andS14)." 2. Interpretation of data in a way that shows the utility of this approach (how does it give more information than one gets in an optical micrograph)?

Answer: We modify the conclusion to indicate that these probe are useful using two imaging modalities More minor concerns: 3. Fig4B is HRTEM, not STEM. If they used a Cs-corrected JEOL-2100F, they should absolutely be able to get a better image than F7C.

Answer: The Figure 4 was modified. It displays a HAADF-STEM of the probe as well as size distribution of a representative population of particles 4. I recommend EDS work with Ag-enhancement to see if particulate would form where gold is and is not located. It's straightforward to do a point-and-shoot spectrum.

Answer: An EDX analysis of the nuclear portion containing contrasting particles was performed (See supporting information Figure S17). The spectrum shows the presence of the gold and silver atoms confirming the assertion from Aurion about selective deposition of silver onto AuNPs.

Introduction

The investigation of protein trafficking and localization is fundamental to understand cell functioning. Fluorescent microscopy (FM) and Electron Microscopy (EM) are two valuable tools in this endeavour. 1 Continuous technological advances in both FM and EM are steadily pushing the resolution boundaries and providing new insights on how cellular constituents precisely organize together and participate in the life and wellbeing of biological organisms. Pinpointing selected elements within the subcellular organization is unfortunately not always feasible due to the lack of contrast differential between the various cellular constituents. Specific labelling methodologies must therefore be developed and optimized to accompany the progress of microscopy technology. The Aequorea victoria Green Fluorescent Protein (GFP) and fluorescent derivatives are popular tags to label chosen proteins through genetically made fusion proteins and to localize them with FM even in living cells and organisms. For EM, gold nanoparticles (AuNPs) are the contrasting agents of choice. 2 The traditional AuNPantibody conjugates first reported by Faulk and Taylor, 3 consisting of 150 kDa antibodies that are physically adsorbed onto colloidal AuNPs of 10-15 nm, are still in use today. Yet, the large size of AuNPs and IgG molecules, as well as the difficult to define bonds between the AuNP and the IgG hamper the efficiency of labelling as well as accurate target localization. 4 Reduction of the AuNP's diameter to less than 1.4 nm led to improved labelling agents with favourable diffusion and penetration abilities into tissues. 5 Replacement of the full 150 kDa IgG with the smaller 50 kDa fragment antigen-binding domain (Fab) was also beneficial. 6,7 In a step toward further optimization, 12-15 kDa single-domain camelid antibody fragments (VHH) or nanobodies (Nbs) appear highly appealing. [8][9][START_REF] Schumacher | This journal is[END_REF] They are interesting not only because of their compactness (molecular weight (MW) of ca. 15 kDa for a size of 4 nm x 2.5 nm), but also due to their bioengineering properties. 11 Point mutations of selected amino-acids to cysteines 12,13 afford precise attachment sites for thiol-reactive fluorophores. The resulting fluorescently-labelled Nbs have known spacing distances between the targeted epitope and the fluorescent signal, 14 affording probes with enhanced target localization accuracy suited for super-resolution microscopy imaging of biological targets. 15,16 Nanobodies have also been applied to EM immunolabelling. Schematic route for the synthesis of the Nb 2 -AuNP conjugates using the strong and mutual associating ability of E3 and K3 dimers originating from a p53 heterotetramer. 17,18 According to the structure of the p53 tetramerization domain (pdb1OLG), 19 the dimers mainly fold into -helixes. The GFP binding nanobody (Nb) mainly folds into -sheets (in blue pdb3OGO). 20 Kijanka and colleagues developed a protocol for the labelling of HER2 using an HER2 binding Nb followed by a secondary anti-Nb antibody and then a protein A-gold particle conjugate. 21 Ariotti and colleagues developed fusion proteins between GFP-binding Nbs and a soybean ascorbate peroxidase to locally produce a stain upon addition of diaminobenzidine (DAB) and H 2 O 2 . 22,23 The brownish stain visible by optical microscopy (OM) could be further converted into an electron dense product through treatment with OsO 4, permitting correlative light and electron microscopy observation. Investigation of the direct conjugation of Nbs to 10-15 nm AuNPs by adsorption has been reported by Goossens and colleagues. 24 Functional conjugates were obtained but maintaining the colloidal stability of the nanobody-AuNP conjugates was clearly extremely challenging because of the huge difference in bulkiness between the Nb and the AuNP. Decreasing the difference can be achieved by decreasing the diameter of the AuNP. 25 However, decreasing the diameters weakens the strength of protein adsorption onto the AuNP surface, leading to highly dynamic protein coronas. 26,27 If the gold-bound protein layer is mandatory for target recognition, linkage of the protein to the AuNP should hence be performed using stronger coordination links such as the Au-S bonds 28,29 and/or functionalization strategies involving covalent bonds. 30 To design Nb-AuNP conjugates with usefulness for gold immunolabelling application of cellular specimen, we explored methods for strongly linking thionitrobenzoates-coated AuNPs (AuG) 31,32 to bioengineered GFP binding Nbs. 8,20 We discovered that the non-covalent conjugation approach based on the complementary associating dimers originating from a p53 heterotetramer variant, 17,18,33 led to Nb:AuNP assemblies with excellent immunogold labelling abilities (Figure 1).

Results and discussion

Synthesis of GFP nanobody-AuNP conjugate

To generate immunogold probes based on Nbs, we selected the GFP binding Nb cAbGFP4 with a previously determined structure. It displays a dissociation constant (K d ) within the nanomolar range. 20 For the gold domain, we used the 2.4 nm AuG that was previously described and extensively characterized. This AuG is water-soluble and contains a single layer of substituted thiobenzoates that can be readily replaced with other thiol-containing molecules via a thiolate-for-thiolate exchange. Functionalized AuNPs with robust Au-S coordination bonds might hence be conveniently synthesized. In our first attempts, we investigated the preparation of nanobody-AuNP conjugates directly from Nbs that were pointmutated to contain an additional thiol. 14 The GFP binding Nb sequence was bioengineered to possess either a C-terminal cysteine (C143-Nb) or a cysteine at the position 7 (C7-Nb). The C143-Nb displayed severe proteolytic instability and was not further used (Supporting Information, Figure S1). The C7-Nb was stable at 4°C for more than six months, had GFP binding ability similar to that of the wild type Nb (Supporting Information, Figure S2) and could be linked to AuG via Au-S coordination. The conjugates could be further purified and characterized by SDS-PAGE (Supporting Information, Figure S3). Unfortunately, in contrast to the covalent appending of fluorescent molecules, 14 the direct coordination of C7-Nb onto the 2.4 nn AuNP dramatically altered its ability to bind GFP and labelling potency (Figures S3). Such alteration of the Nb specificity for its target after conjugation has already been pointed out in the literature. [START_REF] Schumacher | This journal is[END_REF] Even the functionalization of Nbs with fluorophores of M.W. smaller than 1000 Da fluorophores via random coupling to lysines can drastically reduce Nb binding. 14 To work out a Nb-AuNP conjugate without loss of binding affinity of the Nb for its target and loss of protein integrity, we could have played around by comprehensive modification of the peptide sequence at the C-terminus. Instead of undergoing this classical approach, we chose to investigate a non-covalent functionalization approach using the properties of 2 peptides derived from the p53 protein to specifically form a stable heterotetramer composed of two homodimers (Figure 1). Relative to the native p53 tetramerization domain, one sequence was enriched in lysine (K) while replacement of one K with glutamic acid (E) provided a E-enriched sequence (E3). 17 These E-and K-enriched sequences, named E3 and K3 respectively, were recently demonstrated to conveniently favour the selective non-covalent union of 2 distinctly tagged functional proteins into highly stable heterotetramers even in complex biological fluids crowded with all sorts of proteins and diverse compounds. 18,33 First, the Nb was genetically engineered at its C-terminal end with the aspartate-enriched variant of the p53 heterotetramer domain (E3) along with a polyhistidine sequence (Figure 2). 18 The recombinant Nb-E3 fusion protein was then produced in E. coli and purified by immobilized metal affinity chromatography followed by size exclusion chromatography (SEC) with high yield and purity (Figure 2B). The SEC profile showed elution of a species with an apparent Molecular Weight (MW) close to 66 kDa corresponding to the MW of a dimer of Nb-E3 (Figure 2C). The integrity of the Nb-E protein was fully maintained upon storage at -80°C. Proteolytic cleavage was nonetheless Please do not adjust margins Please do not adjust margins observed upon storage > 120 days at 4°C (Supporting information, Figure S4). The K-enriched sequence K3 17,18 was fused to the CALNN motif. 34 According to extrapolation from the structure of the p53 domain, 19 the two cysteines at the N-terminal end are 2 nm apart with little chance to cross react. They can also point toward the same direction, offering possible bidentate coordination to AuG upon thiolate exchange reaction. Functionalization of the AuG with this dimer in 0.1 M HEPES buffer, pH 7.5 was then assayed at increasing dimer/AuG ratios (Supporting Information, Figure S5). After a 3h incubation time at 25°C to allow thiolate-to-thiolate exchange of Au(I)coordinated ligands on the AuG surface, the crude reactions were analysed by SDS-PAGE. Functionalization of the AuNP with the dimer via exchange of ligands proceeded smoothly with an increased number of dimer attachment per particle by increasing the dimer/AuG ratios. At the dimer/AuG ratio of 3, the mixture was devoid of unreacted AuG. Three distinctive AuNP species with different electrophoretic mobility were produced, likely corresponding to AuNPs equipped with 1, 2 and 3 dimers. To note, the masking of the gold surface of AuG by coordination of thiolated PEG 2000 Da was mandatory to minimize non-specific adsorption of the probe onto cellular ultrastructure (Supporting Information, Figure S6). Therefore, this reaction condition was scaled-up and immediately followed by the addition of excess thiolated PEG 2000 Da to ensure full PEG-mediated shielding of the AuNP surface (Figure 3). The end product (K3) 2 AuNP, barely migrated into the polyacrylamide gel, likely because of the properties of PEG to shield the superficial charge of nanomaterial. The non-covalent association of (Nb-E3) 2 to (K3) 2 AuNP was next evaluated by titrating the (K3) 2 AuNP PAGE with increasing amounts of (Nb-E3) 2 . A PAGE analysis in non-denaturing conditions allowed the assessment of binding by detecting free/unbound (Nb-E3) 2 (Figure 4). A small amount of free (Nb-E3) 2 started to be detected at the (Nb-E3) 2 /(K3) 2 AuNP ratio of 2 indicating that each (K3) 2 AuNP can anchor slightly less than 2 (Nb-E3) 2 . In the next experiments, the non-covalent (Nb-E3) 2 :(K3) 2 AuNP assemblies were generated by mixing the modified AuNP with 2 molar equivalents of (Nb-E3) 2 .

The High-Angle annular dark field Scanning Transmission Electron Microscopy (HAADF-STEM) observation of (Nb-E3) 2 :(K3) 2 AuNP assemblies revealed gold particles of roughly spherical shape (Fig. 4 B). Analysis of a TEM image containing 180 gold particles (The sizes of the AuNP were distributed mostly between 2 and 2.6 nm TEM image containing 180 gold particles indicates that the diameters were mostly within the 2.0 to 2.6 nm range (average diameter: 2.31 nm; standard deviation: 0.31 nm) (Supporting information, Figure S7). Moreover, the image was almost devoid of clustered particles (less than 2%) indicating that the used synthetic condition mainly favours coordination of the two thiols of the bidentate K3 dimer onto gold atoms of one single AuNP, as drawn in the illustration (Figure 1).

Binding ability of non-covalent (Nb-E3) 2 :(K3) 2 AuNP to GFP

In order to use the complex as a probe for GFP in microscopy, we had to check whether the (Nb-E3) 2 :(K3) 2 AuNP complex maintained its ability to bind GFP. To do so, we performed an indirect ELISA assay using purified GFP as the plasticimmobilized antigen and compared the binding profiles of the wild type Nb, (Nb-E3) 2 and (Nb-E3) 2 :(K3) 2 AuNP complex (Figure 5). The dose-dependent binding of the various Nbs tested showed similar sigmoidal profiles from which we assessed the apparent Kds. Accordingly, we found a Kd of 0.8 nM for the wild type Nb, 0.4 nM for each Nb of (Nb-E3) 2 and 2 nM for each Nb of (Nb-E3) 2 :(K3) 2 AuNP.

ARTICLE

Please do not adjust margins Please do not adjust margins The Kd values indicated that the E3-mediated dimerization of Nb results in a gratifying avidity effect (four fold increased in apparent affinity when the (Nb-E3) 2 is compared to Nb). 18,37 In contrast, the grafting of about 2 (Nb-E3) 2 onto (K3) 2 AuNP resulted in a 5-fold affinity loss relative to the (Nb-E3) 2 .

The reason behind the apparent affinity loss of (Nb-E3) 2 after anchorage to the (K3) 2 AuNP might be explained by the presence of the PEG. The PEG is required to minimize unspecific association of proteins to the AuNP surface. However, its bulkiness and vicinity to the Nb-anchored AuNP may entail a weakening of the apparent binding affinity of the AuNP-attached nanobodies for GFP.

The efficiency of the (Nb-E3) 2 :(K3) 2 AuNP complex to mark GFPprotein fusions with a AuNP inside cells was then evaluated using immunocytochemistry method followed by bright field microscopy (BFM) (Figure 6). The ability of the (Nb-E3) 2 :(K3) 2 AuNP to detect GFP in cellular specimen fixed with low percentage of glutaraldehyde 39 was preserved although with a loss in sharpness (Supporting information, Figures S9 andS10). For comparison of labelling activity, a mouse anti-GFP antibody-gold particle conjugate made from the same TAB-, TNB-coated gold particle and a monoclonal 150 kDa IgG was prepared (Supporting Information, Figure S11). Its gold labelling ability was similar to the one of (Nb-E3) 2 :(K3) 2 AuNP (Supporting Information, Figures S12-S14). A tendency of the anti-GFP IgG-AuNP conjugate to preferentially label the nuclear periphery of the H2B-GFP HeLa was noticed (Figures S12 andS14). If the (K3) 2 AuNP is preferably mixed with the (Nb-E3) 2 : right before use, the (K3) 2 AuNP complex was seen to remain active 7 days after storage at 4°C (Supporting Information, Figures S15). The selectivity of E3 dimer for the K3 dimer could also be exploited by detecting the specimen-bound (GFP Nb-E3) 2 with (K3) 2 AuNP through sequential incubations (Supporting Information, Figure S15). Altogether, the experiments clearly demonstrate the usefulness of (Nb-E3) 2 :(K3) 2 AuNP as probe to label GFP and fusion proteins with gold particles either inside the cytosol or inside the nucleus.

(Nb-E3) 2 :(K3) 2 AuNP assemblies for immunogold labelling of GFP-protein fusion inside cells by Electron Microscopy

We next evaluated whether (Nb-E3) 2 :(K3) 2 AuNP complexes were suitable for EM labelling of GFP-tagged proteins in cells using a stably transformed HeLa cell line that expresses the nuclear histone protein H2B fused to GFP. 40 First, the stable H2B-GFP HeLa and wild type HeLa cell lines were fixed and permeabilized for immunocytochemistry labelling with extensive silver-mediated amplification of AuNPs for FM and BFM imaging (Figure 7, Images A and B). Since the silver staining correlated well with the GFP signal, the cells were processed for gold immunolabelling and moderate silver enhancement before resin embedding, sectioning and EM observation of the specimen. We also prepared an EM specimen of the immunolabelled HeLa H2B-GFP without the silver enhancement step. This non-silver enhanced specimen was analysed for gold content in the nucleus using Energy Dispersive X-ray (EDX) spectroscopy (Supporting Information, Figure S16). Please do not adjust margins Please do not adjust margins difficulty to find the nucleus in where to zoom in. We therefore performed a mild silver enhancement procedure of the specimen containing (Nb-E3) 2 :(K3) 2 AuNP-labeled H2B-GFP HeLa cells to grow the size of the particle to size below 20 nm. After sectioning, the specimen was analysed by TEM. HAADF-STEM (Figure 7, image C) clearly showed the presence of discrete particles inside the nucleus. EDX spectroscopy confirmed that these particles were composed of silver and gold (Supporting Information, Figure S17). The density of AuNPs in the different subcellular compartments was then measured by compiling data from six images to obtain: 37 AuNPs per µm 2 in the heterochromatin (HC) near the nuclear periphery; 14 AuNPs per µm 2 within the perinucleolar compartment (PC) and; 4 AuNPs per µm 2 within the euchromatin (EC) regions within the nucleus. The nucleoli and the cytosol were almost devoid of probes (0.5 and 0.2 AuNPs per µm 2 for the Nucleoli and cytosol, respectively). A similar H2B-GFP nuclear labelling pattern was also obtained using conventional TEM whereas the wild type HeLa was devoid of any particles (Supporting Information, Figure S18). A preferential localization of H2B-GFP in the heterochromatin near the nuclear or nucleolar periphery was in agreement with published studies. 40,41 We can nonetheless note a discrepancy between nuclear staining seen by OM and inhomogeneous nuclear labelling observed by EM. This variation likely results from the silver staining procedure (sizes of AuNP-seeded silver particles for BFM imaging were made larger than the ones for EM imaging) and from the difference in specimen thickness (BFM images were projections of light intensity variation throughout the whole cell; EM were performed on about 100 nm thick specimen). The density of gold labelling inside the nuclei was lower than the expected calculated density of nearly 10 4 H2B per µm 2 (one human cell nucleus contains approximately 30 million nucleosomes, corresponding to 60 million H2B molecules). 42 However, the immunogold labelling protocol often underestimates the real number because PFA might preclude recognition of a proportion of target epitope. High local concentration of targets might also play a role. 43 In our specific case, the H2B-GFP fusion was moreover expressed in addition to the endogenous non-labelled H2B, likely at a low percentage.

Materials and methods

Genetic engineering of the Nb-E3 construct

The GFP binding nanobody was engineered to contain the E3 peptide sequence 18 and polyhistidine at the C terminal end.

The GFP binding nanobody 20 gene was amplified using PCR and primers of sequences: CGTCAG CCATGG GGTCCC AGGTTCAGC and CCACAG GAATTC ACAATG GTGATG ATGGTG ATGTGCG. The DNA fragment was then inserted into a pETOM vector containing the E3 tag DNA sequence using NcoI and SpeI restriction sites. 18

Expression and purification of (Nb-E3) 2

The Recombinant nanobody was expressed in E. coli BL21(DE3)pLys after induction with 1 mM IPTG in 100 mL LB medium at 20°C for 24h. The bacteria were lysed by ultrasonication. The his-tagged proteins were then purified by immobilized metal affinity chromatography using a HisTrap HP column (1 mL) charged with NiSO 4 and then by gel filtration on a HiLoad Superdex 200PG preparative column operating at a flow rate of 0.5 mL/min. Protein fractions were analysed by SDS-PAGE. Selected fractions were pooled and protein solutions were concentrated with Amicon Ultra 4 mL centrifugal devices (MWCO 3 kDa).

Synthesis of AuNP-nanobody conjugates

Typically, the AuG in 0.1 M HEPES, pH 7.5 solution (70 μL, 3 nmol) was added to the peptide of sequence [CALNNGEYFTLQIRGRERFEMFRKLNKALELKDAQA] (176 μL of a 1 mM solution, 18 nmol) freshly prepared in 0.1 M HEPES, pH 7.5. After 3 h at 25°C the remaining exchangeable thiolates on the AuG surface were then exchanged to polyethyleneglycol 2000 Da by addition of alpha-methoxy-omega-mercapto poly(ethylene glycol) 2000 Da (276 μL of a 1 mM solution, 276 nmol) followed by a 3h incubation time at 25°C. The crude mixture was then purified by ultrafiltration (30 kDa cut-off) (5 times 0.5 mL of 0.1 M HEPES, pH 7.5) to yield 50 µL of the modified AuNP ((K3) 2 AuNP). The concentration of AuNP was determined spectrophotometrically to be 18 µM. 44,45 The (Nb-E3) 2 :(K3) 2 AuNP was finally obtained by addition of (Nb-E3) 2 (20 µL of a 5 µM solution, 100 pmol) with the (K3) 2 AuNP (5 µL of a 10 µM solution).

Transfection experiments

The plasmids encoding Mitochondria Targeting Signal-GFP fusion (MTS-GFP), GFP-β-galactosidase fusion (GFP-β-gal), GFP-PCNA Interacting Peptide fusion (GFP-PIP) were gifts from Prof. Étienne Weiss and Dr. Mariel Donzeau. 44 They were transfected into HeLa cells using PEI derivatives. 46,47 Cells were seeded onto glass coverslips in 24-well plates at 50000 cells/well the day before the transfection experiment.

Transgene expression was analysed after 24 h.

Immunocytochemistry for bright field microscopy

Coverslip-adhered cells were fixed with 4% PFA in PBS for 20 min at 20°C. The coverslips were then washed with PBS (3 x 0.5 mL, 5 min), PBS containing 50 mM glycine (0.5 mL, 20 min) and the cell plasma membranes were permeabilized with 0.05% Triton X-100 in PBS (0.5 mL, 5 min). The coverslips were soaked in PBS containing 10% (w/v) BSA for 1h, washed with 0.2% acetylated BSA (BSA-c) in PBS (2 x 0.5 mL, 5 min) and then incubated with 6 nM (Nb-E3) 2 :(K3) 2 AuNP in 0.2% BSA-c containing 10% FCS, 0.5 mL for 1 h. Next the cells were washed with 0.2% BSA-c (2 x0.5 mL, 5 min) and with 80 mM citrate buffer pH 6.2 (3 x 0.5 mL, 5 min). Finally the AuNPs were made visible using a silver staining protocol modified from the Danscher method as reported previously. Please do not adjust margins Please do not adjust margins

Pre-embedding immunolabelling and sample preparation for EM

Cells were fixed with 4% PFA in PBS for 20 min at 20°C. The coverslips were washed with PBS (3 x 0.5 mL, 5 min), PBS containing 50 mM glycine (0.5 mL, 20 min) and the cell plasma membranes were permeabilized with 0.05% Triton X-100 in PBS (0.5 mL, 5 min). The coverslips were soaked in PBS containing 10% (w/v) BSA for 1h, washed with 0.2% BSA-c in PBS (2 x 0.5 mL, 5 min) and then incubated with (Nb-E3) 2 :(K3)

Conclusion

During our investigation to precisely functionalize AuNPs with the minimal sized Nb, we discovered that the conjugation approach involving the non-covalent assembly between complementary associating peptide tags appended to the AuNP and the nanobody, respectively, yielded probes that specifically bind to GFP. These probes proved highly effective for the visualization of various GFP-protein fusions inside cells by BFM and high resolution HAADF-STEM, thus pointing out the versatility of these gold-tagged Nbs for labelling applications using two different imaging modalities. The synthesis of the probe is convenient to perform: the synthetic steps can be monitored in a straightforward manner and the building blocks can be separately synthesized and stored in accordance with their stability. Moreover, the linkage of the recombinant nanobody to the AuNP at the latest synthetic step (in here after the required pegylation to protected the AuNP from interacting with encountered proteins) is extremely convenient as it limits possible chemical or structural denaturation of the protein. Even if the current probes are mixtures of species (the number of dimerized E3-Nbs per (K3) 2 AuNP oscillates around 2), this approach offers by far more control over stoichiometry and orientation of the attached E3-Nbs than the nanoparticle functionalization via adsorption or EDC chemistry. This non-covalent conjugation approach is clearly implementable for other nanobodies and therefore unlocks the usefulness of future nanobodies targeting proteins for the individual localization of protein inside the cell by EM and possibly cryo EM at unprecedented high resolution.

Nanobodies and thiolate-coated gold nanoparticles of circa 2 nm diameters are attractive elements for building multifunctional assemblies. A specific and non-covalent approach is reported to associate the targeting ability of nanobodies with the detectability of 2.4 nm gold nanoparticles for immunolabelling of cellular effectors using light and high-resolution electron microscopy.

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Supporting Information

Gold Labelling of Green Fluorescent Protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles Nadja Groysbeck, Mariel Donzeau, Audrey Stoessel, Anne-Marie Haeberle, Stéphane Ory, Danièle Spehner, Patrick Schultz, Ovidiu Ersen, Mounib Bahri, Dris Ihiawakrim, Guy Zuber*

Table of content

Materials and Methods (Addition to the main article)

Materials and Methods (Addition to the main article)

Materials: The AuG was synthesized as previously reported with a diameter of 2.4 nm. 1 The peptides were ordered form GeneCust in purity above 80%. The 2000 Da alpha-methoxyomega-mercapto poly(ethylene glycol) (PEG) was ordered from Iris Biotech. Electron Specific equipment: A HI 2210 pH meter was used for measuring the pH of buffer solutions.

Centrifugation of 50 mL tubes was done using an Eppendorf 5810R centrifuge equipped with an A-4-81 rotor. For smaller volumes (0.1 -2 mL) centrifugation was performed in an Eppendorf 5415R centrifuge. AuNP reaction solutions were shaken on a Heidolph Rotamax 120 rocking platform. Functionalized AuNPs were concentrated and purified using Amicon Ultra 0.5 mL centrifugal devices (MWCO 30 kDa, if not stated otherwise).

ARTICLE

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Figure 1 .

 1 Figure 1. Schematic route for the synthesis of the Nb 2 -AuNP conjugates using the strong and mutual associating ability of E3 and K3 dimers originating from a p53 heterotetramer.17,18 According to the structure of the p53 tetramerization domain (pdb1OLG),19 the dimers mainly fold into -helixes. The GFP binding nanobody (Nb) mainly folds into -sheets (in blue pdb3OGO).20

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Figure 2 .

 2 Figure 2. Characterization of the recombinant E3 tagged GFP binding nanobody. A. Peptide sequence. The GFP VHH (blue) is fused to the E-enriched p53 oligomerization domain (red) and to a polyhistidine tag. B. SDS PAGE analysis of the bacteria-expressed and purified protein after Ni-NTA chromatography showed monomeric Nb-E3. C. Size exclusion chromatography of the purified E3-fused nanobody. Elution of a species with an apparent molecular weight of 66 kDa confirmed dimerization.

  The direct Au-S coordination bond at this C7 position may have exposed the nanobody to the AuNP's corona, resulting in obliteration of the nearby GFP-Nb binding interface.

Figure 3 .

 3 Figure 3. PAGE analysis of reaction products obtained by reaction of AuG with 3 molar equivalent of thiolated K3 dimer (Crude A) and then thiolated PEG 2000 Da ((K3) 2 AuNP).

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 4 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Figure 4 .

 4 Figure 4. Characterization of the non-covalent (Nb-E3) 2 :(K3) 2 AuNP assemblies. A. Native PAGE analysis to determine the average stoichiometry for complete assembly of (Nb-E3) 2 with (K3) 2 AuNP. B. Scanning Transmission Electron Microscopy (STEM) analysis of the (Nb-E3) 2 :(K3) 2 AuNP.

Figure 5 .

 5 Figure 5. Apparent binding abilities of the GFP-binding nanobody (green triangle), the dimerized (Nb-E3) 2 (blue dot) and the non-covalent (Nb-E3) 2 :(K3) 2 AuNP assemblies (red square). The binding to GFP was determined using indirect ELISA analysis and plateadsorbed GFP

Figure 6 .

 6 Figure 6. Fluorescent and (Nb-E3) 2 :(K3) 2 AuNP-mediated detection of protein-GFP fusions after transient gene transfection. HeLa cells were transiently transfected with DNA plasmids to express different GFP-protein fusions as indicated: -Galactosidase-GFP (-Gal-GFP), Mitochondria-Targeting Signal tagged GFP (MTS-GFP), GFP tagged-PCNA-Interacting Peptide (GFP-PIP). Upper images: Detection of the GFP signal in PFA-fixed HeLa cells 24 hours after DNA plasmid transfection. Lower images: the cell membrane was permeabilized, the cells were incubated with 6 nM (Nb-E3) 2 :(K3) 2 AuNP, washed and the gold nanoparticles bound to cellular components were detected by extensive silver enhancement visible by BFM

  HeLa cells were transiently transfected with DNA plasmids encoding a -Galactosidase-GFP (-Gal-GFP), a Mitochondria-Targeting Signal tagged GFP (MTS-GFP) and a GFP tagged-PCNA-Interacting Peptide (PIP) (GFP-PIP). The cells expressing various levels of each exogenous protein were then observed by FM (Figure, 6, upper images). The subcellular localization of these GFPprotein fusions were then assayed by immunogold labelling with (Nb-E3) 2 :(K3) 2 AuNP followed by silver enhancement to sizes observable by BFM (Figure6, lower images). Comparison between the FM and BFM images of the transfected HeLa cells showed an excellent correlation between the fluorescent signal and the silver-enhanced AuNPs. The 520 kDa βgalactosidase-GFP fusion protein was seen inside the cytosol but not inside nuclei in agreement with the function of the nuclear envelop to block passive diffusion of macromolecules above 50 kDa. When the 520 kDa β-galactosidase-GFP fusion was further equipped with a Nuclear Localization Signal (NLS), FM and immunogold labelling experiments showed also the expected transport of -Gal-GFP-NLS into the cell nuclei (Supporting Information, FigureS8). The preferential accumulation of MTS-GFP in mitochondria (filamentous ultrastructure near surrounding the nucleus) was also observed by the immunogold labelling protocol. The GFP-PIP can be used as a marker for cell replication.38 Two patterns could be observed in transfected cells: GFP-PIP fluorescent foci in the nucleus corresponding to early DNA replication initiation sites, or the nucleus filled with GFP-PIP with accumulation near the nucleoli corresponding to a more advanced state of DNA replication. Both of these patterns were also observed using (Nb-E3) 2 :(K3) 2 AuNP. 6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins

Figure 7 .

 7 Figure 7. Fluorescent and (Nb-E3) 2 :(K3) 2 AuNP-mediated detection of H2B-GFP fusions inside the nuclei of HeLa cells transformed to stably express H2B-GFP fusions. A. Fluorescence detection of GFP in H2B-GFP HeLa. B. BFM image of H2B-GFP HeLa with (Nb-E3) 2 :(K3) 2 AuNP and extensive silver enhancement. C. HAADF-STEM image of H2B-GFP HeLa after labelling with (Nb-E3) 2 :(K3) 2 AuNP and mild silver enhancement.

  The EDX spectrum clearly demonstrated the presence of gold atoms inside the cell nucleus (Au Ma = 2.123 keV, Au Mb = 2.203 keV, Au La = 9.713 keV, Au Lb = 11.443 keV). Unfortunately, in our hands, the direct visualization of the AuNPs in the cell sections was unfruitful for the moment due to the low contrast of the specimen and This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

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 8 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Figure S1 . 7 Figure S2 . 8 Figure S3 . 9 Figure S4 .Figure S5 . 10 Figure S6 . 11 FigureFigure S8 .Figure S9 .Figure S10 .Figure S11 .Figure S12 . 2 Figure S13 .Figure S14 .Figure S15 .Figure S16 .Figure S17 .

 S17S28S39S4S510S611S8S9S10S11S122S13S14S15S16S17 Figure S1. DNA and amino acid sequences of GFP nanobody constructs and analysis of the construct stability as reported S-7

  microscopy grade paraformaldehyde (20% w/v solution) and glutaraldehyde (25% w/v solution) were purchased from Electron Microscopy Sciences. Other reagents were obtained from Sigma Aldrich, Carl Roth, VWR Chemicals, Euromedex or Honeywell. They were used without further purification unless stated otherwise. All solutions and buffers were made with water purified with a Millipore Q-POD apparatus. Precision Plus Protein Standard Dual Xtra (BioRad) was used as protein ladder for SDS-PAGE analysis. The affinity-purified goat IgG directed against the nanobody domain was purchased from Jackson ImmunoResearch. The donkey anti-goat IgG (H+L) antibody conjugated to Alexa Fluor Plus 594 was purchased from Invitrogen. BSA-c was bought as 10% solution from Aurion. DNA transfection was performed with the in vitro transfection reagent jetOPTIMUS from Polyplus-transfection. Ni NTA agarose beads were purchased from Qiagen.

S 4 8 Figure S2 . 9 Figure

 48S29 Figure S2. Evaluation of the apparent binding affinities of GFP nanobody and mutated Nb-C7 for adsorbed GFP using ELISA. Data revealed apparent Kds of 0.78 and 0.87 nM for the wt Nb and Nb-C7 respectively, suggesting that the point mutation did not alter the apparent affinity of the nanobody for GFP.

10 Figure S4 . 11 Figure S5 . 12 Figure S6 . 13 Figure S7 .Figure S8 . 15 Figure S9 .Figure S10 . 17 Figure S11 .Figure S12 . 19 Figure S13 .Figure S14 . 21 Figure S15 .SFigure S16 . 23 Figure S17 .Figure S18 .

 10S411S512S613S7S815S9S1017S11S1219S13S1421S15S1623S17S18 Figure S4. PAGE Analysis of the Nb-E3 construct integrity after storage at -80°C for > 1 ye ar (line 1) and storage at 4°C for 127 days (line 2).

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Acknowledgements

This research was supported by the CNRS 80prime program attributed to GZ, OE and PS, the ITI Innovec (IdEx (ANR-10-IDEX-0002), SFRI (ANR-20-SFRI-0012)) and La Ligue contre le Cancer CCIR-Est). NG was funded by a PhD fellowship from the IdEX Unistra (Université de Strasbourg and Investissement d'Avenir). Transmission electron microscopy observation was performed at Plateforme Imagerie in vitro, CNRS UPS 3156, Strasbourg. We are grateful to Robert Drillien for style-edition of the text.

Author Contributions

NG, MD, GZ, DS, MB, DI and AMH designed and performed experiments; NG, GZ, PS, SO, OE conceived experiments, analysed the data and revised the manuscript, NG and GZ drafted the manuscript.

Conflicts of interest

There are no conflicts to declare.

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