Name: Live-cell Imaging of Endocytic Transport using Functionalized Nanobodies in Cultured Cells (Video) | JoVE Journal
Uploaded: 2025-10-17T14:40:00.000+00:00
Duration: 8 min 2 s
Description: Learn live-cell imaging of endocytic transport using functionalized nanobodies in HeLa cells. This method enables visualization and quantification of transmembrane protein trafficking dynamics.

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This work was supported by the University of Basel. We thank Prof. em. Dr. Martin Spiess for creating and laying out figures, the Imaging Core Facility (IMCF) of the Biozentrum for support, and PNAS for reprint permission.

The authors have nothing to disclose.

Nanobodies represent a rapidly emerging class of protein-binding scaffolds that offer numerous advantages over conventional antibodies: they are small, highly stable, monomeric, non-crosslinking, lack disulfide bonds, and can be selected for high-affinity binding24. In our previous study, we developed and applied functionalized nanobodies produced by bacterial expression to label surface-exposed proteins and monitor their intracellular trafficking to various compartments, with a particular focus on retrograde transport to the trans-Golgi network (TGN)17. We utilized an anti-GFP nanobody to ensure versatility, enabling the targeting of fusion proteins tagged with extracellular GFP, YFP, or mCerulean -- fluorescent tags that are widely available and often already characterized. Functionalization of the anti-GFP nanobody with mCherry, APEX2, or tyrosine sulfation motifs allowed us to visualize cargo uptake by fixed and live-cell imaging, localize retrograde transport compartments at the ultrastructural level, perform targeted compartment ablation, and quantitatively assess transport kinetics to the TGN. One limitation of this approach is the prerequisite of genetic modification of the target cell lines -- either by stable overexpression or endogenous tagging -- before functionalized anti-GFP nanobodies can be effectively employed. However, this strategy can be readily adapted to the growing repertoire of nanobodies directed against untagged, endogenous target proteins generated through animal immunization or in vitro selection from synthetic VHH libraries using ribosome or phage display technologies34. In addition, nanobodies targeting commonly used epitope tags, such as mCherry, V5, or ALFA, have been developed in recent years35,36,37. Notably, we recently utilized a sulfation-competent anti-mCherry nanobody to investigate retrograde trafficking of CDMPR in GGA2-depleted HeLa cells through rapid functional inactivation28. Small epitope tags offer the advantage of preserving the native conformation and function of the target protein, as they are less likely to interfere with critical structural domains. Nanobodies recognizing these compact epitopes (e.g., ALFA, V5) can be seamlessly integrated into the functional nanobody toolkit to extend its utility in studying endocytic and retrograde trafficking mechanisms.
In the present study, we demonstrate the application of VHH-mCherry in live-cell imaging experiments. Using EGFP-CDMPR and TfR-EGFP as reporter proteins, we were able to visualize receptor-specific endocytic uptake and their progression toward steady-state distribution. Quantitative analysis of the trafficking of these reporters from the plasma membrane to their respective intracellular compartments revealed kinetics consistent with those obtained through biochemical assays presented in Figure 2 of a previous study17. We found that the apparent half-life was considerably longer for EGFP-CDMPR (~9 min) than for TfR-EGFP (~4 min), with the signals becoming saturated after about 43 min and 20 min, respectively. These quantitative findings align well with previously published data on MPR and TfR trafficking dynamics38,39,40. Notably, the VHH-mCherry construct has garnered significant attention from the research community, as evidenced by numerous Addgene distribution requests, and has already been successfully implemented in several independent studies28,41,42,43,44.
Depending on the microscope configuration or experimental requirements, mCherry-tagged anti-GFP nanobodies may not always be optimal. In such cases, alternative fluorophores or fluorescent protein variants may provide more suitable spectral properties when used in combination with GFP reporter cell lines. To address this, we established a library of functionalized nanobodies incorporating a diverse range of fluorescent proteins, including mTagBFP2, mNeptune2.5, mCardinal2, TagRFP657, and iRFP670 (243764-68). These additional nanobody constructs will be made available through Addgene. The use of spectrally distinct fluorophores enables clear separation of excitation and emission signals, thereby facilitating optimal live, real-time imaging of up to three fluorescence channels with minimal spectral overlap.
While monitoring endocytic uptake to steady state may appear straightforward, tracking retrograde transport of proteins to the TGN presents a greater experimental challenge. In addition to VHH-mCherry and a GFP-tagged reporter protein, an additional fluorescent or dye-conjugatable TGN-resident marker is required for visualization and quantification. However, certain Golgi-resident proteins exhibit partial leakiness to the cell surface, which can be further exacerbated by perturbations of intracellular trafficking pathways, such as those introduced by experimental manipulation of transport machinery components. TGN46, for instance, is commonly used as a Golgi-resident marker despite its functional role as a cargo receptor mediating TGN-to-cell surface transport of certain secretory cargoes16,45. Similarly, glycosyltransferases such as &#946;-1,4-galactosyltransferase have also been detected at the plasma membrane41. Although these proteins can cycle to the surface, their predominant steady-state localization remains within the TGN. Tagging such markers with a spectrally distinct fluorescent protein or a dye-compatible tag (e.g., SNAP, Halo, etc.) and stably co-expressing them with a GFP-tagged transmembrane reporter would enable quantitative live-cell imaging of TGN arrival. This could be measured analogously to the workflow described above, with quantification based on the accumulation of mCherry signal within the fluorescence channel corresponding to the Golgi-resident fusion protein. Current strategies for investigating retrograde trafficking in fixed or live cells often rely on chimeric receptor constructs in combination with IgG antibodies (e.g., CD8)46,47. However, the inherent risk of cross-linking due to the bivalency of conventional IgGs during surface labeling can significantly alter the intracellular fate of the labeled protein, frequently redirecting it toward lysosomal degradation after endocytosis48,49. In contrast, nanobodies, due to their monovalent nature and 1:1-binding stoichiometry, do not induce crosslinking, thereby preserving the physiological trafficking routes of the target protein. This makes nanobody-based approaches particularly valuable for studying authentic retrograde transport dynamics without introducing artificial routing artifacts.
Optimal performance of this protocol for live-cell imaging requires careful titration of nanobody concentration, as excessive amounts lead to elevated background fluorescence from unbound nanobody in the medium. Furthermore, differences in expression levels of GFP reporter cell lines must be accounted for during titration to ensure accurate quantitative comparison. For nanobody production, low-temperature induction in E. coli is critical to promote proper folding of the mCherry domain. Higher induction temperatures typically result in misfolded or degraded protein and are, therefore, not recommended.
The success of this protocol critically depends on three key aspects: first, the high-yield expression and purity of functional nanobody constructs to ensure consistent labeling and minimal background; second, the use of well-characterized cell lines stably expressing GFP-tagged reporter proteins with cell surface localization to recapitulate physiological trafficking pathways; and third, optimal handling and configuration of the fluorescence microscopy setup, including imaging parameters and spectral separation, to enable accurate quantitative and qualitative analysis of cargo transport dynamics.
The method and protocol presented here, centered on the use of mCherry-tagged anti-GFP nanobodies, enable both quantitative and qualitative tracking of cargo proteins from the cell surface to endocytic compartments in cultured mammalian cells. When co-expressed with a fluorescently labeled Golgi-resident marker, this approach further allows the analysis of retrograde transport pathways. As such, this tool provides a powerful strategy to monitor the intracellular trafficking of cell surface transmembrane proteins and receptors that were previously inaccessible to labeling due to the absence of specific ligands or compatible monomeric binding reagents.

Endocytosis of receptors and other transmembrane (TM) proteins from the plasma membrane to various intracellular compartments is important for the maintenance of membrane homeostasis1,2. Following internalization by clathrin-dependent or -independent endocytosis, protein cargoes first populate early endosomes from where they are further sorted either along the endo-lysosomal system, recycled to the plasma membrane, or retrogradely shipped to the trans-Golgi network (TGN)3,4,5. Recycling from endosomes and/or the cell surface to the TGN is part of the functional cycle of a number of transmembrane cargo receptors, such as the cation-dependent and cation-independent mannose-6-phosphate receptors (CDMPR and CIMPR), delivering newly synthesized lysosomal hydrolases from the TGN to late endosomes and lysosomes6,7,8, Wntless (WLS) transports Wnt ligands to the cell surface9,10,11,12, or TGN46 that escorts soluble secretory cargo proteins, including pancreatic upregulated factor (PAUF) and other CARTS cargo, from the TGN13,14,15,16. While all these cargo receptors commute through the TGN for the collection of new client proteins, the transferrin receptor (TfR) that internalizes iron-bound transferrin is excluded from the Golgi return17,18,19,20,21.
To investigate endocytic and retrograde traffic, we previously established a nanobody-based toolkit designed to label and trace cargo proteins from the cell surface to intracellular compartments17. Nanobodies constitute a novel class of protein binders derived from homodimeric heavy-chain-only antibodies (hcAbs) naturally found in camelids and cartilaginous fishes22,23. They represent the variable heavy-chain domain (VHH) of hcAbs and offer numerous advantages over conventional antibodies (e.g., IgGs): they are monomeric, small (~15 kDa), highly soluble, lack disulfide bonds, can be bacterially expressed, and are amenable to selection for high-affinity binding24. To enhance the versatility and broad applicability of this nanobody toolkit, we employed functionalized anti-GFP nanobodies to surface-label and track proteins tagged with GFP tags on their extracellular or lumenal domains. Through recombinant conjugation of nanobodies with mCherry, ascorbate peroxidase 2 (APEX2), or tyrosine sulfation sequences, the retrograde trafficking of bona fide transmembrane cargo proteins can be analyzed by fixed and live-cell fluorescence microscopy, electron microscopy, or biochemical assays using autoradiography. Given that tyrosine sulfation, catalyzed by tyrosylprotein sulfotransferases TPST1 and TPST2, is a posttranslational modification confined to the trans-Golgi/TGN, this strategy enables direct assessment of transport dynamics and kinetics from the cell surface to the Golgi compartment25,26,27. More recently, we have expanded this repertoire of functionalized protein binders to include derivatized anti-mCherry nanobodies. These reagents have proven instrumental in dissecting machinery-dependent transport pathways of MPRs, particularly of the CDMPR17,28, to the TGN through biochemical analyses.
While the previous study mainly focused on the application of nanobodies modified with tyrosine sulfation sequences to biochemically assess TGN arrival by autoradiography19, this methods article describes the generation of fluorescently labeled, functionalized nanobodies (VHH-mCherry) and reporter cell lines for live-cell imaging of endocytic trafficking. In combination with an additional Golgi-resident fluorescent reporter protein, this protocol can be further adapted to serve as a robust workflow for quantitatively analyzing live-cell imaging of endosome-to-TGN transport of selected cargo proteins.
Before applying this protocol, researchers should ensure that the target protein is expressed as a GFP-tagged fusion construct, typically generated by standard molecular cloning approaches. Functionalized nanobodies can be readily produced in bacteria at yields sufficient for surface labeling experiments, with working concentrations in the low nanomolar range being suitable for most live-cell imaging assays. Users should also consider that transport kinetics may vary between cargo proteins, requiring adjustment of labeling duration or imaging frequency for optimal visualization of endocytic or endosome-to-TGN trafficking.

<table><tbody><tr><td>Anti-GFP antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>118144600001</td><td>Product is distributed by Sigma-Aldrich, but manufactured by Roche</td></tr><tr><td>100-mm cell culture dishes</td><td>TPP</td><td>TPP93100</td><td></td></tr><tr><td>Anti-actin antibody</td><td>EMD Millipore&amp;nbsp;</td><td>MAB1501</td><td></td></tr><tr><td>Anti-HA antibody</td><td>from lab</td><td>made from 12CA5 hybridoma</td><td></td></tr><tr><td>Anti-His6 antibody</td><td>Bethyl Laboratories&amp;nbsp;</td><td>A190-114A</td><td></td></tr><tr><td>Anti-T7 antibody&amp;nbsp;</td><td>Bethyl Laboratories&amp;nbsp;</td><td>A190-117A</td><td></td></tr><tr><td>BL21(DE3) Competent&amp;nbsp;E. coli</td><td>NEB</td><td>C2527H</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Merck Millipore</td><td>102382</td><td>dissolved in sterile water, stock is 1 M</td></tr><tr><td>Carbenicillin disodium salt</td><td>Applichem</td><td>A1491</td><td>dissolved in sterile water, stock is 100 mg/mL</td></tr><tr><td>Coomassie-R (Brilliant Blue)</td><td>Sigma-Aldrich</td><td>B-0149</td><td></td></tr><tr><td>D-biotin</td><td>Sigma-Aldrich</td><td>B4501</td><td>dissolved in sterile 500 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;or DMSO</td></tr><tr><td>Disposable PD10 desalting columns</td><td>GE Healthcare</td><td>GE17-0851-01</td><td></td></tr><tr><td>DMEM GlutaMAX-I</td><td>Thermo Fisher Scientific</td><td>61965026</td><td></td></tr><tr><td>DMEM, high glucose, no glutamine, no phenol red</td><td>Thermo Fisher Scientific</td><td>31053028</td><td></td></tr><tr><td>DNase I</td><td>Applichem</td><td>A3778</td><td>dissolved in sterile water</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS) w/o Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>A5256701</td><td></td></tr><tr><td>Filtropur S, PES, Porengr&amp;ouml;&amp;szlig;e: 0.45 &amp;micro;m</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>FuGENE HD transfection reagent</td><td>Promega&amp;nbsp;</td><td>E2311</td><td></td></tr><tr><td>Glass coverslips (No. 1.5H)</td><td>VWR</td><td>631-0153</td><td></td></tr><tr><td>Goat anti-mouse HRP</td><td>Sigma-Aldrich</td><td>A-0168</td><td></td></tr><tr><td>Goat anti-rabbit HRP</td><td>Sigma-Aldrich</td><td>A-0545</td><td></td></tr><tr><td>His buffer kit</td><td>Cytvia</td><td>11003400</td><td></td></tr><tr><td>His GraviTrap columns</td><td>Cytvia</td><td>11003399</td><td></td></tr><tr><td>ibidi &amp;micro;-Slide 4 well, ibiTreat</td><td>80426-IBI</td><td>ibidi</td><td></td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranosid (IPTG)</td><td>Applichem</td><td>A1008</td><td>dissolved in sterile water, stock is 1 M</td></tr><tr><td>Kanamycin sulfate</td><td>Applichem</td><td>A1493</td><td>dissolved in sterile water, stock is 100 mg/mL</td></tr><tr><td>L-glutamine</td><td>Applichem</td><td>A3704</td><td></td></tr><tr><td>Lysozyme&amp;nbsp;</td><td>Sigma-Aldrich</td><td>18037059001</td><td>Product is distributed by Sigma-Aldrich, but manufactured by Roche</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Merck Millipore</td><td>105833</td><td>dissolved in sterile water, stock is 1 M</td></tr><tr><td>Mini-Protean TGX gels, 4-20%, 15-well</td><td>Bio-Rad</td><td>4561096</td><td></td></tr><tr><td>Modified HeLa Kyoto</td><td>from lab</td><td></td><td></td></tr><tr><td>NEB5-alpha Competent&amp;nbsp;E. coli</td><td>NEB</td><td>C2987U</td><td></td></tr><tr><td>NucleoBond Xtra Midi Plus, 10 preps</td><td>Macherey-Nagel&amp;nbsp;</td><td>740412.1</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Bioconcept&amp;nbsp;</td><td>4-01F00-H</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Applichem</td><td>A0999.0025</td><td>dissolved in 40% DMSO 60% isopropanol, stock in 500 mM</td></tr><tr><td>Phoenix ampho cell line</td><td>Nolan lab</td><td></td><td></td></tr><tr><td>Polybrene (hexadimethrine bromide)</td><td>Sigma-Aldrich</td><td>H9268</td><td></td></tr><tr><td>Puromycin</td><td>Invivogen</td><td>ant-pr-1</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Merck Millipore</td><td>106404</td><td>dissolved in sterile water, stock is 5 M</td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11360039</td><td></td></tr><tr><td>Trans-Blot Turbo Pack, mini</td><td>Bio-Rad</td><td>1704158</td><td></td></tr><tr><td>Tryptone</td><td>Applichem</td><td>A1553</td><td></td></tr><tr><td>Yeast extract&amp;nbsp;</td><td>Applichem</td><td>A1552</td><td></td></tr></tbody></table>

live-cell imaging of endocytic transport using functionalized nanobodies in cultured cells

Live-cell Imaging of Endocytic Transport using Functionalized Nanobodies in Cultured Cells

Endocytosis of receptors and other transmembrane proteins from the cell surface to endosomes and beyond is critical for homeostasis, physiology, and function. To investigate endocytic uptake and retrograde protein trafficking, we have established a versatile toolkit comprising functionalized nanobodies to monitor transport from the cell surface to the trans-Golgi network (TGN) by means of fixed and live-cell imaging, electron microscopy, and gel electrophoresis combined with autoradiography. We developed derivatized nanobodies targeting green fluorescent protein (GFP) or mCherry - monomeric, non-crosslinking, high-affinity protein binders - that can be added to cell lines expressing membrane proteins of interest bearing the corresponding extracellular fluorescence tags. Upon binding to GFP- or mCherry-tagged transmembrane reporters, the nanobodies are specifically internalized and trafficked in parallel with the reporters' endogenous sorting routes. These nanobodies were functionalized with selected fluorophores to track retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to resolve ultrastructural localization by electron microscopy, and with tyrosine sulfation motifs to quantitatively assess TGN arrival kinetics. In this methodological study, we detail the general protocol for bacterial expression and purification of functionalized nanobodies, as well as the generation of stable GFP-reporter cell lines. We exemplify the utility of this approach for live-cell imaging by employing an mCherry-modified anti-GFP nanobody (VHH-mCherry) to analyze the endocytic uptake of the transferrin receptor (TfR) and the cation-dependent mannose-6-phosphate receptor (CDMPR).

To investigate retrograde protein trafficking to various intracellular compartments, we have recently established an anti-GFP nanobody-based tool for labeling and tracking recombinant fusion proteins from the cell surface17. Here, we describe the bacterial production of such derivatized nanobodies and demonstrate their utility in monitoring endocytic uptake by live-cell imaging. In combination with a Golgi-resident fluorescent reporter, this protocol offers a robust platform to quantitatively study the retrograde transport of selected cargo proteins to the trans-Golgi network (TGN) in real time.
We have generated a collection of distinct functionalized anti-GFP nanobodies sharing a common modular architecture, using standard molecular cloning techniques17. Although the current study focuses on the fluorescent variant VHH-mCherry, we also include additional derivatized nanobodies to illustrate the versatility of this toolset and highlight their potential for future applications. Our most basic construct, VHH-std (std for standard), comprises the VHH domain, T7 and HA epitopes for antibody-based detection, a C-terminal hexahistidine (His6) tag for purification, and a biotin acceptor peptide (BAP) sequence to enable enzymatic biotinylation and high-affinity streptavidin-based pulldown assays (Figure 1A). From this core design, various nanobody derivatives were developed to facilitate the study of endocytic and retrograde trafficking through biochemical analysis, fixed and live-cell imaging, and electron microscopy.
For live-cell imaging of endocytic transport, we engineered a fluorescent anti-GFP nanobody incorporating a fluorophore with excitation and emission spectra distinct from those of GFP, thereby ensuring optimal spectral separation during fluorescence microscopy. Based on its superior folding properties in IPTG-induced E. coli and its well-characterized photophysical properties, we selected mCherry as the fluorescent tag of choice. Other red-shifted fluorophores, such as monomeric red fluorescent protein (mRFP), would also be suitable alternatives in principle, but mCherry proved particularly robust and effective in this system.
What is the potential of other functionalized nanobodies than VHH-mCherry? To investigate protein trafficking from the plasma membrane to the trans-Golgi network (TGN), we leveraged the compartment-specific localization of TPSTs, which reside exclusively in the TGN and trans-Golgi cisternae. To this end, we modified VHH-std with a tandem tyrosine sulfation motif (2xTS) derived from rat procholecystokinin29, thereby enabling a biochemical readout of cargo arrival at these compartments. While fusion of VHH-std to mCherry allows for direct visualization of retrograde transport by fixed or live-cell imaging, functionalization with peroxidases such as APEX230 permits ultrastructural localization by electron microscopy, targeted cytochemical ablation, or proximity-based biotinylation assays. Moreover, incorporation of a tobacco etch virus (TEV) protease cleavage site into the nanobody scaffold provides a biochemical means to distinguish between internalized and surface-bound nanobodies (Figure 1A). We previously employed the VHH-tev construct to monitor recycling kinetics of nanobody-bound EGFP-CDMPR and TfR-EGFP17. The reappearance of nanobody-tagged receptors at the cell surface could be readily detected by applying recombinant TEV protease extracellularly, resulting in a specific loss of the nanobody's C-terminal epitope cassette. Analogously, the inclusion of a TEV cleavage site within the here presented mCherry-functionalized nanobody VHH-mCherry allows for dynamic assessment of EGFP reporter recycling by live-cell imaging. Functionalization with alternative protein domains - such as additional fluorophores, enzymatic tags, or sequence motifs for posttranslational modification - can be readily accomplished by subcloning desired inserts into the VHH-std backbone by the SpeI and EcoRI restriction sites. All functionalized anti-GFP nanobody constructs used in the original study17 have been deposited with Addgene for public distribution.
Using the protocol described above, all nanobody variants illustrated here were purified to high yield and purity (Figure 1B). Only the mCherry fusion exhibited minor proteolytic clipping of protein domains following purification (Figure 1B, lane 5). The two observed degradation products most likely correspond to the individual VHH and mCherry domains, as inferred from their apparent molecular weights and verified by epitope-specific immunoblot detection (Figure 1C). In the absence of co-expressed BirA, VHH-mCherry typically recovered at yields of approximately 20 mg per preparation. Based on our experience, co-expression of BirA consistently reduces nanobody yield by roughly 1/3rd to 1/2. Biotinylation was fairly complete because the nanobodies from a 1:1 mixture with BSA were fully recovered by streptavidin-agarose (Figure 1D).
To evaluate the suitability of this nanobody toolkit for studying endocytic transport, we generated stable HeLa cell lines expressing EGFP-tagged surface receptor proteins with distinct intracellular trafficking routes. These included TfR, which cycles between the plasma membrane and early (sorting and recycling) endosomes; TGN46, which traffics between the plasma membrane and the TGN by early endosomes; and both MPRs, which shuttle between the TGN, plasma membrane, and both early and late endosomes17. EGFP was fused to the extracellular domain of each receptor - specifically, inserted between the signal peptide and the receptor sequence for CDMPR, CIMPR, and TGN46 - and to the C-terminus of TfR. This design preserved the native cytoplasmic domains, ensuring that all known sorting signals remained intact and the EGFP tag accessible for binding by extracellular anti-GFP nanobodies (Figure 1E). For CIMPR, whose extracellular domain is unusually large, a truncated version was used, consistent with previous studies demonstrating that such truncation preserves normal trafficking behavior31,32.
Stable cell lines were established by retroviral transduction, followed by fluorescence-activated cell sorting (FACS) to isolate homogeneous cell populations with moderate and comparable expression levels. Of note, EGFP-CDMPR consistently appeared as a double band in immunoblots, similar to its endogenous counterpart33, indicative of heterogeneous glycosylation. To assess whether the EGFP fusion proteins recapitulate the steady-state localization and expression patterns of the endogenous proteins, the stably expressing cells were co-cultured with parental HeLa cells and analyzed by confocal fluorescence microscopy (Figure 2B). The EGFP signal faithfully mirrored the distribution of the corresponding endogenous proteins. As expected, CDMPR, CIMPR, and their EGFP-tagged versions localized predominantly to the perinuclear region - reflecting TGN and late endosomal compartments - with additional labeling in peripheral endosomes. Both endogenous and EGFP-tagged TGN46 were found almost exclusively in the perinuclear TGN, while TfR and TfR-EGFP displayed the characteristic early endosome distribution, with prominent peripheral sorting and perinuclear recycling endosomes. Although the antibodies used detected both the endogenous and EGFP-tagged forms (except for CIMPR), the overall staining intensity was not markedly increased in cells expressing the fusion proteins, suggesting that the EGFP constructs were not substantially overexpressed. We included EGFP-TGN46 and EGFP-CIMPR in Figure 2B for direct comparison with EGFP-CDMPR and TfR-EGFP. The protocols used for cell fixation and fluorescence microscopy imaging are outlined in previous studies17,19.
Using VHH-mCherry, endocytic transport of EGFP-tagged reporter proteins can be monitored by live-cell imaging. For this, we used as examples cells expressing EGFP-CDMPR and TfR-EGFP. The cells were imaged over time with an inverted widefield fluorescent microscope upon addition of VHH-mCherry to the medium (Video 1 and Video 2). Still images at various time points are shown in Figure 3. Uptake was quantified by measuring the signal in the mCherry channel, subtracting the autofluorescence background, and normalizing to the EGFP signal to eliminate fluctuations due to the movement of labeled compartments or potential small shifts in the focal plane. The fluorescence of VHH-mCherry in the medium at 25 nM was negligible and did not interfere with the measurements. Analysis of transport of the reporters from the cell surface to their intracellular compartments, to the steady-state distribution, yielded the same kinetic results as the biochemical experiments shown in Figure 2 of the previous publication17, with apparent half-lives of uptake of ~9 min for EGFP-CDMPR and ~4 min for TfR-EGFP, and saturation after ~43 min and ~20 min, respectively. These values were comparable with values obtained from biochemical uptake experiments using immunoblotting assays17. In principle, the kinetics of retrograde transport into subcellular regions of interest, such as the perinuclear region of the highest concentration of MPRs, can also be analyzed. However, the perinuclear region contains not only Golgi/TGN but is also enriched in late endosomes and recycling endosomes. The kinetics of nanobody uptake into the perinuclear region are not sufficiently specific to analyze retrograde transport to a defined organelle. To more reliably assess plasma membrane-to-TGN transport, another fluorescent protein, a TGN-resident transmembrane protein, must be stably co-expressed along with the GFP reporter protein, in a way that there is no spectral overlap of fluorophore properties. Using this extra marker allows the detection of reporter-imported VHH-mCherry, analogous to tyrosine sulfation mediated by TPSTs, as previously documented19.
<img alt="Protein analysis via SDS-PAGE and Western blot; schematic shows VHH variants tagging; electrophoresis results." class="xfigimg" src="/files/ftp_upload/69284/69284fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Design and production of derivatized nanobodies for tracking EGFP-tagged cell surface proteins. (A) Schematic overview of the derivatized nanobodies. The standard nanobody construct comprises a GFP-specific VHH domain, T7 and HA epitope tags, a biotin acceptor peptide (BAP), and a C-terminal hexahistidine (His6) tag for purification. Additional nanobody variants include modifications with tandem tyrosine sulfation sites (2xTS), the engineered peroxidase APEX2, or the fluorescent protein mCherry. Scale bar indicates amino acid (aa) length. (B) Bacterially expressed and affinity-purified nanobodies (20-50 µg) were analyzed by gradient SDS-PAGE and visualized by Coomassie staining. Molecular weight standards (in kDa) are indicated on the left. Minor proteolytic clipping was observed only for VHH-mCherry, likely occurring between the VHH and mCherry domains. (C) Immunoblot analysis of nanobody preparations (10 ng) using antibodies directed against T7, HA, or His6 epitopes, or detected by streptavidin-HRP (SA-HRP) for biotinylation assessment. (D) The extent of nanobody biotinylation was evaluated by incubating nanobodies at a 1:1 ratio with BSA, followed by streptavidin-agarose pulldown, pelleting, and washing of the beads. Equal volumes of the supernatant (S) and the bead-bound material (B) were subsequently analyzed by SDS-PAGE and Coomassie staining. Quantitative recovery of the nanobody in the bead-bound fraction indicates complete biotinylation. The presence of both VHH and mCherry fragments in the bound fraction suggests partial degradation, likely occurring during sample preparation for SDS-PAGE analysis. The white line between lanes 2 and 3 indicates the deletion of two unrelated lanes. This figure has been modified from17. <a href="https://www.jove.com/files/ftp_upload/69284/69284fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="EGFP-tagged protein localization; microscopy and Western blot analysis; protein expression; diagram and experiment results." class="xfigimg" src="/files/ftp_upload/69284/69284fig2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Expression and intracellular localization of fluorescently labeled EGFP-tagged cargo receptors. (A) Schematic representation of the EGFP fusion constructs. Sequences derived from secretory transmembrane proteins are depicted in black, with N-terminal signal peptides and internal transmembrane domains highlighted in yellow. The EGFP moiety is illustrated in green. Full-length constructs were generated for CDMPR, TfR, and TGN46, while a well-characterized truncated variant was used for CIMPR to preserve normal trafficking behavior. Scale bar indicates amino acid (aa) length. EGFP-CDMPR and TfR-EGFP have been described previously17. (B) To assess subcellular localization, HeLa cells stably expressing EGFP fusion proteins were co-cultured with parental HeLa cells and analyzed by fluorescence microscopy. Scale bar represents 10 µm. (C) HeLa cells stably expressing EGFP-tagged reporter proteins were lysed, and protein extracts were subjected to SDS-PAGE followed by immunoblotting using antibodies against GFP and actin. Molecular weight markers (in kDa) are indicated on the right. This figure has been modified from17. <a href="https://www.jove.com/files/ftp_upload/69284/69284fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="EGFP-CDMPR and TfR-EGFP fluorescence microscopy, time-lapse protein colocalization, intensity graphs." class="xfigimg" src="/files/ftp_upload/69284/69284fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Live-cell imaging of nanobody endocytic uptake kinetics mediated by EGFP-CDMPR and TfR-EGFP. Live-cell imaging was performed following the addition of 25 nM VHH-mCherry to HeLa cells stably expressing (A) EGFP-CDMPR or (B) TfR-EGFP in phenol red-free complete medium at 37 °C. Cells were imaged in the GFP and mCherry channels using a semi-automated widefield fluorescence microscope at 36 s intervals. Representative merged still images are shown, accompanied by enlarged views of the perinuclear region (magnification: 2.2x) in individual channels below. (Scale bars, 10 µm.) See also Video 1 and Video 2. Quantitative analysis of nanobody uptake kinetics in (C) EGFP-CDMPR and (D) TfR-EGFP was performed using data from three independent experiments, each capturing approximately 40 individual cells. To account for organelle movement and focal plane variability, the mCherry fluorescence intensity was normalized (norm) to the GFP signal and plotted as the mean ± SD across all cells from the three experiments. The average maximal uptake signal was normalized to 1. Uptake kinetics for each cell were individually fitted using a first-order kinetic model. The plotted lines represent the averaged curves derived from the mean of the corresponding rate constants. This figure has been modified from17. <a href="https://www.jove.com/files/ftp_upload/69284/69284fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Live-cell imaging of mCherry-nanobody uptake by EGFP-CDMPR. HeLa cells stably expressing EGFP-CDMPR were incubated at 37 °C in complete medium containing 25 nM VHH-mCherry and imaged in both the EGFP and mCherry fluorescence channels at 36 s intervals. The movie was rendered at a playback rate of 5 frames/s, corresponding to a real-time compression of 3 min/s. This movie has been modified from17. <a href="https://www.jove.com/files/ftp_upload/69284/MOVIE%201.mp4" target="_blank">Please click here to download this Video.</a>
Video 2: Live-cell imaging of mCherry-nanobody uptake by TfR-EGFP. HeLa cells stably expressing TfR-EGFP were incubated at 37 °C in complete medium supplemented with 25 nM VHH-mCherry and imaged for EGFP and mCherry fluorescence at 36 s intervals. The movie was rendered at 5 frames/s, representing a time-lapse rate of 3 min/s. This movie has been modified from17. <a href="https://www.jove.com/files/ftp_upload/69284/MOVIE%202.mp4" target="_blank">Please click here to download this Video.</a>

Endocytic and retrograde transport of proteins from the plasma membrane to the trans-Golgi network is essential to maintaining membrane homeostasis and regulating signaling. Here, we describe a method to image and quantify endocytic transport of transmembrane cargo proteins by live-cell microscopy using derivatized anti-GFP nanobodies in HeLa cells.

Endocytosis of receptors and other transmembrane proteins from the cell surface to endosomes and beyond is critical for homeostasis, physiology, and ...

We are studying the endocytic and retrograde traffic of transmembrane proteins as well as the machinery that confers the transport. To investigate protein traffic from the cell surface, conventional antibodies are typically used, but they might promote cross-linking and lysosomal targeting. To begin, obtain a pellet of Escherichia coli bacteria transformed with VHH-mCherry.Add 30 milliliters of ice cold binding buffer containing 20 millimolar imidazole in PBS to the bacterial cell pellet. With a pipette, resuspend the pellet thoroughly by pipetting up and down. Transfer the resuspended mixture into a labeled 50 milliliter centrifuge tube.Supplement the resuspended cells with 200 micrograms per milliliter lysozyme, 20 micrograms per milliliter DNase I, one millimolar magnesium chloride and one millimolar phenylmethylsulfonyl fluoride. After incubating the tube at room temperature for 10 minutes, place it on an end over end rotator and continue incubation at four degrees Celsius for one hour. Next, insert a six millimeter solid probe tip of the sonicator directly into the cell suspension.Mechanically disrupt the bacterial cells using three one minute sonication pulses at 40%amplitude and a duty cycle of one second on and one second off. Allowing a one minute cooling interval between each pulse. After centrifugation, keep the cleared lysate on ice while preparing for immobilized metal affinity chromatography using pre-packed, single-uses tag purification columns designed for gravity flow operation.Secure nickel-nitrilotriacetic acid columns onto a metal stand or an appropriate column holder. Then drain the storage buffer from the column completely. Equilibrate the column by adding 10 milliliters of binding buffer containing 20 millimolar imidazole in PBS.Allow the buffer to pass through by gravity. Gradually apply approximately 30 milliliters of the cleared bacterial lysate to the equilibrated column. Allow it to pass through by gravity and discard the flow through.Then wash the column by applying two consecutive 10 milliliter volumes of binding buffer containing 20 millimolar imidazole in PBS. Elute the bound nanobodies by applying two milliliters of elution buffer containing 500 millimolar imidazole in PBS into a two milliliter microcentrifuge tube. Equilibrate a desalting column seeded in a 50 milliliter tube adapter by rinsing five times with five milliliters of PBS.Let the buffer fully enter the packed resin, then discard the flow through. After the final rinse, centrifuge the column at 1000 x g for two minutes. Now move the column with its adapter onto a new 50 milliliter collection tube after discarding the flow through.Load two milliliters of the eluted functionalized nanobody onto the pre-equilibrated desalting column. Then centrifuge again at 1000 x g for two minutes to collect the eluate before validating VHH-mCherry expression and purity. Launch the software of the automated live cell imaging system.Transfer the ibidi microscopy chamber onto the microscope stage. Set up two imaging channels using excitation and emission filters for EGFP and mCherry. Choose a short exposure time and low illumination intensity to avoid photo bleaching.Keep the settings constant for all experiments. Settings might differ between reporters. For each condition, store coordinates of 10 different fields of view containing three to four cells in focus in a point list.Then capture a single snapshot of each position in the point list to serve as the reference image before adding VHH-mCherry. To initiate endocytosis, gently add 350 microliters of prewarmed VHH-mCherry solution to each well while minimizing disturbance to the monolayer. Then proceed with image acquisition.Perform a time-lapse acquisition for 100 frames at 32nd intervals while maintaining 37 degrees Celsius and 5%carbon dioxide. To analyze endocytic uptake of VHH-mCherry, load the time-lapse image sets into the image analysis software. Apply segmentation and tracking tools to quantify internalized fluorescence over time across different regions of interest.All functionalized nanobody variants were purified to high-yield and purity, with only VHH-mCherry exhibiting minor proteolytic degradation. The degradation products of VHH-mCherry were confirmed as individual VHH-mCherry domains using epitope-specific immunoblot detection. In the absence of BirA, VHH-mCherry was recovered at approximately 20 milligrams per preparation.And biotinylation was confirmed by streptavidin agarose pulldown of nanobody BSA mixtures. EGFP-tagged fusion proteins expressed in HeLa cells showed subcellular localization patterns consistent with their endogenous counterparts, including perinuclear localization of EGFP-CDMPR and EGFP-CIMPR. Exclusive perinuclear localization of EGFP-TGN46 and peripheral localization of TfR-EGFP.VHH-mCherry enabled live cell visualization of endocytic transport of EGFP-CDMPR, showing increasing colocalization over 60 minutes. Uptake of VHH-mCherry into EGFP-CDMPR expressing cells reached a plateau after approximately 43 minutes, with a half-life of nine minutes. In TfR-EGFP-expressing cells, VHH-mCherry rapidly accumulated intercellularly with a half-life of four minutes and saturation after 20 minutes.We have established versatile nanobody-based toolkit to analyze transport of any transmembrane protein from the cell surface. We use fluorescent nanobodies to label surface cargo proteins and then we study their endocytic trafficking by live cell microscopy. With our nanobody-based live cell imaging approach, we want to uncover pathways that drive surface to trans-Golgi network cargo transport.

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Research

JoVE Journal

Biochemistry

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking.  Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Source: Zhang, Y., et al. Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria. J. Vis. Exp. (2015)This video demonstrates the use of fluorescent nanoparticles to visualize endo-lysosomal remodeling in human epithelial cells infected by intracellular bacteria. Following bacterial invasion, the pathogens manipulate the host cytoskeleton and endo-lysosomal system, forming vacuoles with tubular projections that alter vesicle transport.

Nanoparticle-Mediated Visualization of Endo-Lysosomal Remodeling in Bacterially-Infected Cells

This video demonstrates a method to study the uptake and retrograde transport of functionalized nanobodies to understand their intracellular trafficking pathways. By introducing radiolabelled sulfate and utilizing isolation and detection techniques, this method enables the study of endocytic uptake and trans-Golgi network arrival of cargo proteins.

Monitoring the Arrival of GFP-Tagged Surface Proteins to the Trans-Golgi Network Using Functionalized Nanobodies

This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles' movement.

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Learn live-cell imaging of endocytic transport using functionalized nanobodies in HeLa cells. This method enables visualization and quantification of transmembrane protein trafficking dynamics.

Live-cell Imaging of Endocytic Transport using Functionalized Nanobodies in Cultured Cells

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