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

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.