Background

The fluorescent labeling of proteins to follow in real time their spatiotemporal localization in living cells was mainly achieved until now by using transgenic or overexpression-based approaches. However, the labeling of specific endogenous proteins or even posttranslational modifications in living cells is not yet routinely possible. Imaging of cellular structures and processes is typically performed by either immunofluorescence (IF) labeling on fixed cells or by exogenously overexpressing fluorescent fusion proteins in living cells. Although these well-established techniques showed to be very powerful to locate or follow proteins inside the cells, they inherit also some important drawbacks. In IF, the cells need to be chemically fixed and permeabilized to be able to incubate them with specific primary and secondary antibodies. Despite many variables and potential artifacts (Schnell et al., 2012; Teves et al., 2016) like fixation-related protein denaturation or permeabilization efficiency, IF is still often used to visualize target proteins in fixed cells or tissues. Otherwise, imaging of proteins in living cells is mainly achieved through the exogenously expression of fluorescent fusion proteins (Ellenberg et al., 1999; Betzig et al., 2006; Schneider and Hackenberger, 2017) or by knock-in of a fluorescent tag into the endogenous locus using the CRISPR/Cas9 technology (Ratz et al., 2015). Although fluorescent fusion proteins have been proven to be very powerful, they often do not behave as their endogenous counterparts due to their increased levels when exogenously overexpressed (Burgess et al., 2012). On the other hand, endogenous fusion proteins containing knocked-in tags are difficult to obtain as knock-in efficiencies are often very low. Consequently, there is a need for new and easy to implement imaging approaches to visualize endogenous target proteins in single living cells. Previous studies and methods, like FabLEM or the expression of mintbodies, showed that intracellular labeling of proteins with fluorescently labeled antibody fragments can give new insights into the dynamics of histone modifications (Hayashi-Takanaka et al., 2009; Hayashi-Takanaka et al., 2011; Sato et al., 2013). However, these techniques suffer from lower delivery efficiencies into living cells, or potential poor solubility of the intracellular expressed mintbodies. Recently, another method achieved fluorescent labeling of endogenous proteins by using a bacterial toxin called streptolysin O, which creates pores in the membrane of cells and allows for the delivery of fluorescent probes into living cells (Teng et al., 2016). However, this method requires additional steps to be able to reseal the membrane after treatment which can be quite harmful for the cells and can decrease cell viability. In contrast, our versatile antibody-based imaging approach (VANIMA) uses fluorescent dye-conjugated antibodies or Fabs, which are delivered into the cells by electroporation (Freund et al., 2013; Brees and Fransen, 2014). The antibody labeling reaction is highly efficient and can result in up to 5-7 fluorescent dyes per molecule of antibody depending on the antibody and the labeling kit used. The transduction of the antibodies has a very high delivery efficiency and viability of the cells is above 90% in human cancer cell lines such as U2OS. Afterwards, the transduced antibodies will bind to the endogenous target protein inside the cell and for nuclear targets they will be transported with the target protein into the nucleus (piggyback mechanism). Otherwise, for faster delivery into the nucleus of the cells, the antibodies can be digested to produce Fabs which can freely diffuse into the nucleus to find and bind their target. Thus, even proteins with posttranslational modifications in the nucleus can be visualized specifically using fluorescently-labeled Fabs against the target. Considering that there are several thousands of commercially-available antibodies that specifically recognize intracellular target proteins with high affinity, VANIMA can be used to uncover the dynamical behavior of a plethora of targets in living cells (Conic et al., 2018). Besides nuclear targets, the antibodies could also be used to label and image cytoplasmic structures/proteins. However, it is important to note that only proteins that are either directly accessible for the antibodies/Fabs or that can be reached through the piggyback mechanism can be labeled using this technique. We were already able to label α-tubulin in the cytoplasm but other accessible targets like the mitochondrial membrane or cytoplasmic vesicles could also be tested for labeling with VANIMA. However, specific labelling of cytoplasmic targets would only be possible if the target molecules are highly expressed. If their abundance in the cell is below the one of the introduced antibodies, a large fraction of the antibodies does not bind and will generate background staining. Additionally, the method is easy to implement in any laboratory and can also be used to perform multicolor imaging with different targets just by labeling two different antibodies with different dyes or by combining it with an already established endogenous knock-in clone. Finally, VANIMA can also be used with identified inhibiting antibodies to disrupt protein functions inside living cells.