Background

Protein complexes execute fundamental functions involved in various biological processes. To have these molecular machines assembled in a timely manner in a precise cellular location is critical for cellular activities, including protein synthesis, chromosome segregation, nuclear transport, signaling, motility and other diverse functions. Typically, the formation of many multimeric complexes is thought to occur in a stepwise manner via recruitment and integration of subunits or subcomplexes, which are bridged to existing molecules within the larger assembly by a physical protein-protein contact. Understanding the assembly of large macromolecular complexes is historically the job of biochemists and crystallographers who employ an arsenal of techniques: purification of complexes to determine the identity of subunits, co-purification of proteins, yeast two-hybrid screening, in vitro binding assays, chemical cross-linking studies and structural analysis to understand protein-protein interactions, and finally, reconstitution of the complex to determine the hierarchy of assembly.

While this traditional approach has been successful in many cases, a major challenge is incorporating knowledge from studies on a handful of proteins to the larger and more relevant problem of macromolecular complex assembly within the cell. In cells, the timing of protein production, protein localization and post-translational modifications affect distribution, local concentrations and interactors that in turn influence complex assembly. A number of imaging-based techniques have been developed to study protein-protein interactions within cells, including fluorescent resonance energy transfer (FRET), which examines energy transfer between pairwise donor-acceptor fluorophores linked to proteins of interest; fluorescence cross-correlation spectroscopy (FCCS), which measures the diffusion pattern of two fluorescent molecules through the focal volume; and bimolecular fluorescence complementational (BiFC), which is based on ability of proteins in close proximity to self-complement and create a functional fluorescent protein from two non-fluorescent protein fragments (Bacia et al., 2006; Slaughter et al., 2007; Kerppola, 2008; Miller et al., 2015; Unruh et al., 2018). These techniques can be used in live cells to study endogenously expressed protein-protein interactions in a pairwise manner provided the molecules of interest can be fused to fluorescent proteins (FP). In yeast, genes can be easily tagged at the N- or C-termini using PCR-based methods (Gardner and Jaspersen, 2014). With the development of CRISPR/Cas9 methodologies, genome editing is becoming increasingly feasible such that these approaches can be applied in a wide array of systems. The use of endogenous proteins, rather than transgenes, greatly minimizes false positives caused by high levels of protein expression. In comparison to in vitro assays, FRET, FCCS and BiFC have the advantage of indicating where within a cell the protein-protein interaction occurs (for example, see Muller et al., 2005; Cabantous and Waldo, 2006; Kerppola, 2006; Slaughter and Li, 2010; Sung et al., 2013; Chen et al., 2014; Smoyer et al., 2016; Hennen et al., 2018). However, these methods are limited in their spatial resolution due to the diffraction limit (~200 nm for most FPs), and combining FRET or FCCS with super-resolution methods in cells is extremely challenging.

Structured illumination microscopy (SIM), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM) are examples of super-resolution microscopy (SRM) approaches that are pushing cell biology in new directions once thought impossible by beating the diffraction limit (Huang et al., 2009; Schermelleh et al., 2010). The ~10-100 nm resolution achievable by these methods has enhanced our overall understanding of cell biology, particularly at the level of organelles, membranes and other cellular structures. The application of SRM to protein-protein interactions within nanosized protein complexes has been slower to develop, in part because it is difficult to study endogenous proteins with many of these methods, particularly if multiple fluorophores/FPs are needed to monitor interactions and to provide orientation of where within the complex or the assembly cycle the interaction is occurring. While a plethora of FPs have been developed, linear unmixing to image multiple fluorophores has not been integrated into SRM yet, limiting the use of many combinations of FPs. To overcome this obstacle, we combined a version of BiFC based on split-GFP to assay protein-protein interactions (Figure 1).To provide spatial and temporal information as to where and when binding within our complex of interest occurs at sub-diffraction resolution, we developed a three-color imaging strategy using HyVolution with 140 nm lateral resolution (Chen et al., 2019). Importantly, our protocol allows for imaging of endogenously expressed proteins in live cells fused to mTurquoise2 and GFP, along with mCherry, without spectral cross-talk. While the method was developed in Saccharomyces cerevisiae, it is suitable for a wide variety of systems, and it greatly expands our ability to inspect the step-wise assembly pathway of heteromeric macromolecular machines by elucidating protein-protein interactions in vivo at the nanoscale level.