Background

Cell-sonar is an easy-to-implement method that allows indirect intracellular tracking of target proteins. The elucidation of secretory pathways has long been the primary objective of most research fields. Available methods for this are diverse microscopy techniques [1–4] or “omic” screenings [5]. These specific procedures result in high-definition data but, in almost all applications, they are complex, complicated, expensive, and elaborate. Our illustrated protocol is an alternative to these existing techniques, with the difference that cell-sonar is inexpensive, simple, and quick to apply. The results provide a first insight into protein localization.

The cell-sonar concept works universally for any application due to the principle of proteostasis, which regulates the protein balance in cells [6]. Several evolutionarily conserved signaling cascades for protein expression are controlled by proteostasis [6–8]. These protein networks are organized via signaling by recognition of stress responses and the maintenance of cellular proteins [7–9]. Internally, the endoplasmic reticulum (ER) controls the unfolded protein response (UPR) to each protein change [7] during its biosynthesis by regulating the ER capacity of nascent protein levels [7]. Signaling to the nucleus for the expression of required proteins is also orchestrated by UPR [10–12]. The signaling network located outside the ER is controlled by the mitogen-activated protein kinase (MAPK), and they interact in a synergistic manner [7]. The recognition of intracellular stress by MAPK-induced nucleus signaling is essential for gene expression [13]. This ability to dynamically detect changes, such as transgenic protein insertion or compound exposure, allows the detection of induced cellular protein expression. Exposure to a compound or expression of a transgenic target protein serves as an initiator of a stress response. Comparison of two cell lines that differ only in compound exposure or protein expression allows the detection of expression changes of interacting proteins, as shown in Figure 1. Altered intracellular protein levels provide a tracking indicator to observe secretory or other pathways of interest.

Figure 1. Schematic explanation of the cell-sonar principle. Mitogen-activated protein kinase (MAPK) and unfolded protein response (UPR) synergistically orchestrate signaling throughout the cell with subsequent induction of gene expression in the nucleus. (See details in Background section.) Highlighted in green are the components of the proteostasis network, which balances the synthesis, folding, and degradation of proteins to maintain the count of cellular proteins. MAPK and UPR are signaling networks that regulate any protein change that affects proteostasis processes. Highlighted in blue are the treatments that affect these balances. Chemical compounds or transgene proteins, which are not typically present in the cell, can result in an imbalance of the affected proteins as they interact (represented by blue dashed arrows). The proteostasis network regulates by altering gene expression to restore the balance. This is the reason why expression changes of interacting protein levels can be observed in direct comparison with untreated (green boxplot) and treated (blue boxplot) cellular proteins.

If there is a detectable expression change, it is the result of a protein interaction; if there is none, no protein interaction has happened. The results obtained from a few selected marker proteins provide a comprehensive overview of the fate of the target protein without the need to demonstrate the co-localization between a target and a marker protein. The advantages of cell-sonar are that it is adaptable to a wide range of proteins, easy to apply, and applicable to almost any cell line while it provides insights into the secretory pathway tracking in a short time.

The limitations include the fact that cell-sonar remains an overview. In order to make a reliable statement, it is crucial to use at least two marker proteins for one aspect and possess a detailed knowledge of the pathway and the proteins involved.