Intercellular communication is a fundamental process of all living things. Cells of the same, or different species, exchange molecular messages to perform basic functions like response to environmental conditions, social behaviors in microbes, differentiation and development in multicellular organisms, homeostatic maintenance, etc. The most studied mechanism is the binding of extracellular ligands to membrane receptors. For example, quorum sensing in gram-negative species of bacteria [1], pheromone-based communication in fungi [2], or paracrine, autocrine and endocrine communication [[3], [4], [5]]. The molecular nature of the ligands is very diverse, they can be metabolites, peptides, proteins, polysaccharides, nucleic acids, etc., which can transmit signals diffusing in the extracellular media or acting attached to the surface of one of the interacting cells (communication by direct cell-cell or cell-extracellular matrix contact). When the ligand binds to the corresponding receptor, an intracellular signal is transduced, and a cellular response is triggered. These types of receptor-ligand interactions are typically extracellular and non-covalent.

Molecular messengers can also be molecules that are internalized into the cytoplasm or integrated into the phospholipid bilayer at the plasma membrane of the recipient cell, these messages are exchanged through processes like secretion and internalization of extracellular vesicles (EVs) e.g. exosomes or exophers [6,7], movement of cytoplasmic components through pores connecting adjacent cells (gap junctions in animals, plasmodesmata in plants or septum in filamentous fungi [[8], [9], [10]], the extraction of a fragment of one cell by the other [11], or by connecting cells through long membrane protrusions (of which the most studied type in animal cells is tunneling nanotubes) [12]. The result is the intercellular transfer of metabolites, RNAs, proteins and organelles whose functions contribute to the resultant physiological changes in the recipient cells. The importance of this transfer is illustrated by the evolution of various mechanisms to perform this task in diverse phylogenetic groups.

In particular, the intercellular transfer of proteins and mitochondria has been reported to have prominent effects on the recipient cells. For example, making cells responsive to inflammatory stimulus [13], contributing to the adaptive immune response [14,15], extragenetic acquisition of drug-resistance in cancer cells [[16], [17], [18], [19]], progression of cancer [6,20], survival of damaged endothelial cells [21] or neurons after spinal cord injury or stroke [22,23]. The therapeutic potential of the intercellular transfer of proteins, nucleic acids and organelles is a subject of active research [[24], [25], [26]]. However, the mechanisms used to share proteins among cells can also contribute to the dissemination of viral particles [[27], [28], [29]], pathogenic bacteria [30,31] or proteins that lead to neurodegenerative diseases [[32], [33], [34], [35]].

Many questions remain unanswered regarding the exchange of proteins and mitochondria among cells. For example, what are the molecular signals that trigger the transfer? What determines which cell is the donor and which recipient? How are the transferred proteins selected and what are the functions they perform in the recipient cells? What mechanisms regulate the amount of transferred material? To answer these questions extensive research is needed using experimental systems that allow differentiation of proteins from donor and recipient cells. The vast majority of studies have used fluorescent tags for protein detection or fluorescent dyes for organelle detection by confocal microscopy or flow cytometry. These approaches, however, are suited for the analysis of a few selected proteins or organelles in each experiment. Mass spectrometry-based proteomic analyses allow an unbiased, untargeted general analysis of the set of transferred proteins. Yet, this methodology has not been used extensively in this field. Here, we provide an overview of the different mechanisms of intercellular transfer of proteins and mitochondria, discussing the experimental methodologies used to study them and emphasizing the contributions of proteomic studies.