The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communications and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediates a long range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.

HIV-1 particle production is driven by the Gag precursor protein Pr55^Gag. Despite significant progress in defining both the viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of assembly in the infected cell remains to be elucidated. The Gag trafficking itinerary in primary monocyte-derived macrophages is especially poorly understood. To define the site of assembly and characterize the Gag trafficking pathway in this physiologically relevant cell type, we have made use of the biarsenical-tetracysteine system. A small tetracysteine tag was introduced near the C-terminus of the matrix domain of Gag. The insertion of the tag at this position did not interfere with Gag trafficking, virus assembly or release, particle infectivity, or the kinetics of virus replication. By using this in vivo detection system to visualize Gag trafficking in living macrophages, Gag was observed to accumulate both at the plasma membrane and in an apparently internal compartment that bears markers characteristic of late endosomes or multivesicular bodies. Significantly, the internal Gag rapidly translocated to the junction between the infected macrophages and uninfected T cells following macrophage/T-cell synapse formation. These data indicate that a population of Gag in infected macrophages remains sequestered internally and is presented to uninfected target cells at a virological synapse. The viral Gag protein is both necessary and sufficient for the assembly of a new generation of virus particles. There has been a significant amount of debate in recent years regarding the site in the cell at which HIV-1 assembly takes place. Of particular interest has been the site of assembly in macrophages, a cell type that serves as an important target for HIV-1 infection in vivo. In this study, we examine the site of Gag localization and virus assembly in primary human macrophages in living cells by using biarsenical dyes that become fluorescent when they bind a small target sequence introduced into HIV-1 Gag. We observe Gag localization both at the plasma membrane and in an apparently internal compartment that bears markers characteristic of multivesicular bodies (MVBs). Significantly, when infected macrophages are cocultured with uninfected T cells, the apparently internal Gag moves rapidly to the contact site, or synapse, between the macrophage and the T cell. These findings support the hypothesis that infected macrophages sequester assembled HIV-1 particles in an internal compartment and that these particles move to synapses where cell–cell transmission can occur.

Summary: Cell-to-cell communication is essential in multicellular organisms. Tunneling nanotubes (TNTs) have emerged as a new type of intercellular spreading mechanism allowing the transport of various signals, organelles and pathogens. Here, we study the role of the unconventional molecular motor myosin-X (Myo10) in the formation of functional TNTs within neuronal CAD cells. Myo10 protein expression increases the number of TNTs and the transfer of vesicles between co-cultured cells. We also show that TNT formation requires both the motor and tail domains of the protein, and identify the F2 lobe of the FERM domain within the Myo10 tail as necessary for TNT formation. Taken together, these results indicate that, in neuronal cells, TNTs can arise from a subset of Myo10-driven dorsal filopodia, independent of its binding to integrins and N-cadherins. In addition our data highlight the existence of different mechanisms for the establishment and regulation of TNTs in neuronal cells and other cell types.