Cell-to-cell communication: Mechanism of tunneling nanotube formation and function

1: Recent findings of tunneling nanotube-like structures in vivo suggest that they fulfill important roles under physiological conditions. To develop markers to trace TNTs in vivo and to interfere with TNT-dependent diseases such as the spread of pathogens between cells, we first focused on key regulators of filopodium outgrowth in PC12 cells. This revealed exciting insight into the sequential changes during filopodium-to-TNT conversion: The filopodial tip marker myosin X, a motor protein, was shown to be an integral part of TNTs. Myosin X induced filopodia and concentrated an adhesion molecule at the tip of filopodia and the TNT-target cell interface. Myosin X knockdown and dominant negative mutant expression counteracted these properties. Other tip-complex proteins were found to be removed from the distal end of the nascent TNTs. Their overexpression induced filopodia outgrowth, but impaired TNT formation. In contrast, another protein was recruited to nascent TNTs. In summary, our results demonstrate that although TNTs derive from filopodia they evolve into distinct F-actin-based adherent structures ("Transformation of filopodia into tunneling nanotubes involves attenuation of actin dynamics and adherens junction assembly?, Ivan Rios-Mondragon, Julia Schölermann, Nickolay Bukoreshtliev, Xiang Wang, Richard Cheney and Hans-Hermann Gerdes (Manuskript er i revision i Journal of Cell Science). 2: To further analyze the role of cell adhesion molecules in establishing the contact of TNT precursors with target cells, we focused on the cell adhesion mediating Cadherins, whose expression patterns correlate with epithelial-mesenchymal transition in embryonic development and cancer. Using CHO cells which normally do not express Cadherins we were able to distinguish Cadherins involved in TNT connection and organelle transfer, and a dependence on calcium of these processes. We were able to demonstrate this connection with the help of different cancer cell lines. ("Role of E- and N-cadherin in the formation of TNTs and intercellular organelle transfer in the framework of EMT». Julia Schölermann, Tanja Kögel, Dominik Michael Frei, Katarzyna Wnuk-Lipinska, Ivan Rios-Mondragon, David R Micklem, Carien Niessen, and Hans-Hermann Gerdes. Manuscript in preparation). 3: Direct investigation of TNTs is possible, but difficult and labor intensive because TNTs are delicate structures which rupture easily. Since there are many myosin motors and G-proteins that potentially could be involved in TNT-dependent transfer we aimed at developing a more robust read-out that would be suitable for screening. We measured intercellular organelle transfer with a microscopy based protocol that allows us to differentiate between contact-dependent and -independent transfer. With a set of siRNAs targeting 36 GTPases, myosins and other F-actin interacting proteins, we screened for their involvement in contact-dependent intercellular transfer of DiD. In order to automatically evaluate the transferred material, we developed a program that affiliates all voxels of a given confocal 3D image to the area of individual cells or background(Hodneland E et al. Source Code Biol Med 2013). The first round of screening revealed 16 candidates that affected the transfer. Expression of fluorescently tagged proteins corresponding to the candidates of the screen confirmed the effect of several candidates. Some were found to transfer significantly themselves. Additionally, we showed that the proto-oncogen H-Ras transfers in our system, and that we could inhibit this transfer. Finally, we automated the image acquisition to make the system suitable for higher throughput screening such as cancer or HIV drug screening ("Novel microscopy-based screening method reveals regulators of contact-dependent intercellular transfer." Dominik Michael Frei, Erlend Hodneland, Ivan Rios-Mondragon, Anne Burtey, Beate Neumann, Jutta Bulkescher, Julia Schölermann, Rainer Pepperkok, Hans-Hermann Gerdes and Tanja Kögel. (Manuscript in revision in Nature Scientific Reports). 4: Embryonic development: Finally, to investigate the potential physiological implication of TNTs in embryonic development, we showed that immature hippocampal neurons generated short protrusions towards astrocytes resulting in TNT formation. Our findings suggest that within a limited maturation period developing neurons establish electrical coupling and exchange of calcium signals with astrocytes resulting in TNT formation. Our findings suggest that within a limited maturation period developing neurons establish electrical coupling and exchange of calcium signals with astrocytes via TNTs, correlating with a high neuronal expression level of connexin 43. This novel cell-cell communication pathway between cells of the central nervous system provides new concepts in our understanding of neuronal migration and differentiation (Wang X et al. PLoS One 2012).

Tunnelling nanotubes (TNTs) were discovered only a few years ago as conduits for a previously unrecognised type of cell-to-cell communication. Since then, a growing number of cell types have been found to use TNTs to exchange diverse cargoes ranging from cytoplasmic signalling molecules such as calcium ions to small vesicles. In the past year, the human immunodeficiency virus, HIV, and prion proteins, the infectious agent that causes diseases such as scrapie in sheep and Creutzfeld-Jakob syndrome in human s, were found to spread between cells through these intercellular membrane channels. Due to the emerging wide range of implications of TNTs in the field of biological and biomedicial research, it is necessary to first learn the basic principles and mecha nisms of TNT-dependent cell-cell interactions by focusing on a few basic models. We here propose to focus on three main questions: First, in light of our data showing that filopodia are the precursors of TNTs, we will investigate how outgrowing filopodia evolve into TNT structures. Second, we will characterize/identify cell surface molecules as the likely candidates for establishing the contact/recognition of TNT precursors with the target cells. Third, we will identify the motor proteins facilitating the delivery of vesiclular cargo through TNTs. Detailed mechanistic insights into TNT-dependent cell-to-cell communication at the cellular level will allow to address the potential physiological implication of TNTs such as embryonic development, immune defen se and cancer. Furthermore, it will enable to develop molecular strategies to interfere e.g. with TNT-specific diseases including the spread of pathogens between cells.