The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis

Abstract

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.

An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion

“…In contrast, TNTs that also form by extending long protrusions remain stable structures that connect two cells. Furthermore, while TNTs have been reported to form in short 4-hour cultures of RAW/LR5 macrophages (Hanna et al, 2017), fusion of peritoneal macrophages begins 9 hours after addition of IL-4 (Faust et al, 2017). Finally, as revealed in our studies, short rather than long protrusions predominantly initiate macrophage fusion.…”

“…TNTs are thin long membranous tubes with diameters of 50-800 nm connecting two cells that have been reported in numerous cell types, including macrophages (Rustom et al, 2004;Kimura et al, 2012;Onfelt et al, 2006;Hanna et al, 2017). Formation of TNTs requires F-actin and, as recently shown in macrophages, depends on the activity of Rac1, Cdc42 and WASp (Hanna et al, 2017). Despite the general requirement for F-actin and the activators of actin polymerization, there seems to be clear distinctions between TNTs and fusion-competent protrusions.…”

Role of connexin 43 in different forms of intercellular communication – gap junctions, extracellular vesicles and tunnelling nanotubes

“…Not surprisingly, cytoskeleton regulators, such as N-WASP, Rac1, ezrin and Cdc42, have been linked to TNTs (Arkwright et al, 2010;Hanna et al, 2017;Hase et al, 2009;Lachambre et al, 2014). However, and despite structural similarities, a recent study revealed that TNT formation and TNT-mediated vesicle transfer is negatively regulated by the filopodia-promoting complex Cdc42-IRSp53-VASP (IRSp53 is also known as BAIAP2), suggesting that the formation of filopodia and TNTs involves different mechanisms that may depend on the cell or stimulus (Delage et al, 2016).…”

Amyloid-β induced membrane damage instigates tunneling nanotubes by exploiting p21-activated kinase dependent actin remodulation

“…In addition, the role of CDC42 and Rho-GTPases in TNT formation is not fully investigated. A report of Hanna et al, (2017), demonstrated that the activity of CDC42 and Rho-GTPases positively contributes to the formation of TNTs in macrophages. In contrast, Delage et al, (2016); found that CDC42/IRSp53/VASP negatively regulates the formation of TNTs.…”

“…Some of these studies also reveal that vesicle transfer, recycling vesicles, lysosomes and molecule involved in membrane expansion play a role in the formation of TNTs, the actin membrane protrusions (Bhat et al, 2018;Victoria and Zurzolo, 2017;Hase et al, 2009). Actin depolymerization and actin modulations have been shown to inhibit the formation of these structures (Hanna et al, 2017).…”