Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells

Abstract

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.

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“…Due to their thin, tubular nature and their actin dependence, it has been tempting to view IMTs as specialized filopodia. However, evidence from a neuronal cell line suggests distinct and even opposite molecular characteristics between IMTs and filopodia (Delage et al, 2016), although IMTs in this system still contain parallel actin filaments that run the length of the IMT, similar to filopodia (Sartori-Rupp et al, 2019).…”

“…Originally termed cytonemes (Ramírez-Weber and Kornberg, 1999) or tunneling nanotubes (TNTs) (Önfelt and Davis, 2004;Rustom et al, 2004), these protrusions are thin (<500 nm width) and can extend over 100 m. Given their dimensions, and the fact that they are not on the basal surface but are extended between cells like a tightrope, the protrusions are easily destroyed by certain fixation procedures (Rustom et al, 2004;Sartori-Rupp et al, 2019), perhaps contributing to their relatively late identification.…”

“…One difference between the structures concerns cytoskeletal composition. While the initially identified structures were shown to contain actin but not microtubules (Ramírez-Weber and Kornberg, 1999;Rustom et al, 2004;Sowinski et al, 2008;Sartori-Rupp et al, 2019), a number of subsequently identified structures contain microtubules (Önfelt et al, 2006;Gerdes et al, 2013;Osswald et al, 2015;Kumar et al, 2017;Resnik et al, 2018;Kim et al, 2019). Where examined, intermediate filaments have also been identified (Iglič et al, 2007;Ady et al, 2014;Sáenz-de-Santa-María et al, 2017;Resnik et al, 2018).…”

“…A second difference concerns the size of the structures. TNTs are generally defined as having a diameter less than 500 nm (Rustom et al, 2004;Ariazi et al, 2017;Sartori-Rupp et al, 2019), but other structures can be several microns wide (Vidulescu et al, 2004;Osswald et al, 2015;Kumar et al, 2017). These variations have led to use of additional names such as 'microtubes' to reflect the structural differences.…”

“…IMTs have been identified in vivo in both Drosophila (Ramírez-Weber and Kornberg, 1999;Roy et al, 2011Roy et al, , 2014Huang et al, 2019) and mammals (Chinnery et al, 2008) suggesting physiological significance. A number of communication functions have been attributed to IMTs, including targeted growth factor transmission (Roy et al, 2011(Roy et al, , 2014, neurotransmitter signaling from epithelial cells (Huang et al, 2019), and direct exchange of cytoplasmic materials including: mitochondria (Kumar et al, 2017;Kretschmer et al, 2019), endosomes/lysosomes (Rustom et al, 2004;Kumar et al, 2017;Sartori-Rupp et al, 2019), plasma membrane proteins (Önfelt and Davis, 2004), mRNA (Haimovich et al, 2017), and microRNAs (Thayanithy et al, 2014).…”