Tunneling Nanotubes and the Eye: Intercellular Communication and Implications for Ocular Health and Disease

Abstract

Cellular communication is an essential process for the development and maintenance of all tissues including the eye. Recently, a new method of cellular communication has been described, which relies on formation of tubules, called tunneling nanotubes (TNTs). These structures connect the cytoplasm of adjacent cells and allow the direct transport of cellular cargo between cells without the need for secretion into the extracellular milieu. TNTs may be an important mechanism for signaling between cells that reside long distances from each other or for cells in aqueous environments, where diffusion-based signaling is challenging. Given the wide range of cargoes transported, such as lysosomes, endosomes, mitochondria, viruses, and miRNAs, TNTs may play a role in normal homeostatic processes in the eye as well as function in ocular disease. This review will describe TNT cellular communication in ocular cell cultures and the mammalian eye in vivo, the role of TNTs in mitochondrial transport with an emphasis on mitochondrial eye diseases, and molecules involved in TNT biogenesis and their function in eyes, and finally, we will describe TNT formation in inflammation, cancer, and stem cells, focusing on pathological processes of particular interest to vision scientists.

Figure 1

Morphogen transport and the drunken sailor analogy. (a) The transport of morphogens from a source establishes a gradient in the target field. (b–h) Five major morphogen transport models are illustrated using the drunken sailor analogy, in which drunken sailors move by random walks from a ship into a city. In this analogy, morphogen molecules are represented by sailors and cells are represented by buildings. (b) In the case of free diffusion, sailors (green dots) leave the ship (blue oval) and disperse into the city (white square). Inset: sailors take steps of the indicated fixed size, and the direction of each step is random. This “random walk” describes the diffusive behavior of molecules in solution. (c) In the tortuosity-mediated hindered diffusion model, buildings (gray) act as obstacles that sailors must move around, thus increasing the tortuosity of the environment. (d) In the case of diffusion that is hindered by tortuosity and transient binding, the sailors stop in pubs (negative diffusion regulators, yellow) located at the periphery of buildings. Note that, in contrast to effects from tortuosity alone, sailors congregate at the periphery of buildings, and there are relatively few freely moving sailors. (e, f) The shuttling model does not require a localized source of sailors. Instead, sailors are initially present mostly in pubs (negative diffusion regulators, yellow) and uniformly distributed in the city (e). Police officers (positive diffusion regulators, red) disperse from a source on the right side, pick up sailors from pubs, and escort them through the city by preventing further pub visits (f). When police officers disappear (not shown), sailors can reenter the pubs. Over time, this results in the concentration of sailors on the left. (g) In the transcytosis model, the sailors travel through the buildings. (h) During directed transport-mediated by cytonemes, the sailors travel through subway tunnels (orange), which deposit the sailors in buildings (reproduced from Muller et al. (2013) with no alterations under the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/)) [1].

Figure 2

Cytonemes and tunneling nanotubes. (a) Cytonemes were originally identified in Drosophila imaginal discs but have also been detected in developing chick limb buds. These cellular structures transport ligands and receptors over long distances from one cell to another in the complex tissue environment. Ligands cluster at the tip of a cytoneme and interact with clustered receptors on the surface of the recipient cell. (b) Tunneling nanotubes (TNTs) transport cellular organelles such as mitochondria, endosomes, and lysosomes, as well as other cargoes such as viruses, prions, and Ca²⁺ signals. TNTs form a tube through which these cargoes are directly transported between cells.

Figure 3

TNTs connecting inflammatory macrophages adhering to the corneal endothelium. (a) Confocal Z-stack of Iba-1⁺ (red) CD68⁺ (green) macrophages on the posterior surface of the corneal endothelium in mice one week after topical exposure to synthetic DNA, which causes accumulation of keratic precipitates [136] (asterisks denote nuclei of the endothelial cell monolayer). Interconnecting cytoplasmic TNTs are clearly visible between some of the macrophages (b, inset of dashed box in a), with punctate intracellular CD68⁺ signal visible between two cells (arrows; which represent approximately 3 μm distance from the endothelium). (c) Rotated view of Z-stack showing nanotube connecting the two cells (white arrow in c), which appears above the layer of the endothelium (blue arrow in c). Scale bar is 25 μm.

Figure 4

Cellular protrusions formed by ocular trabecular meshwork (TM) cells. (a, b) The cell membrane of TM cells was labeled with CD44 antibodies (cyan), and vesicles were labeled with DiO (green) or DiD (red). DiD-labeled vesicles are clearly observed in a TNT connecting to a DiO-labeled cell. (c, d) A long cellular protrusion (130 μm) extends between two TM cells in culture (CD44 immunostaining = green; SiR-actin = red). At higher magnification, the tips of the protrusion are not fused to form a continuous tunnel so this structure may be a cytoneme. (e–g) TM cells were labeled with DiO (e; green) or with MitoTracker (f; red) and CD44 cell membrane immunostaining (cyan). Red mitochondria are transferred into a DiO-labeled cell via TNTs. Scale = 20 μm.

Figure 5

Phenotype of two Myo10 knockout mouse models. tm1d completely ablated all Myo10 forms, whereas tm2 selectively knocked out the full-length, actin-binding Myo10, but “headless” Myo10 expression was unaffected. (a) Fluorescence image of flat mounted retinas showing retinal vasculature at P5. Dissected retinas were stained with a PECAM-1 antibody (green) and counterstained with phalloidin (red). The retinal vasculature extended to similar positions in the control and Myo10^tm1d/tm1d eyes. The image represents a stitch of micrographs taken at 20x and is best visualized if the images are enlarged on a digital display. (b) High-resolution images of the angiogenic expansion front from the P5 retinas in (a) showing filopodia radiating from endothelial tips cells. Loss of Myo10 results in a decreased number of filopodia and leads to a less dense vascular network. Images were captured as Z-stacks at 60x and displayed as maximum projections with the PECAM-1 channel displayed in inverted grayscale to highlight endothelial filopodia. (c) High-resolution MRI (magnetic resonance imaging) of enucleated and fixed eyes from adult wild-type (WT) and Myo10^tm2/tm2 mice, where representative of 6 eye scans for each genotype reveals persistence of the hyaloid vasculature in mutant mice. The hyaloid artery emerges from the optic disc and extends toward the lens, as schematically illustrated on the right. Scale bars: 1 mm (reproduced from (a, b) Heimsath et al. 2017 and (c) Bachg et al. 2019 under the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/)) [105, 106].

Figure 6

Schematic of the eye summarizing the current findings on TNTs in various anterior and posterior ocular cells and tissues. TNTs are found in (1) mouse corneal stroma [40], (2) trabecular meshwork cells [50], (3) retinal pigment epithelial cells [52], and (4) cornea sclera margin, ciliary margin, and retina following transplantation of hematopoietic stem cells [44]. All images are reproduced with permission under the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/) or from the indicated citations ([40] AAI is the copyright holder and [44] ARVO is the copyright holder).