Tunneling nanotubes: emerging view of their molecular components and formation mechanisms

Cell-to-cell communication is essential for the development and maintenance of multicellular organisms. The tunneling nanotube (TNT) is a recently recognized distinct type of intercellular communication device. TNTs are thin protrusions of the plasma membrane and allow direct physical connections of the plasma membranes between remote cells. The proposed functions for TNTs include the cell-to-cell transfer of large cellular structures such as membrane vesicles and organelles, as well as signal transduction molecules in a wide variety of cell types. Moreover TNT and TNT-related structures are thought to facilitate the intercellular spreading of virus and/or pathogenic proteins. Despite their contribution to normal cellular functions and importance in pathological conditions, virtually nothing is known about the molecular basis for their formation. We have recently shown that M-Sec (also called TNFaip2) is a key molecule for TNT formation. In cooperation with the RalA small GTPase and the exocyst complex, M-Sec can induce the formation of functional TNTs, indicating that the remodeling of the actin cytoskeleton and vesicle trafficking are involved in M-Sec-mediated TNT formation. Discovery of the role of M-Sec will accelerate our understanding of TNTs, both at the molecular and physiological levels.     

Cell communication by tunneling nanotubes: Implications in disease and therapeutic applications

Intercellular communication is essential for the development and maintenance of multicellular organisms. Tunneling nanotubes (TNTs) are a recently recognized means of long and short distance communication between a wide variety of cell types. TNTs are transient filamentous membrane protrusions that connect cytoplasm of neighboring or distant cells. Cytoskeleton fiber-mediated transport of various cargoes occurs through these tubules. These cargoes range from small ions to whole organelles. TNTs have been shown to contribute not only to embryonic development and maintenance of homeostasis, but also to the spread of infectious particles and resistance to therapies. These functions in the development and progression of cancer and infectious disease have sparked increasing scrutiny of TNTs, as their contribution to disease progression lends them a promising therapeutic target. Herein, we summarize the current knowledge of TNT structure and formation as well as the role of TNTs in pathology, focusing on viral, prion, and malignant disease. We then discuss the therapeutic possibilities of TNTs in light of their varied functions. Despite recent progress in the growing field of TNT research, more studies are needed to precisely understand the role of TNTs in pathological conditions and to develop novel therapeutic strategies.     

Tunneling nanotubes: A possible highway in the spreading of tau and other prion-like proteins in neurodegenerative diseases

The mechanisms of intercellular spreading of amyloidogenic proteins involved in neurodegenerative diseases have yet to be fully elucidated. While secretion has been implicated in the transfer of many proteins, including prions and α-synuclein, tunneling nanotubes (TNTs) have also been demonstrated for prions and mutant Huntingtin. Here, we provide further evidence that Tau aggregates, which have been demonstrated to predominantly be transferred via secretion, can also be found in TNTs. Additionally, cells that have taken up Tau have increased TNT formation. Coupled with previous evidence that other amyloidogenic aggregates also induce TNT formation we propose that misfolded protein aggregates can, through a common mechanism, promote the formation of TNTs and thereby their own intercellular transfer, contributing to the propagation of pathology.     

Modeling Intercellular Transfer of Biomolecules Through Tunneling Nanotubes

Tunneling nanotubes (TNTs) have previosly been observed as long and thin transient structures forming between cells and intercellular protein transfer through them has been experimentally verified. It is hypothesized that this may be a physiologically important means of cell–cell communication. This paper attempts to give a simple model for the rates of transfer of molecules across these TNTs at different distances. We describe the transfer of both cytosolic and membrane bound molecules between neighboring populations of cells and argue how the lifetime of the TNT, the diffusion rate, distance between cells, and the size of the molecules may affect their transfer. The model described makes certain predictions and opens a number of questions to be explored experimentally.     

Selective block of tunneling nanotube (TNT) formation inhibits intercellular organelle transfer between PC12 cells

Organelle exchange between cells via tunneling nanotubes (TNTs) is a recently described form of intercellular communication. Here, we show that the selective elimination of filopodia from PC12 cells by 350 nM cytochalasin B (CytoB) blocks TNT formation but has only a weak effect on the stability of existing TNTs. Under these conditions the intercellular organelle transfer was strongly reduced, whereas endocytosis and phagocytosis were not affected. Furthermore, the transfer of organelles significantly correlated with the presence of a TNT-bridge. Thus, our data support that in PC12 cells filopodia-like protrusions are the principal precursors of TNTs and CytoB provides a valuable tool to selectively interfere with TNT-mediated cell-to-cell communication.     

MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking

Tunneling nanotubes (TNTs) are actin‐rich structures that connect two or more cells and mediate cargo exchange between spatially separated cells. TNTs transport signaling molecules, vesicles, organelles, and even pathogens. However, the molecular mechanisms regulating TNT formation remain unclear and little is known about the endogenous mechanisms suppressing TNT formation in lung cancer cells. Here, we report that MICAL2PV, a splicing isoform of the neuronal guidance gene MICAL2, is a novel TNT regulator that suppresses TNT formation and modulates mitochondrial distribution. MICAL2PV interacts with mitochondrial Rho GTPase Miro2 and regulates subcellular mitochondrial trafficking. Moreover, down‐regulation of MICAL2PV enhances survival of cells treated with chemotherapeutical drugs. The monooxygenase (MO) domain of MICAL2PV is required for its activity to inhibit TNT formation by depolymerizing F‐actin. Our data demonstrate a previously unrecognized function of MICAL2 in TNT formation and mitochondrial trafficking. Furthermore, our study uncovers a role of the MICAL2PV‐Miro2 axis in mitochondrial trafficking, providing a mechanistic explanation for MICAL2PV activity in suppressing TNT formation and in modulating mitochondrial subcellular distribution.     

Tunneling nanotubes spread fibrillar α‐synuclein by intercellular trafficking of lysosomes

Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α‐synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α‐synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co‐cultured neurons, here we show that α‐synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α‐synuclein fibrils are able to seed soluble α‐synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α‐synuclein aggregates in lysosomes dispose of this material by hijacking TNT‐mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies.     

Automated detection of tunneling nanotubes in 3D images.

BACKGROUND: This paper presents an automated method for the identification of thin membrane tubes in 3D fluorescence images. These tubes, referred to as tunneling nanotubes (TNTs), are newly discovered intercellular structures that connect living cells through a membrane continuity. TNTs are 50-200 nm in diameter, crossing from one cell to another at their nearest distance. In microscopic images, they are seen as straight lines. It now emerges that the TNTs represent the underlying structure of a new type of cell-to-cell communication. METHODS: Our approach for the identification of TNTs is based on a combination of biological cell markers and known image processing techniques. Watershed segmentation and edge detectors are used to find cell borders, TNTs, and image artifacts. Mathematical morphology is employed at several stages of the processing chain. Two image channels are used for the calculations to improve classification of watershed regions into cells and background. One image channel displays cell borders and TNTs, the second is used for cell classification and displays the cytoplasmic compartments of the cells. The method for cell segmentation is 3D, and the TNT detection incorporates 3D information using various 2D projections. RESULTS: The TNT- and cell-detection were applied to numerous 3D stacks of images. A success rate of 67% was obtained compared with manual identification of the TNTs. The digitalized results were used to achieve statistical information of selected properties of TNTs. CONCLUSION: To further explore these structures, automated detection and quantification is desirable. Consequently, this automated recognition tool will be useful in biological studies on cell-to-cell communication where TNT quantification is essential.     

Tunneling nanotubes: Reshaping connectivity

Tunneling nanotubes (TNTs), open membranous channels between connected cells, represent a novel direct way of communication between distant cells for the diffusion of various cellular material, including survival or death signals, genetic material, organelles, and pathogens. Their discovery prompted us to review our understanding of many physiological and pathological processes involving cellular communication but also allowed us to discover new mechanisms of communication at a distance. While this has enriched the field, it has also generated some confusion, as different TNT-like protrusions have been described, and it is not clear whether they have the same structure–function. Most studies have been based on low-resolution imaging methods, and one of the major problems is the inconsistency in demonstrating the capacity of these various connections to transfer material between cells belonging to different populations. This brief review examines the fundamental properties of TNTs. In adult tissues, TNTs are stimulated by different diseases, stresses, and inflammatory signals. ‘Moreover’, based on the similarity of the processes of development of synaptic spines and TNT formation, we argue that TNTs in the brain predate synaptic transmission, being instrumental in the orchestration of the immature neuronal circuit.     

Exosomes and nanotubes: Control of immune cell communication

Cell–cell communication is critical to coordinate the activity and behavior of a multicellular organism. The cells of the immune system not only must communicate with similar cells, but also with many other cell types in the body. Therefore, the cells of the immune system have evolved multiple ways to communicate. Exosomes and tunneling nanotubes (TNTs) are two means of communication used by immune cells that contribute to immune functions. Exosomes are small membrane vesicles secreted by most cell types that can mediate intercellular communication and in the immune system they are proposed to play a role in antigen presentation and modulation of gene expression. TNTs are membranous structures that mediate direct cell-cell contact over several cell diameters in length (and possibly longer) and facilitate the interaction and/or the transfer of signals, material and other cellular organelles between connected cells. Recent studies have revealed additional, but sometimes conflicting, structural and functional features of both exosomes and TNTs. Despite the new and exciting information in exosome and TNT composition, origin and in vitro function, biologically significant functions are still being investigated and determined. In this review, we discuss the current field regarding exosomes and TNTs in immune cells providing evaluation and perspectives of the current literature.     

Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes

Background

Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis.

Methodology and Principal Findings

Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²⁺ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells.

Conclusions and Significance

Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic.     

Long-distance electrical coupling via tunneling nanotubes

Tunneling nanotubes (TNTs) are nanoscaled, F-actin containing membrane tubes that connect cells over several cell diameters. They facilitate the intercellular exchange of diverse components ranging from small molecules to organelles and pathogens. In conjunction with recent findings that TNT-like structures exist in tissue, they are expected to have important implications in cell-to-cell communication. In this review we will focus on a new function of TNTs, namely the transfer of electrical signals between remote cells. This electrical coupling is not only determined by the biophysical properties of the TNT, but depends on the presence of connexons interposed at the membrane interface between TNT and the connected cell. Specific features of this coupling are compared to conventional gap junction communication. Finally, we will discuss possible down-stream signaling pathways of this electrical coupling in the recipient cells and their putative effects on different physiological activities. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Rab11a mediates cell-cell spread and reassortment of influenza A virus genomes via tunneling nanotubes

Influenza A virus [IAV] genomes comprise eight negative strand RNAs packaged into virions in the form of viral ribonucleoproteins [vRNPs]. Rab11a plays a crucial role in the transport of vRNPs from the nucleus to the plasma membrane via microtubules, allowing assembly and virus production. Here, we identify a novel function for Rab11a in the inter-cellular transport of IAV vRNPs using tunneling nanotubes [TNTs]as molecular highways. TNTs are F-Actin rich tubules that link the cytoplasm of nearby cells. In IAV-infected cells, Rab11a was visualized together with vRNPs in these actin-rich intercellular connections. To better examine viral spread via TNTs, we devised an infection system in which conventional, virion-mediated, spread was not possible. Namely, we generated HA-deficient reporter viruses which are unable to produce progeny virions but whose genomes can be replicated and trafficked. In this system, vRNP transfer to neighboring cells was observed and this transfer was found to be dependent on both actin and Rab11a. Generation of infectious virus via TNT transfer was confirmed using donor cells infected with HA-deficient virus and recipient cells stably expressing HA protein. Mixing donor cells infected with genetically distinct IAVs furthermore revealed the potential for Rab11a and TNTs to serve as a conduit for genome mixing and reassortment in IAV infections. These data therefore reveal a novel role for Rab11a in the IAV life cycle, which could have significant implications for within-host spread, genome reassortment and immune evasion.     

Nucleolar protein GLTSCR2 stabilizes p53 in response to ribosomal stresses

p53 is a key regulator of cell growth and death by controlling cell cycle progression and apoptosis under conditions of stress such as DNA damage or oncogenic stimulation. As these processes are critical for cell function and inhibition of tumor development, p53 regulatory pathways are strictly monitored in cells. Recently, it was recognized that nucleolar proteins, including nucleophosmin/B23, ribosomal protein L11, and alternate reading frame (ARF), form the nucleolus-ARF-murine double minute 2 (MDM2) axis in p53 regulatory pathways, which increases p53 stability by suppressing the activity of MDM2. In this work, we show that nucleolar protein glioma tumor-suppressor candidate region gene 2 (GLTSCR2) translocates to the nucleoplasm under ribosomal stress, where it interacts with and stabilizes p53 and inhibits cell cycle progression without the involvement of the major upstream p53 regulator, ARF. Furthermore, ectopic expression of GLTSCR2 significantly suppressed growth of cancer cells in a xenograft animal model via p53-dependent pathway. Our data identify GLTSCR2 as a new member of the nucleolus-nucleoplasmic axis for p53 regulation. ARF-independent direct regulation of p53 by GLTSCR2 may be a key mechanism and therapeutic target for cell death or growth inhibition when nucleolus-ARF-p53 pathways are inactivated by genetic or epigenetic modifications of ARF, which are the second most common types of genetic change observed in human cancers.     

Tunneling nanotube formation is essential for the regulation of osteoclastogenesis

Osteoclasts are the multinucleated giant cells formed by cell fusion of mononuclear osteoclast precursors. Despite the finding of several membrane proteins involving DC‐STAMP as regulatory proteins required for fusion among osteoclast precursors, cellular and molecular events concerning this process are still ambiguous. Here we identified Tunneling Nanotubes (TNTs), long intercellular bridges with small diameters, as the essential cellular structure for intercellular communication among osteoclast precursors in prior to cell fusion. Formation of TNTs was highly associated with osteoclastogenesis and it was accompanied with the significant induction of the M‐Sec gene, an essential gene for TNT formation. M‐Sec gene expression was significantly upregulated by RANKL‐treatment in osteoclast precursor cell line. Blockage of TNT formation by Latrunclin B or by M‐Sec siRNA significantly suppressed osteoclastogenesis. We have detected the rapid intercellular transport of not only the membrane phospholipids labeled with DiI but also the DC‐STAMP‐GFP fusion protein through TNTs formed among osteoclast precursors during osteoclastogenesis. Transportation of such regulatory molecules through TNTs would be essential for the process of the specific cell fusion among osteoclast precursors. J. Cell. Biochem. 114: 1238–1247, 2013. © 2012 Wiley Periodicals, Inc.     

Active Generation and Propagation of Ca Signals within Tunneling Membrane Nanotubes

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca²⁺ signaling; however, our simulations indicate that passive diffusion of Ca²⁺ ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP₃)-evoked Ca²⁺ signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca²⁺ signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP₃ receptors along the TNT. We propose that IP₃ receptors may actively propagate intercellular Ca²⁺ signals along TNTs via Ca²⁺-induced Ca²⁺ release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials.     

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.Apoptosis is an important regulatory mechanism of tissue homeostasis. It is triggered by the extrinsic pathway through the activation of proapoptotic receptors or by the intrinsic pathway through the destabilization of mitochondria in response to various forms of cell injury or stress.¹ Notably, stressed cells are also strongly influenced by intercellular communicative networks. This includes diffusible growth factors, cytokines and other small molecules secreted from neighbouring cells, which can modulate the fate of distressed cells. For example, stem cells release growth factors to protect dysfunctional neurons in the brain.² In tumour stroma, activated fibroblasts are thought to promote tumour progression by secreting growth factors that act in a paracrine manner.³ Moreover, contact-dependent signalling, for example, via adhesion molecules, can trigger contact inhibition or protection of endothelial cells.⁴ In addition, gap junctions have been shown to be involved in the transfer of death or survival molecules in different cell types.⁵ Therefore, the signals transferred from neighbouring cells influence the viability of target cells through different pathways.In 2004, our group described a previously unrecognized form of cell-to-cell interaction based on nanoscaled, F-actin-containing membrane tubes.^{6, 7} These tubes, referred to as membrane or tunneling nanotubes (TNTs), were subsequently found in numerous cell types in culture and in tissues.^{8, 9, 10, 11} Importantly, TNTs facilitate the intercellular exchange of diverse cellular signals and components ranging from electrical signalling to organelles.^{12, 13, 14, 15} Moreover, pathogens such as human immunodeficiency virus (HIV) and prions can spread between cells along TNTs.^{16, 17} Consistent with the model that TNTs are involved in cell-to-cell communication, apoptosis regulators may be transferred via TNTs between apoptotic and healthy cells to alter the fate of recipient cells. Indeed, it has been shown that TNTs can propagate the death signal Fas ligand between T lymphocytes to induce cell death.^{18, 19} TNTs have been also proposed to participate in the rescue of injured cardiomyoblasts or endothelial cells by mesenchymal stem cells (MSCs) through transferred mitochondria.^{20 ,21} However, the rescue mechanism by how and when this event was accomplished remains elusive.In this study, we found that PC12 cells stressed by ultraviolet (UV) radiation were rescued from apoptosis when cocultured with untreated, healthy PC12 cells. Single-cell analysis showed that stressed cells in the early stages of apoptosis form a new type of TNT to interact with untreated cells. These TNTs have a distinct cytoskeletal composition and biophysical properties when compared with TNTs interconnecting normal PC12 cells. We also observed the presence and transport of mitochondria in the TNTs formed by stressed cells. Notably, the rescue effect was inhibited when the formation of TNTs were impaired by incubating with an F-actin-depolymerizing drug, or when the mitochondria of rescuer cells were damaged. Our results suggest that the delivery of functional mitochondria via TNTs mediates the recovery of PC12 cells in the early stages of apoptosis.     

p53 negatively regulates Pin1 expression under ER stress

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.