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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Wiley Interdiscip Rev Nanomed Nanobiotechnol. Author manuscript; available in PMC 2017 September 18.
Published in final edited form as:
PMCID: PMC5602582
NIHMSID: NIHMS905116

Intercellular nanotubes: insights from imaging studies and beyond

Abstract

Cell-cell communication is critical to the development, maintenance, and function of multicellular organisms. Classical mechanisms for intercellular communication include secretion of molecules into the extracellular space and transport of small molecules through gap junctions. Recent reports suggest that cells also can communicate over long distances via a network of transient intercellular nanotubes. Such nanotubes have been shown to mediate intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, intercellular nanotubes have been observed in vivo and have been shown to enhance the transmission of pathogens such as human immunodeficiency virus (HIV)-1 and prions in vitro. These studies indicate that intercellular nanotubes may play a role both in normal physiology and in disease.

Classical mechanisms of intercellular communication include those that are relatively well characterized such as the secretion of molecules (e.g., neurotransmitters or chemokines) into the extracellular space, and the transport of small molecules through gap junctions formed between neighboring cells. In addition, it has been suggested that cells outside the nervous system can communicate via long cellular extensions. For example, long and dynamic filopodia have been observed to extend from developing sea urchin embryos1 and Kornberg et al. observed filopodia-like cytoplasmic extensions of cells, which they termed cytonemes, in Drosophila wing imaginal discs2 (Figure 1.1). These studies suggest that some of the signaling previously thought to be mediated by diffusible signals may instead be the result of direct interactions mediated by cellular extensions.3

Figure 1
Intercellular nanotubes. (1) Growing cellular nanotubes (cytonemes) in culture of fragments cut from Drosophila wing discs. Fluorescence microscopy initially revealed round green fluorescent protein (GFP)-containing cells, (a), but after approximately ...

Long-range cell–cell communication between mammalian cells has recently been complemented with a newly discovered class of intercellular structures. Rustom et al. reported that cultured neuronal rat pheochromocytoma cells (PC12) cells could be connected by tubular structures containing membrane and filamentous (F) actin4 (Figure 1.2). These structures were suspended in the medium between connected cells and, thus, did not rest on the substratum. Furthermore, they were shown to mediate the transfer of subcellular organelles between cells by actin-dependent mechanisms and membrane-bound proteins were seen diffusing between cells, suggesting a seamless transition of the plasma membranes of the two connected cells. Because of the unexpected properties displayed by these tubular structures, they were initially called tunneling nanotubes.

Since the first report, it has become clear that similar structures, here collectively called intercellular nanotubes (ICNs), readily form between a variety of cell types, including natural killer (NK) cells and Epstein–Barr virus (EBV)-transformed B-cells5 (Figure 1.3), cultured DU154 prostate cancer cells,7 THP-1 monocytes,8 endothelial progenitor cells, rat cardiac myocytes,9 human and murine macrophages,10,11 and astrocytes.12 Studies have shown that ICNs can support transfer of organelles,4,9,13 membrane-bound components, or cytoplasmic molecules.8,14 Furthermore, the growing number of reports implicating ICNs as pathways for pathogens, such as bacteria,11 virus,1517 and prions18 indicates that ICNs may also play a role in disease. The possibility that ICNs are only a consequence of in vitro cell culture conditions has been ruled out by recent observations of ICNs in vivo.19

Prior to the description of ICNs, artificial nanotube–vesicle networks (NVN) had been reported in liposome systems20 and it was demonstrated that NVNs could be functionalized with membrane proteins.21 More recently, it was shown that NVNs could be constructed directly from the plasma membrane of cultured cells6 (Figure 1.4). The similarities between cellular and artificial nanotubes suggest that NVNs could act as model systems to study some aspects of the formation and transport mechanisms of ICNs.22

The mechanisms for cell–cell communication supported by nanotubes are still poorly understood. Studies have revealed a substantial heterogeneity in structure, formation process, and functional properties.17,2325 There is also some confusion regarding the terminology and definitions of ICNs, illustrating the immaturity of this research field. In this review, we discuss the literature of these cellular structures, nanotubular support for long-range intercellular communication, and other functional roles of intercellular nanotubes in biology. We also cover what can be learned from artificial lipid model systems and describe new ultrasensitive analytical techniques that could provide insight into their compositions and functions. We start by reviewing the recent literature suggesting a biological function of ICNs.

Biological Function

Signaling Across ICN

Watkins and Salter recently demonstrated that ICNs could facilitate calcium signaling between myeloid cells8 (Figure 9.1). Calcium fluxes influence many cellular events and are known to be transmitted between adjacent cells through gap junctions or through autocrine activity of secreted adenosine triphosphate. Coordinated intercellular calcium oscillations offer a rapid mechanism of local intercellular communication and are thought to be critical in synchronizing cellular activities. Watkins and Salter8 observed that dendritic cells (DCs) at the receiving end of signals delivered by nanotubes rapidly underwent morphological changes, demonstrating how important messages might be delivered through ICNs. A physical disruption of ICNs between the cells prevented cell-to-cell transmission of calcium fluxes, indicating that, at least in some situations, ICNs can function as an alternative to established intercellular calcium signaling pathways. Based on the speed by which the calcium flux propagates, action potential was ruled out. Instead, as is the case for gap junction-dependent intercellular calcium transmission,26 ICNs may allow cell-to-cell transport of the second messenger inositol trisphosphate (IP3), which induces calcium release from internal stores. Here, heterogeneity among different cell types is apparent as, for example, ICNs between T-cell do not facilitate intercellular calcium signaling.17

Figure 9
Transport of cargo in intercellular nanotubes (ICNs). (1) Diffusional translocation of molecules is effective on the cellular scale. (a) and (b), Time sequence of the spread of calcium signal (pseudocolored) on the left and the corresponding differential ...

ICN-mediated Protein Transfer

Another potential functional role of ICNs could be to mediate the exchange of proteins between cells.27 It has also been shown by Niu et al.28 that intercellular transfer of proteins, lipid transfer, and cytoplasmic component transfer can occur simultaneously and that a direct cell–cell contact is required. Several reports have shown that immune cells can swap proteins during transient encounters.29,30 For example, T-cells can acquire antigen from target cells, making them susceptible to being killed by other cytotoxic T lymphocytes (CTL), in a mechanism that has been termed fratricide killing.31 Similarly, it has recently been shown that NK cells that have acquired activating ligands during encounters with target cells can trigger activation in other NK cells.101 The mechanisms for membrane transfer are largely unknown, as discussed in recent reviews.24,32 Potential routes of transfer include membrane bridges that have been observed in the immune synapse between CTL and target cells,33 membrane protrusions within and surrounding the immune synapse in NK cells,34 and ICNs.23 Flux of membrane proteins over membrane nanotubes has been used to demonstrate that NVNs can be reconstituted with plasma membrane lipids and proteins of cultured cells.6

ICN-Mediated Pathogen Transfer

Virus

In recent years, HIV has been found to transfer between cells directly via ICNs by mechanisms that may facilitate the evasion of the immune defense. Lehman et al. showed that viruses can undergo inward trafficking along filopodia similar to the myosin II-dependent retrograde flow observed in several other systems.35 As the virus reached the cell body, it could be endocytosed, thus infecting the cell. In a following study, it was shown that uninfected cells could extend filopodia toward infected cells, establishing an ICN, or viral cytoneme.16 It was shown that viruses could undergo retrograde flow along this newly formed ICN, leading to infection of the previously uninfected cell [see labels a–c in Figure 2.1], and that the stability of the ICN was dependent on a receptor–ligand interaction.

Figure 2
Pathogens exploit intercellular nanotubes (ICNs). (1) Viral particles. ICNs mediate cell-to-cell transmission of retroviruses. (a), Superimposed video frames illustrate the opposing movements of a receptor-expressing filopodium (red) and the movement ...

In another study, Sowinski et al. proposed17 that HIV-1 spread using ICNs formed by short-term intercellular unions between T-cells. Here, transfer was also observed to be receptor dependent, as T-cells lacking CD4 were not infected in cocultures with infected T-cells. The ICNs between T-cells appear to be distinct from those observed by Mothes and coworkers35 as a dynamic junction was observed along T-cell nanotubes or at their contact with cell bodies. Unlike the report from Sherer et al.16, images of T-cell ICNs indicate that these could extend from the infected cells allowing ‘forward’ transfer of virus particles along the tube toward the uninfected cell.

Recently, it was reported that an HIV infection of primary human macrophages induces the formation of ICNs with a time course that correlates with that of viral replication15 [see label d in Figure 2.1]. The authors reported that several classes of ICNs and viruses seemed to be localized both inside and on the surface of these nanotubes. It has also been shown that HIV infection modifies cell-cell interaction, enhancing the number of viral synapses, filopodial bridges and ICNs in a manner that is dependent on lymphocyte function associated antigen 1 (LFA-1), an integrin previously known to also facilitate HIV replication.37 Taken together, these reports have shown that ICNs are accessible to viruses and that there are mechanisms for transmission between cells and possibly even upregulation of ICNs during infection. However, the details of these processes are largely unknown.

Bacteria

Mycobacterium bovis bacillus calmette-Guerin (BCG) has been observed to bind and surf along ICNs, connecting human macrophages. This surfing was mediated by a constitutive flow of nanotube membrane that appeared to be similar to the retrograde flow suggested for viral transport.16 Önfelt et al. demonstrated that bacteria could be transported along the nanotube to the cell body where they were phagocytosed11 [see labels a and b in Figure 2.2], but it is unclear if this process was dependent on specific receptors mediating binding to the bacteria and anchoring to F-actin inside the ICNs. However, for human neutrophils Galkina et al.38 proposed a mechanism by which nitric oxide-induced ICNs enable neutrophils to bind and aggregate bacteria at a distance from the neutrophil cell body. Experiments in NVN systems have also shown similar surfing of bacteria, both on the outer and inner leaflet of the bilayer membrane, with the aid of tension induced membrane flow [see label c in Figure 2.2].36 Thus, these studies suggest that ICNs can capture bacteria in the extracellular space and transport them to cell bodies for phagocytosis. One of the most intriguing processes relating to ICNs is cell-to-cell transfer of the bacteria Listeria monocytogenes.39 The bacterium takes over of the host cell's cytoskeleton machinery and polymerizes a so-called comet tail, using host-produced actin filaments and is pushed through the host cell's membrane to invade neighboring cells, creating a trailing nanotube-like structure between the cells in the process.

Prions

How prions spread to and through the central nervous system has been a long-standing question. Reports have indicated that cell–cell contact enhances the infection process; for example, it has been shown that scrapie prions transfer by cell–cell contact40 and observed that prion proteins incorporated in vesicles are capable of intercellular transfer through neurites.41 Gousset et al.18 have now demonstrated that prion protein can travel through ICNs between neuronal cells, or connecting primary neurons with bone marrow-derived DCs, suggesting a potential route for prion spread from the peripheral site of entry to the nervous system via ICNs (Figure 2.3).

Wild-type prion protein traveling through ICN suggests that prion can spread via these structures.18 Similarly, the misfolded, diseased form of the prion protein (PrPSc) could be transported through ICNs and the speed and pattern of migration indicated a vesicular transport mechanism. It was shown that diseased prions only spread when cells were connected by ICNs as other types of cell–cell contact did not allow spreading of PrPSc.

In Vivo

In vitro model systems play a crucial role in the investigation of the molecular mechanisms for the formation and function of ICNs. To rule out the possibility that ICNs are merely an artifact of in vitro culture conditions, the community has also started to address the issue of in vivo observation of ICNs. Cellular extensions that resemble ICNs were observed in tissue before the report by Rustom et al. in 2004. Among them is the report of cytonemes in the fruit fly imaginal discs2 (Figure 1.1) and interconnecting filopodia in mouse blastocyst.42 The first example of ICNs formed in animals was demonstrated in mouse corneas19 (Figure 3). These were generally short and straight and corresponded closely to previous descriptions of membrane nanotubes in vitro. In this study, the frequency of ICNs was significantly increased in corneas subjected to trauma and lipopolysaccharides, which suggests that nanotubes have an important role in cell–cell communication between widely spaced DCs during inflammation. The authors speculated that ICNs between DCs in vivo could represent a significant means of transmitting antigen, thus amplifying local immune surveillance in an environment like the mammalian cornea where antigen-presenting cells are scarce.

Figure 3
In vivo

Structure and Dynamics of Icns

Within the lamellipodia of migrating cells are actin structures, which, when they spread beyond the lamellipodium frontier, are called filopodia. As a cell migrates along a surface, it extends filopodia at the leading edge, attaching the cell to the substratum at focal adhesion spots further down the migratory pathway.43 Connected with the ability to generate mobility, the filopodia participate in fundamental physiological processes like wound healing, developmental processes such as neurite outgrowth, serving as precursors for dendritic spines in neurons,44 and in cell signaling.45

Mechanisms for the up- and downregulation of the actin filaments46 [see label b in Figure 4.1] perform a wide range of important functions in cell motility, as well as in locating and transporting protein complexes in the cell. The dynamic assembly and disassembly of actin structures, such as lamellipodia and filopodia, are controlled by protein binding to existing actin filaments to form a branching filament network.47,48 This F-actin regulatory pathway is likely to also affect formation of ICN.49

Figure 4
Cytoskeletal structure and formation of ICNs (1) Actin structure of ICNs illustrated with macrophages, (a), differential interference contrast micrograph, (b), micrograph showing a 3D rendered confocal stack. F-actin stained by phalloidin (white) and ...

Cytoskeletal Structure of ICNs

Several reports have shown that actin is a main cytoskeletal content of ICNs5,13 (Figure 4.1), and studies on fixed samples have revealed F-actin organization with an implantation pattern into the cell resembling structures seen in filopodia.50 Interrupting actin polymerization reduces transfer efficacy via ICNs.18,49 There are also examples of ICNs that contain microtubuli, such as in macophages,11 urothelial cell lines,51 and in human prostate cancer cells.7

Formation of ICNs

Researchers have observed different mechanisms for ICN formation. For example, it has been shown that ICNs can form de novo, growing from filopodial structures.4,16 Membrane tube bundles emanate from a single cell and dynamically protrude out into the surrounding media seeking contact with neighboring cells, [see labels a and b in Figure 4.2], and occasionally three-way junctions (bifurcation) can be observed, [see label c in Figure 4.2].

ICN can also form during separation after tight cell–cell contacts [see label d in Figure 4.2]. Immune synapses between NK cells and EBV-transformed B-cells were observed to result in nanotube formation as cells separated. Similarly, ICNs have been observed after transient contacts between macrophages5 and T-cells.17,52 When the membranes of the two cells detach, extensions containing both actin and cytokeratin filaments form as the cells move apart. Cytokeratins provide these nanotubes with stronger mechanical properties, preventing rupture because of cell migration or environmental stresses.53

Recently, putative molecular mechanisms have been proposed to explain the formation of ICNs. The formation of ICNs between T-cells, for example, was proposed to occur through LFA-1, and integrin activation by the cysteine protease cathepsin X was suggested to cause the elongation of nanotubes toward target cells as well as to support ICN formation when cells move apart52(Figure 4.4).

Membrane Continuity

An unresolved question is which mechanism regulates whether the ICNs are open-ended, with a continuous membrane between the connected cells4,8 (Figure 5(a)), or contain an intercellular junction17 (Figure 5(b)). Rustom reported evidence that green fluorescent protein (GFP) bound to the plasma membrane could transfer between cells connected by ICNs4 and Önfelt et al. reported the colocalization of membrane components at the base of an ICN between two EBV-transformed B-cells, possibly indicating fused plasma membranes.5 In T24 cells, actin–GFP have been observed to spread into nontransfected neighboring cells51 connected by ICNs. However, in that case, lipid material was not observed to pass through the junction, which would be expected if the tube had a continuous membrane. Several other observations suggest that ICNs are not open-ended tunnels, but instead contain a distinct junction between the two connected cells. For instance, two reports show that T-cell nanotubes contain such a junction17,52 (Figure 5(b)). Thus, there is compelling evidence that ICNs are heterogeneous with regard to the structure of the interface between connected cells. One important future challenge is to resolve under what circumstances and by which mechanisms membranes can fuse, leading to open-ended ICNs.

Figure 5
Membrane continuity of intercellular nanotubes. (a) Transmission electron microscope (TEM) ultrastructure of a tunneling nanotube (TNT) between rat pheochromocytoma cells where a serial sectioning showed that, at any given point along TNTs, the membrane ...

Model Nanotubes and Formation of NVNs

Methods based on pipette aspiration can be used to probe the physical and chemical properties in single lipid nanotube extensions and nanotube three-way junctions in model systems like NVNs. Formation of model nanotubes from vesicles can be performed by extracting membrane material with methods that apply a point force to the lipid membrane, for instance by micromanipulation54 (Figure 6), optical tweezers55 (Figure 7.1), or by using motor proteins56 [see label (e-g) in Figure 9.3].

Figure 6
Schematic illustration of the nanotube-vesicle network fabrication principle. (a), By a combination of mechanical deformation and electric pulses across the liposome, a microinjection needle is inserted into a unilamellar liposome connected to a multilamellar ...
Figure 7
Membrane dynamics and three-way junction formation. (1) Direct nanotube extraction from cell by laser trap manipulation. (a), As a membrane nanotube is extracted from the cell, force versus microscope stage displacement, (b), is recorded, where the dashed ...

With a method based on the principle of electroporation,58 a microinjection technique can be utilized to create biomimetic NVNs22 (Figure 6). The networks are composed of surface-immobilized phospholipid bilayer vesicles, interconnected with vesicles 1–50 μm in diameter and 10–15 – 10–12 L in volume. The width of these synthetic lipid nanotubes is approximately 25–300 nm in diameter, similar to the dimension observed for ICNs. By electromechanical insertion of a pipette tip into a unilamellar vesicle, followed by lateral pulling of the micropipette away from the vesicle, a nanotube is formed. Buffer solution in the pipette is injected into the nanotube orifice, forming a vesicle of controlled size that can be immobilized on the surface. The networks have controlled connectivity and precise topography with regard to the container size, the angle between nanotube extensions, and nanotube length.59 The internal fluid composition of individual vesicles is defined during the formation of the network by the selection of the solution within the micropipette.60 The protocols allow for the formation of NVNs of high geometrical complexity,61 where each node within a network can have a unique chemistry62 and material can actively be transported in the nanotubes.63 In addition, the NVNs can also be created from live cells based on these techniques,6 (Figure 1.4) to study transport phenomena such as protein transfer onto nanotubes.

Membrane Dynamics of Nanotubes

Upon the formation of ICNs, the plasma membrane must undergo substantial adaptive changes. The plasma membrane is a fluid lipid bilayer that responds elastically to applied mechanical forces, and these forces will become distributed throughout the entire surface area. To buffer against changes in membrane tension cells maintain a plasma membrane reservoir in the form of ruffles or by addition of lipids from internal stores.64 This reservoir of excess cell membrane makes the extraction of nanotubes feasible.

To understand the behavior and geometry of ICNs, a physical description of membrane mechanics is beneficial. One approach suggested by Helfrich in the 1970s is to use the thin elastic shell theory.65 The contribution to the elastic free energy of the lipid membrane is described by a set of independent shape deformations of the membrane surface. Any such membrane deformation must increase the total energy compared to that at equilibrium. With the development of the theories of lipid membrane mechanics, the principles of vesicle bilayer and NVN formation, based on properties such as thermal transitions, elasticity, rigidity, cohesion, and colloidal interactions, have become better understood.66,67 It is now generally accepted that the shape of a lipid vesicle is determined primarily by the bending elasticity and curvature of the vesicle.

Once formed, artificial membrane nanotubes can remain stable.56 NVNs are also stable in a local energy minima as long as the network is anchored to a substrate.20 It has been suggested that the stability of the tubular membrane protrusions without the inner supporting rod-like cytoskeleton is a consequence of the accumulation of anisotropic membrane components, or nanodomains, [see label (a) in Figure 4.3], in the bilayer membrane of nanotubular protrusions.68 These properties of lipid bilayers and plasma membranes provide insight into the stability of ICNs once formed, even if actin filaments are disassembled.51

Membrane dynamics of nanotubes formed from cells, for example, the nanotubes observed in human peripheral blood69 and tubulovesicular extensions in human neutrophils70 acquired with measurements of nanotube extraction forces, provide information on their structure and elastic properties. Overall, despite their actin content50 (Figure 7.1), the elastic dynamics of ICN do not appear to be fundamentally different from those observed for hollow tubes in endoplasmatic reticulum, Golgi apparatus71 or artificial systems.57 Considering these similarities, understanding the dynamics of pure lipid membrane networks, the principles of their reorganization and the transport inside them will facilitate understanding the organization and trafficking inside ICNs.

Three-way Junctions

In coalescence experiments in model systems, in which two nanotubes are pulled from the same vesicle and brought closer to each other until they merge,72 the force required to extract a tether and the angle between tethers at coalescence directly yield the bending rigidity and the membrane tension of the membrane nanotubes, and can therefore provide information on the physical dimensions of nanotubes. For instance, such measurements with soybean lipid composition have yielded nanotube radii of 110 ± 26 nm.73 As the tubes coalesce during micromanipulation, they rearrange into three-way junctions (Y-junction) and a similar mechanism can induce self-organization and restructuring of the NVN.74,75 Similarly, three-way junctions51 and V–Y transitions10,50 have been observed in ICNs (Figure 7.2). Surface free energy considerations show that three-way junctions appear spontaneously in NVN systems when two adjacent nanotubes overcome the critical coalescence distance.76 In relaxed NVN systems, three-way junctions are positioned so that connected tubes form 120° relative to each other, thus minimizing the distance required to connect the vesicles.

Interestingly, the three-way junctions observed for ICNs5,50 also often display 120° angles between connected nanotubes (Figure 7.2). In model systems, the path minimization mechanism is well understood, since the lipid networks are fluid and numerical models can be used to study the dynamics of self-organization57 (Figure 7.3). However, considering that ICNs often contain F-actin, the mechanism behind dynamically moving three-way junctions is less clear for cellular systems. Furthermore, three-way junctions in NVNs are fully open, allowing molecules to diffuse in all directions. Whether this can also be the case for similar junctions in ICNs is unknown. Judging from the observed heterogeneity of ICNs, it is unlikely that the answer is straightforward.

Materials and Methods of Nanotubular Studies

Techniques employed for the study of ICNs have mostly been based on microscopy. Fluorescence microscopy77 (Figure 8.1) offers spatiotemporal information of protein trafficking and signal propagation, while electron microscopy78,79 (Figure 8.2) gives high resolution information about cellular structures. Most of our understanding of ICNs has been provided by fluorescence or electron microscopy studies, both on live cells using various fluorescent markers and on fixed cells using immunofluorescence or electron microscopy. Unfortunately, microscopy techniques offer little biochemical information that bulk biochemical analysis methods (e.g., mass spectrometry80) can provide.

Figure 8
Materials and methods of nanotubular studies. (1) Fluorescent imaging techniques such as confocal imaging provide spatiotemporal information based on fluorescent staining. Labeling of cells is based on, (a), genetically modified cell lines expressing ...

Development of ultrasensitive bioanalytical methodologies for the analysis of single cells and organelles could help to address this issue.81 One particular suite of developed tools, for example, should be able to extract detailed chemical information from subcellular structures, organelles, and ICNs from live cells while preserving the spatiotemporal information offered by high-resolution fluorescence microscopy81 For instance, single-cell nanosurgery82,83 could be used to isolate single or small groups of ICNs. Once isolated, ICNs could be encapsulated in aqueous droplets and used as nanolabs for the biochemical manipulation84 and analysis of the composition.8486 A schematic work flow involved in using a droplet nanolab approach for the analysis of single cells, organelles, and ICNs is depicted in Figure 8.3.

Besides the development and application of new sensitive analytical techniques to the study of ICNs, improvements of existing techniques will also enhance our ability to study ICNs. For example, microscopy techniques, such as stimulated emission depletion,87,88 can improve resolution beyond the diffraction limit; computer-based methods, such as the automated detection of nanotubes,89 facilitate data gathering and analysis. As a complement, model systems such as NVNs21,22 may provide fundamental understanding of mechanistic principles, for instance, of transport phenomena36,90,91 and the regulation of reactions.92,93

Transfer and Transport Mechanisms

Recent studies have shown that ICNs can transport a range of cargo, including proteins and organelles, by several different mechanisms. Clearly, in ICNs containing cytoskeletal structures and associated motor proteins, there are potential mechanisms for active transport that are lacking in artificial systems. However, studies of NVN systems have shown that more passive means of transport, for example, diffusion and Marangoni flow, can have functional properties.

Diffusion Inside Nanotubes

There are some indications that diffusion could be a functional mechanism for communication via ICNs. For instance, the calcium signals transmitted between macrophages8 could be mediated by the diffusion of IP3. This mode of transport would require an open-ended tubular structure. Mixing and transport by diffusion could be efficient considering the small length scales of cells networked by ICNs as well as NVNs. Theoretical studies modeling nanotube–vesicle topologies show that network geometry influences the result and can be used to describe how content concentrations in involved vesicles evolve over time.94 The effects of compartmentalization on diffusional modification have been investigated in NVNs95 (Figure 9.1), illustrating how diffusion can be harnessed.

Passive Membrane Transport Along the Nanotube Surface

Cell-surface proteins and/or patches of membrane might transfer via nanotubular connections.4,5 In addition to biological ATP-dependent mechanisms such as the previously mentioned retrograde flow, it has been demonstrated both theoretically and experimentally that a gradient or difference in membrane tension across a lipid membrane surface can drive the lipids to flow from regions of low tension to regions of high tensions, in order to eliminate the tension difference97 (Figure 9.2). In the case of bilayer membranes, nonuniform lipid distributions can be a result of, for example, fluid convection, temperature gradients, electric fields, laser light, and mechanical means like cell movement or the extension of membrane protrusions through actin polymerization or through mechanical force in model systems. The spatial variation in interfacial tension created by such protrusions generates tension gradients over membrane regions that produce a membrane flow directed toward regions of higher surface tension.90 In NVNs, it has been shown that this tension driven surface flow of the lipid membrane induces a Marangoni plug flow of the solution inside the nanotube21,98 which in turn transports contents from one vesicular compartment to another.36

Formation of Bulges or Gondolas

Related to the transport of membrane material over nanotubes, several separate reports have identified moving vesicles, bulges, or gondolas integrated into ICNs.4,5,7 Such expansions of intercellular nanotubes (Figure 9.2) have in some studies been interpreted as cargo too large to fit the inner diameter of the tube.69 Formation of bulges in ICNs is reminiscent of observations in model systems.91 Where a sudden tension difference has been shown to be a mechanism for gondola formation or pearling. In cellular systems, such a change in tension could be caused, for example, by diverging cells. The anchoring of a structural polymer to the membrane99 has also been linked to pearling behavior in model systems. It has been suggested that a similar mechanism may also take place in cells.51 Rearrangement of local constituents, such as lipids and proteins along the nanotubes, enable and favor the formation of the dilatations in cellular nanotubes. Pearling behavior in cells to create gondolas69 might hence be caused by a gradual disruption of the actin cytoskeleton.100

Molecular Motor Dependent Transport

Organelle transport along actin inside nanotubes is mediated by, for example, the translation of myosin V along F-actin. For instance, unidirectional vesicular traffic for PC12 cells4 has been reported, while bidirectional trafficking11 along microtubules was shown for human primary macrophages. In normal rat kidney (NRK) cells and PC12 cells, nanotubes mediate the transfer of various cellular components, including endocytotic organelles.4 The observation of unidirectional transport along ICNs has been suggested to be linked to active transport by myosin motors along actin filaments for organelle transfer13 and viral spread.35The observed reduction of viral infection and organelle transfer in the presence of F-actin depolymerizing agents suggests that transport along nanotubes and filopodia is mediated by the underlying actin cytoskeleton and is controlled by myosin. Because myosin II is a plus-end motor that mediates minus end motility toward the cell body, it must regulate the movement of entire actin filaments, a process called retrograde F-actin flow.35 In addition, a reduction of organelle transfer was also observed after microtubules were depolymerized with nocodazole, although less pronounced as compared to F-actin depolymerizing conditions. This observation might suggest that NRK cell microtubules do not participate in the intercellular translocation itself; instead they convey a function of targeting endosomal organelles to nanotubular entry sites in the cell periphery.

Actin-driven Surfing

Surfing, where cargo is transported along the outside surface of nanotubes (Figure 9(c)) has been demonstrated by the intercellular trafficking of bacteria11 or viral particles.16,17 Surfing is caused by retrograde flow that may be dependent on myosin II. The mechanism is a two-step process, in which the cargo first attaches to the membrane through anchors, such as transmembrane receptors that bind to a virus,16 bacteria,11 or similar object on the outer leaflet of the cell membrane. The binding event is followed by a flow of membrane material toward the target cell transporting the attached cargo.

Conclusion

The discovery of intercellular nanotubes as a novel form of intercellular communication has triggered a lot of attention and new research. However, further research is needed to understand the fundamental properties of ICNs, establish if they are as common in vivo as they are in vitro and, thus, could play a role in intercellular communication and disease on the systemic level.

This review has focused on a variety of topics connected to ICNs including their structure and biological function, which transport processes they support. We also suggest that an interdisciplinary approach, involving studies in artificial systems such as NVNs and the use of novel ultrasensitive bio-analytical methodologies could be a route toward understanding more about ICNs. However, analysis of the biochemical compositions and biomechanical properties of ICNs involves technical challenges. Such analysis includes implementation of novel capabilities offered by, for example, droplet microfluidics and nanosurgery. Application of such new methodologies suggests exciting new possibilities for ICN research.

Acknowledgments

This work was supported by a fellowship from the Knut & Alice Wallenberg Foundation (to J. Hurtig), by the National Institutes of Health and the National Science Foundation (to D. T. Chiu), and from Swedish Foundation for Strategic Research and Swedish Research Council (to B. Önfelt).

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Further Reading

On the following topics we recommend these reviews as further reading.

Intercellular nanotubes

  • Gurke S, Barroso JFV, Gerdes HH. The art of cellular communication: tunneling nanotubes bridge the divide. Histochemistry and Cell Biology. 2008;129(5):539–550. doi: 10.1007/s00418-008-0412-0. [PMC free article] [PubMed] [Cross Ref]
  • Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, Köhler K, Oddos S, Eissmann P, Brodsky FM, Hopkins C, Önfelt B, Sattentau Q, Davis DM. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nature Cell Biology. 2008;10:211–219. doi: 10.1038/ncb1682. [PubMed] [Cross Ref]
  • Davis DM. Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nature Reviews Immunology. 2007;7(3):238–243. doi: 10.1038/nri2020. [PubMed] [Cross Ref]
  • Gerdes HH, Carvalho RN. Intercellular transfer mediated by tunneling nanotubes. Current Opinion in Cell Biology. 2008 doi: 10.1016/j.ceb.2008.03.005. [PubMed] [Cross Ref]

Nanotube-Vesicle Networks

  • Karlsson M, Davidson M, Karlsson R, Karlsson A, Bergenholtz J, Konkoli Z, Jesorka A, Lobovkina T, Hurtig J, Voinova M, Orwar O. Biomimetic Nanoscale Reactors and Networks. Annual Review of Physical Chemistry. 2004;55:613–649. doi: 10.1146/annurev.physchem.55.091602.094319. [PubMed] [Cross Ref]
  • Jesorka A, Orwar O. Liposomes: Technologies and Analytical Applications. Annual Review of Analytical Chemistry. 2008;1:801–832. doi: 10.1146/annurev.anchem.1.031207.112747. [PubMed] [Cross Ref]

HIV and ICNs

  • Sherer NM, Mothes W. Cytonemes and tunneling nanotubules in cell-cell communication and viral pathogenesis. Trends in Cell Biology. 2008;18(9):414–420. doi: 10.1016/j.tcb.2008.07.003. [PMC free article] [PubMed] [Cross Ref]
  • Hladik F, McElrath MJ. Setting the stage: host invasion by HIV. Nature Reviews Immunology. 2008;8(6):447–457. doi: 10.1038/nri2302. [PMC free article] [PubMed] [Cross Ref]
  • Jones S. Cell Biology: Tube travel for HIV? Nature Reviews Microbiology. 2008;6(3):175. doi: 10.1038/nrmicro1866. [Cross Ref]
  • Sattentau Q. Avoiding the void: cell-to-cell spread of human viruses. Nature Reviews Microbiology. 2008;6(11):815–826. doi: 10.1038/nrmicro1972. [PubMed] [Cross Ref]