Had Amin Rustom not messed up, he would not have stumbled upon one of the biggest discoveries in biology of recent times. It all began in 2000, when he saw something strange under his microscope. A very long, thin tube had formed between two of the rat cells that he was studying. It looked like nothing he had ever seen before.

His supervisor, Hans-Hermann Gerdes, asked him to repeat the experiment. Rustom did, and saw nothing unusual. When Gerdes grilled him, Rustom admitted that the first time around he had not followed the standard protocol of swapping the liquid in which the cells were growing between observations. Gerdes made him redo the experiment, mistakes and all, and there they were again: long, delicate connections between cells. This was something new - a previously unknown way in which animal cells can communicate with each other.

Gerdes and Rustom, then at Heidelberg University in Germany, called the connections tunnelling nanotubes. Aware that they might be onto something significant, the duo slogged away to produce convincing evidence and eventually published a landmark paper in 2004 (Science, vol 303, p 1007).

cellular tunnelling nanotubes
© Paul McMenamin / UWA
Tunnelling nanotubes seem to play a major role in anything from how our immune system responds to attacks, to how damaged muscle is repaired after a heart attack.

A mere curiosity?

At the time, it was not clear whether these structures were anything more than a curiosity seen only in peculiar circumstances. Since their pioneering paper appeared, however, other groups have started finding nanotubes in all sorts of places, from nerve cells to heart cells. And far from being a mere curiosity, they seem to play a major role in anything from how our immune system responds to attacks to how damaged muscle is repaired after a heart attack.

They can also be hijacked: nanotubes may provide HIV with a network of secret tunnels that allow it to evade the immune system, while some cancers could be using nanotubes to subvert chemotherapy. Simply put, tunnelling nanotubes appear to be everywhere, in sickness and in health. "The field is very hot," says Gerdes, now at the University of Bergen in Norway.

It has long been known that the interiors of neighbouring plant cells are sometimes directly connected by a network of nanotubular connections called plasmodesmata. However, nothing like them had ever been seen in animals. Animal cells were thought to communicate almost entirely by releasing chemicals that can be detected by receptors on the surface of other cells. This kind of communication can be very specific - nerve cells can extend over a metre to make connections with other cells - but it does not involve direct connections between the interiors of cells.

Quite different

The closest animal equivalents to plasmodesmata were thought to be gap junctions, which are like hollow rivets joining the membranes of adjacent cells. A channel through the middle of each gap junction directly connects the cell interiors, but the channel is very narrow - just 0.5 to 2 nanometres wide - and so only allows ions and small molecules to pass from one cell to another.

Nanotubes are something different. They are 50 to 200 nanometres thick, which is more than wide enough to allow proteins to pass through. What's more, they can span distances of several cell diameters, wiggling around obstacles to connect the insides of two cells some distance apart. "This gives the organism a new way to communicate very selectively over long range," says Gerdes.
"It is a previously unknown way in which cells can communicate over a distance."
Soon after they first saw nanotubes in rat cells, he and Rustom saw them forming between human kidney cells too. Using video microscopy, they watched adjacent cells reach out to each other with antenna-like projections, establish contact and then build the tubular connections. The connections were not just between pairs of cells. Cells can send out several nanotubes, forming an intricate and transient network of linked cells lasting anything from minutes to hours. Using fluorescent proteins, the team also discovered that relatively large cellular structures, or organelles, could move from one cell to another through the nanotubes.

Tube travelling

The first clue to how membrane nanotubes, as some researchers prefer to call them, might be used by cells came from the US. Simon Watkins of the University of Pittsburgh, Pennsylvania, and his colleagues were studying dendritic cells, the sentinels for the immune system. When a dendritic cell detects an invader, it gets ready to sound the alarm. One sign of this activation is a change in calcium levels in the cell.

While Watkins was poking a dendritic cell with a micro-needle filled with bacterial toxins, he noticed a calcium fluctuation in a dendritic cell far away from the one that was touched. "Wow, that's pretty cool," thought Watkins. Information about the toxins was somehow being passed from the cell being poked to a distant cell. Nothing in his experience could explain the phenomenon.

When Watkins dived into the literature, he discovered Gerdes's paper. His team then took another look at the dendritic cells. Sure enough, they found the cells were connected by a network of tunnelling nanotubes.

Recruiting an army

Watkins thinks that the dendritic cells could be using nanotubes to recruit other cells. Conventional wisdom says that once a dendritic cell is activated, it migrates to the lymph nodes to alert the immune system. Sometimes, it might have to travel from the tip of one's finger to the armpit - a long and perilous journey that could result in failure. But if a dendritic cell first recruits other sentinels, and all of them march towards the lymph nodes simultaneously, there is much less chance of the message being lost. "It allows you to amplify the response," says Watkins. "That's all hypothesis really. We have to prove it."

Meanwhile, Stefanie Dimmeler of the University of Frankfurt in Germany and her team have been studying how so-called progenitor cells can be transformed into heart muscle cells. In mice at least, progenitor cells injected after heart attacks seem to turn into new heart muscle cells, replacing dead tissue.

When Dimmeler's team mixed heart muscle and progenitor cells in a dish, they found that the two populations established connections via nanotubes. They even observed the transfer of organelles such as mitochondria (Circulation Research, vol 96, p 1039). Dimmeler suspects that the transfer via nanotubes of signalling molecules and proteins called transcription factors helps to transform the progenitor cells.

Beware hijackers

The best way to prove this would be to show that without nanotubes, progenitor cells mixed with heart muscle cells do not turn into heart muscle cells as well. The trouble is that no one has yet found a way of destroying nanotubes without also damaging the cells that they are attached to.

While the evidence for the normal roles of tunnelling nanotubes remains circumstantial, it is becoming clear that the networks they form can be hijacked. In a study published earlier this year, Daniel Davis's team at Imperial College London infected some immune cells with an HIV modified to express a fluorescent protein, and mixed the infected cells with healthy ones. "We could literally see clumps of that protein moving from the infected to the uninfected cell, along the strand of the nanotube," says Davis. This strongly suggests that the infection may spread from cell to cell in this way (Nature Cell Biology, vol 10, p 211).

This might help to explain why some HIV-infected people, though they carry antibodies to the virus, cannot seem to get rid of it. "Somehow viruses are avoiding immune-system recognition, and one way they could do that is if they could get from one cell to another through direct cell contact," says Davis.

Another infectious agent that could spread via tunnelling nanotubes are prions, the cause of mad cow disease. "One of the key unresolved issues in prion-disease research is how prions pass from one cell to another, as they have to do to spread throughout the nervous system and cause disease," says Byron Caughey of the US National Institutes of Health in Hamilton, Montana.

The prion connection?

Caughey's team has already uncovered one mechanism unrelated to tunnelling nanotubes by which certain prions spread, but it is probably not the only way. "At this point, we are very intrigued by the possibility that tunnelling nanotubes are partly responsible for the spread of prion infectivity from cell to cell," he says. "Once you know the mechanism of cell-to-cell transfer, hopefully it opens up new therapeutic targets."

Nanotubes may also play a role in tumours becoming resistant to chemotherapy. Much of this resistance is due to a class of proteins called ABC transporters, which pump the anti-cancer drugs out of cells. It was recently discovered that tumour cells without these proteins can acquire them from other tumour cells. But how?

Prostate cancers cells have recently been spotted swapping material via a network of tunnelling nanotubes. It is possible to imagine cancer cells using tunnelling nanotubes to exchange ABC transporters and spread drug resistance throughout the tumour, says Gerdes. "If this were to be true, and if I could find a drug which inhibits the growth of these nanotubes, I could reduce the resistance to chemotherapy."

Many shapes and sizes

That is easier said than done, though, because so little is known about these nanotubes. They seem to come in many shapes and sizes, of varying thickness and lengths, and differ from cell type to cell type. Precisely how they function is not yet clear. There are some hints, however. Gerdes's team, for instance, discovered that the nanotubes they studied contained myosin Va, a type of motor protein. Elsewhere in cells, objects attached to myosins move along tracks made of proteins called actins, so Gerdes thinks that this kind of process might help move things through nanotubes.

Characterising exactly what these nanotubes are made of will be crucial. Gerdes's team, for instance, is trying to find proteins that are specific to nanotubes. Once identified, such proteins can be tagged with fluorescent markers, making it much easier to see nanotubes. Such work could also make it possible to destroy or manipulate these structures, and thus provide solid proof of their importance - and a lot of researchers still need convincing.

If tunnelling nanotubes really are ubiquitous, the critics ask, how come they managed to evade discovery until so recently? And why have they only been seen in cells grown outside the body?

Elusive structures

There may be several reasons why nanotubes have eluded notice for so long. For starters, they are extremely fragile: merely shaking a dish of cells or changing the medium - as Rustom failed to do - can rupture these tubes, as can certain chemicals used to fix cells for observation, including those used with electron microscopes. Even prolonged exposure to light can destroy them. (This extraordinary susceptibility to chemicals and light may one day provide a means to selectively destroy nanotubes.)

In addition, when biologists observe cells in culture, they usually focus on the bottom of the dish or slide, where delicate structures such as nanotubes will be obscured by debris. Finally, although nanotubes are elusive, many researchers have spotted them over the years without realising it.

At the University of Western Australia in Crawley, for example, Paul McMenamin's team has been studying dendritic cells in mouse corneas. McMenamin's graduate student Holly Chinnery kept seeing something unusual. "She kept noticing these cells with big, long processes," says McMenamin. "She'd show me the pictures, and I'd say, 'Gosh, I haven't seen anything like that before'." And so it went until a colleague told Chinnery about nanotubes. "That immediately set us off," says McMenamin. "We realised that we had the first evidence of them in vivo."

Unbelievable

Their work, published in May, shows that nanotubes are not just an artefact of the methods used to grow cells in culture, as some have suggested. And what they have seen is spectacular: some of the longest tunnelling nanotubes ever observed, more than 300 micrometres long, connecting dendritic cells in the cornea (The Journal of Immunology, vol 180, p 5779). "We can see them their whole course, spindling all the way through the cornea," says McMenamin. "It's fantastic."

"I'll bet you that within weeks to months, people will start noticing them in other tissues. It's just a case of how you look," he adds. "You've got to know what you are looking for. It's a bit like being a good bird-watcher. A hundred people will see a flock of seagulls, and it's only a very good bird-watcher who will spot this one tern flying in that flock."

Gerdes, meanwhile, continues to marvel at what is unravelling before his very eyes. "Whatever one can think of has been done by nature," he says. "It is unbelievable what the cell is able to do."
It pays to network

The discovery of tunnelling nanotubes (see main story) has led to speculation about just how far their influence extends. Some think they might play a crucial role in development.One of the key outstanding questions in biology has to do with how cells communicate with each other as an embryo develops. We know that some cells release molecules called morphogens, which diffuse through the intercellular medium. The resulting concentration gradient tells cells where they are in the embryo and they can then develop accordingly. But this does not explain everything.For instance, two very distant cells can show similar patterns of gene expression while other cells nearby do not. If a morphogen gradient is responsible, then surely the cells closer to the one releasing the morphogen should also be responding.Such observations could be explained if morphogens are distributed directly to certain cells through a network of nanotubes, speculates Hans-Hermann Gerdes of the University of Bergen in Norway. "It would be much more appealing for nature to have this direct line," he says.Plants cells are known to use their version of nanotubes, called plasmodesmata, to transfer microRNAs that influence gene activity from one cell to another during embryogenesis. "These microRNAs lead to certain expression patterns in adjacent and also very remote cells," says Gerdes. Mammalian cells could also be doing the same.Even more speculative is the idea that the development of organs could be influenced by tunnelling nanotubes. It is not well understood how organs "know" how big they should get, or the exact shape to take. "These are all open questions," says Gerdes. He speculates that if the cells in a given organ were connected by nanotubes, then these connections could help establish a feedback mechanism that provides the necessary information for the organ to grow to the right size and shape.