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TNT and Talking Cells

Biologists have long known that cells "talk" to each other, but there are a limited number of ways in which this conversation can happen. Some cells speak with neurotransmitters or hormones. Other neighboring cells encode their chatter in ions that pass through the holes in their shared membrane known as gap junctions. In plants, a babble of water and small molecules flows through the plasmodesmata between cells. These forms of communication have been known for decades.

So cell scientists can be excused for their surprise when graduate student Amin Rustom at the University of Heidelberg recently described his evidence for a new conversational medium: entire organelles. This finding generated excitement because these intracellular compartments have the potential to carry cumbersome message packets, such as the fat–loving signaling molecules that don't dissolve in the cell's watery contents, and RNA, which dictates the assembly of new proteins. More novel still, a peculiar kind of delivery service—a long, thin, arrow–straight filament suspended between two cultured cells—ships these complex missives.

Rustom called the long strands tunneling nanotubes (TNTs for short) in a detailed report published in the February 13, 2004, issue of Science (303:1007–1010). With coauthors from Heidelberg and the Universities of Belgrade, Ulm and Bergen, he listed the defining features of a TNT: a tube 50–200 nanometers thick, up to several cell diameters long, and made from a membrane–sheathed bundle of actin protein. Despite the moniker, TNTs appeared to be solid rather than hollow; investigators found no evidence that they contained cytoplasm (cell fluids), and the small, diffusible, fluorescent molecule calcein couldn't pass through.

The tunneling nanotube...Click to Enlarge Image

The group soon found that nanotubes transport only specific types of cellular organelles. Cargo seems to be restricted to endosomes—membranous spheres that have been pinched off from the outer surface of the cell. These organelles are large compared to the usual diameter of the TNT, which must swell and contract around them as they scoot along. During this process, the TNT resembles a slim snake distended around its last big meal.

The authors aren't sure how cargo actually moves along the TNT, but they have two theories. Cell biologists know that actin filaments, which also form parts of the cytoskeleton, are inherently directional, having distinct "plus" and "minus" ends. Not surprisingly, the TNT highway is a one–way street. One possible explanation for the movement is a molecular "motor" such as myosin, which might carry the endosome as it walks—always in the same direction—down the nanotube. In fact, the myosin Va protein is found in TNTs. Unfortunately, the lack of cytoplasm presents a problem: Motors require energy in the form of adenosine triphosphate (ATP) molecules, which usually diffuse freely through the cell. No cytoplasm means no source of energy. To get around this obstacle, it's hypothetically possible that some sort of ATP "lunch box" could accompany the freight, but this is conjecture.

The second mechanism harnesses the continuous "treadmilling" of actin filaments, which prefer to polymerize at one end and depolymerize at the other end (hence the plus and minus designation). If the cargo were linked to an actin molecule in the long chain, then it might be carried along passively as one cell doggedly constructed filaments and the other cell relentlessly gnawed at the opposite end.

TNTs have intrigued many cell biologists, but the ones who study development may be most excited: TNTs could illuminate some long–standing mysteries in that field. For example, many morphogens—molecules that direct cells to grow and differentiate during embryogenesis—are not water–soluble, so biologists know little about how they spread out during early development. TNTs could be responsible for establishing morphogen gradients—which are known to exist—within growing embryos by successive endosomal transfers from a single source. The senior author on the paper, Hans–Hermann Gerdes, is clearly energized by this idea, but he also cautions that TNTs have not yet been seen in intact tissue.

Rustom first noticed the strands in a common line of cells called PC12 (a "tall" cell at approximately 15 microns) made from the adrenal gland. He later saw TNTs in two types of kidney cells in culture, raising the possibility that the mechanism may be widespread. But if TNTs are in many types of cells, why haven't they been seen before in hundreds of laboratories?

The answer may have to do with the particular fragility of the strands, which are broken by mechanical stress, by fixation—even by light. In fact, their discovery in this case was an accident, a fluke: Rustom had forgotten to change the media that nourished the cells (sparing them the mechanical shearing forces), and he was working in a darkened room (less chance of disruption by ordinary light) to prolong the life of a membrane–specific fluorescent marker. The microscope used to examine live–cell preparations was highly sensitive, so it didn't use a strong light source to illuminate the sample. And fortuitously, he began the experiment at a focal depth near the middle of the tall PC12 cells rather than the bottom, as he usually did. All in all, it was a serendipitous result. Rustom's advisor Gerdes talks about his part in the story with the ease of having told it many times:

"He phoned me from the imaging center in the cellar, saying, ‘I see something, and I find it somehow interesting, but I don't know exactly what it is. Would you come and have a look?' I saw a horizontal line between two cells … [but the] peculiarity was that it was hanging in the media. So I told him to work on the project for two months."

Of course, the presence of an entirely new paradigm wasn't felt right away. After the two–month progress report, "Our group had many questions. They were not enthusiastic," states Gerdes. Ironically, the objection at that time was that this curiosity had little to do with the main business of the lab—the investigation of membrane exchange between cells. But Rustom's persistence—plus well–timed negligence, a lucky choice of cells, a highly sensitive microscope for live–cell imaging and a keen eye—prevailed. Says his advisor of the paper's percussive impact, "we got a huge response." Explosive, you might say.


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