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
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 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.