Rieder was probing the origin of the microtubules that make up the kinetochore fibers (K-fibers), which attach chromosomes to the spindle during early prometaphase. In vitro data supported both that kinetochores trapped microtubules (MTs) growing from the pole and that kinetochores could nucleate microtubules directly (Mitchison and Kirschner, 1985a,b).
In 1981, Rieder and Gary Borisy had shown that during recovery from cold treatment, kinetochores did not nucleate MTs, and that those closest to the centrosomes attached first (Rieder and Borisy, 1981). But such in vivo studies were confounded by several difficulties, including that kinetochores are lost in the tangle of early prometaphase chromatin and attach very rapidly to the spindle.
Rieder and his colleagues found a system, cultured newt lung cells, that overcame these obstacles. Chromosomes would sometimes become “lost”—positioned far away from poles after nuclear envelope breakdown—and be significantly delayed in spindle attachment. He began filming such cells and saw the lost chromosomes attaching to MTs and moving polewards at speeds ∼30 times faster than anaphase movements.
Intrigued by the attaching chromosomes' speed, he set up a technically demanding set of experiments, called same cell correlative light and electron microscopy, to investigate and capture the mechanism of K-fiber formation. It went something like this: find a cell with a lost chromosome; place it in a perfusion chamber on the light microscope; and watch and film it until the chromosome begins rapid motion toward a pole. Then immediately fix the cell with glutaraldehyde and process it for immunofluorescence with antitubulin antibodies and, in the best cases, for serial section electron microscopy.
“There were so many places where things could go wrong” in the week-long process, says Rieder. But the grueling work paid off in stunning images of a chromosome zipping along a single MT toward a pole, with the MT extending laterally beyond the kinetochore (Rieder et al., 1990).
It showed that the “kinetochore could move across the surface of the MT and not on the MT's end,” says Rieder. The clocked speeds were faster than the rate of MT disassembly in newt cells and more in line with those of the microtubule motor dynein. “The take-home message,” he says, “was that the motors for movement during chromosome attachment are at the kinetochore.”
The group followed up with another video study showing a single astral MT growing out and snagging a lost chromosome's kinetochore (Hayden et al., 1990)—a discovery that had co-author Sam Bowser “hooting and hollering” in the lab before immediately backing up his videotape. A few months later, the result was confirmed in mammalian cells by another group's thick-section electron microscopy (Merdes and De Mey, 1990). Finally, Rieder's group showed a similarity between two movements—that of attaching chromosomes as they moved toward the minus ends of MTs, and of vesicles in the cytoplasm of dividing cells—which further argued for dynein-mediated attachment (Alexander and Rieder, 1991).