Marc Kirschner recalls that the mid-1980s “was a very innovative time.” While the era was giving rise to stonewashed jeans and rap music, cell biology was entering new territory as it finally went molecular. For cell biologists who, like Kirschner, were interested in the cytoskeleton, there was an even more exciting possibility: the modification of interesting proteins to form reagents that could be used to follow dynamics inside of cells. Kirschner's attention turned specifically to biotinylated tubulin.
By 1986, in vitro experiments had given rise to several models for microtubule (MT) dynamics, most notably treadmilling at steady-state (Margolis and Wilson, 1978) or the unusual growing and shrinking MT behavior termed “dynamic instability” (Mitchison and Kirschner, 1984). In a few preliminary in vivo experiments, it appeared that interphase MTs exchanged tubulin subunits rapidly, on the order of every 20 min (Salmon et al., 1984; Saxton et al., 1984), but without spatial resolution it was impossible to tell how they were being exchanged.
To address in vivo dynamics, Eric Schulze and Kirschner microinjected biotin-labeled tubulin into interphase fibroblasts for direct measurement of kinetics. With a growth rate of 3.6 μm/min, the team calculated that 80% of a cell's MTs would turn over in 15 min.
EM immunolabeling of the biotinylated tubulin allowed them to show that new subunit addition occurred in a directional manner, with one clear junction between unlabeled and labeled regions on all MTs (Schulze and Kirschner, 1986). The contiguity of old and new subunits demonstrated growth without the need for spontaneous assembly, and the speed and stochastic nature of the assembly (occurring at different times for different MTs) argued against universal treadmilling. Instead, the study's stunning images supported dynamic instability, with shrinking MTs providing the needed subunits for both growing MTs and new MTs being nucleated at the centrosome.
In a side note, the paper mentions the presence of a stable population of MTs, resistant to turnover. A subsequent study showed that the stable MTs are posttranslationally modified (Schulze et al., 1987), but Kirschner admits the role of this population remains a mystery. “Does the modification further stabilize the MTs?” he asks. “If MTs hit some region and become stabilized and modified, is this a way of generating polarity?” Some of the secrets of MT turnover, it seems, are yet to be revealed.