Interview for Faculty Position

Single-molecule studies of the microtubule depolymerase MCAK and the microtubule polymerase XMAP215

Gary Brouhard

Max Planck Institute

Microtubules are long, slender polymers of the protein tubulin, with which cellular structures such as the cytoskeleton, the mitotic spindle, and the axoneme are built. These structures are not static. Rather, they are broken down and rebuilt when a cell moves, changes shape, or progresses through the cell cycle. Microtubule polymerizers promote microtubule growth and increase polymer levels, while microtubule depolymerizers promote microtubule shrinkage and decrease microtubule lengths. To study the biophysical mechanism of these enzymes, we developed a single-molecule total-internal-reflection fluorescence assay to study the interaction of single polymerizing and depolymerizing molecules with microtubules. Our research has focused on two such enzymes. MCAK, a member of the kinesin family of motor proteins, is a microtubule depolymerase that catalyzes the removal of tubulin subunits from both ends in an ATP-dependent fashion. XMAP215 belongs to a conserved family of proteins that accelerate microtubule growth ten-fold. We find that XMAP215 is a polymerase that directly catalyzes the addition of tubulin dimers to the growing plus end. We found that both enzymes undergo a random motion on the microtubule equivalent to a one-dimensional random walk; for MCAK, the diffusion constant of this motion is 0.38 μm²/s, and the half-life of lattice binding is 0.8 s. The 1D diffusion creates a reduction in dimensionality that allows rapid targeting to microtubule ends.