Complications of In vitro Stabilization of GTP-Containing Microtubules with Glutaraldehyde

Nyanza Rothman, Vassar College ’11 and Prof. Zachary Donhauser

Microtubules (MTs), a component of the cellular cytoskeleton, serve as tracks along which motor proteins transport cell organelles; furthermore, MTs contribute to the movement of chromosomes during mitosis. We used fluorescence microscopy and atomic force microscopy (AFM) to characterize guanosine triphosphate (GTP)-containing MTs polymerized in vitro from tubulin. GTP MTs, which are inherently unstable at room temperature, were stabilized with glutaraldehyde, an exogenous chemical cross-linking reagent that readily reacts with proteins. Previous studies have described structural deformation of MTs as a result of exposure to glutaraldehyde, and have also reported quenching of glutaraldehyde reactions by introduction of excess amines into solution. To control the glutaraldehyde reaction with MTs, we studied the quenching reaction, using a large excess of either the amino acid glycine or Tris buffer, aiming to remove any free glutaraldehyde from solution. Kinking and aggregation of MTs was observed immediately when high concentrations of glutaraldehyde were used (>0.1%) or where the glutaraldehyde stabilization proceeded for >15 minutes before the quench reagent was added. In time-based studies of MTs stabilized with glutaraldehyde under a variety of experimental conditions, kinking and aggregation of MTs developed in <8 hours, including those samples that we quenched. Our results suggest that glutaraldehyde is not an ideal cross-linker for MTs because the MTs do not remain stable for more than several hours and that quenching steps were ineffective in stopping the cross-linking reaction. Future studies will involve quantitative characterization of the change in stiffness of glutaraldehyde-stabilized MTs as a function of time via radial indentation experiments with the AFM and will explore other cross-linking reagents whose interactions with proteins are better understood or more easily characterized.