Isolation of circumferential microtubules from platelets without simultaneous fixation

Abstract

Circumferential bands of microtubules (MT) support the discoid shape of resting platelets and participate with the contractile apparatus in shape change and internal contraction following activation. Elucidation of interactions between the circumferential coils and proteins of the stable and contractile cytoskeleton is essential for understanding MT function in platelet physiology. A previous investigation demonstrated that the circumferential rings can be isolated intact from resting platelets following simultaneous exposure to glutaraldehyde and Triton X-100. However, the use of fixation prevented the characterization of protein interactions. The present study has circumvented this problem by developing a procedure for isolating intact microtubule coils from detergent-treated platelets without the use of fixative agents. Incubation of the platelets for intervals of 30 to 60 minutes with the microtubule-stabilizing agent taxol preserved the circumferential bundle after extraction with Triton X-100 even after washing five times. The procedure has made it possible to carry out protein studies on isolated microtubule rings and associated proteins.