Regulatory factors governing PLT formation and size. Top left: rapid-freeze electron microscopy image and illustration of major cytoskeletal components in the barbell-proPLT end. Boxed regions highlight specific regulatory factors governing PLT formation and size. For rapid-freeze electron microscopy, cells were placed in a solution of 0.75% Triton X-100 in piperazine-N,N-bis-2-ethanesulfonic acid, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, ethyleneglycoltetraacetic acid, and MgCl2 (PHEM) containing 0.1% glutaraldehyde, 5μM phalloidin, and 30μM taxol, and attached to the surface of poly-L-lysine–coated coverslips by centrifugation at 280g for 5 minutes. The cytoskeleton was rinsed in PHEM solution and fixed for 15 minutes in 1% glutaraldehyde in PHEM. Coverslips were washed in distilled water, rapidly frozen in a liquid helium–cooled copper block, transferred to a liquid nitrogen–cooled stage, freeze-dried at −90°C, and metal cast with 1.2 nm of tantalum-tungsten with rotation at 45° and 3 nm of carbon at 90° without rotation. Replicas were floated, picked up on formvar-carbon–coated grid, and examined in a JEOL 1200-EX transmission electron microscope at 80 kV. Professional illustration by Alice Y. Chen.
