Nucleation of microtubules from salt-stripped centrosomes is restored by cytoplasmic extract of myotubes.

<p>(A) Isolated centrosomes from Jurkat cells, treated with 1M potassium iodide (KI) to strip the pericentriolar material. An immunoblot is shown, probed for gamma-tubulin in unstripped centrosomes (centr.), as well as in centrosomes after KI-treatment for 15, 30, 45, and 60 minutes. Treated centrosomes were fractionated by centrifugation, to separate the extracted pericentriolar material (supernatants, S), and the insoluble centrioles (pellets, P). (B) Purified centrosomes, centrifuged onto glass coverslips and stained for immunofluorescence of pericentrin (B1) and gamma-tubulin (B2). Centrosomes were incubated with purified tubulin at 5 mg/ml for 10 minutes, either directly (B3), or following incubation with cytoplasmic extract from undifferentiated myoblasts (B4) or from myotubes (B5). (C) Equivalent experiments as in (B), using centrosomes that had been stripped with KI for one hour. (D) The number of microtubules/aster in a focal plane, as well as the average microtubule length were quantified in each case (n≥34). Bar in (C), 10 µm.