Immunodepletion of Prp19 directly in mitosis compromises spindle assembly.

Spindle assembly was monitored using sperm nuclei in egg extracts after a complete cell cycle. Immunodepletion was performed only after rearrest in mitosis. (A): schematic overview of experimental setup. (B): Immunoblot to show depletion and rescue using a Prp19 specific antibody. (C): Quantification of normal (grey) and weak spindles (red). (D): Representative images of normal and weak spindles in control, depleted (dePrp19) and reconstituted extracts (rescue). (E): 30 spindles were analyzed under the conditions indicated, aligned and averaged (z-projection, average, ImageJ). upper panels (microtubules): average microtubule intensity projections. Graphs show average intensity distributions along the pole-to-pole axes indicated in projections in blue (middle panels). Lower panels: chromatin distributions as determined from the DAPI signal. Scale bars: 20 µm. (F): Model to explain the defects in spindle formation after Prp19 knock-down or depletion. Left: control situation: Prp19 modifies an unknown Spindle Assembly Factor (SAF), which directly contributes to spindle formation by modifying properties of microtubules as a microtubule associated protein, or working as a kinetochore-associated protein in stabilizing kinetochore to microtubule attachments. Right: after knock-down or immunodepletion of the Prp19 complex, the regulated SAF looses its function in spindle formation.