APC depletion changes chromatin appearance in <i>Xenopus</i> egg extract.

<p>Demembranated sperm chromatin was incubated with CSF extract in the presence of a small amount of Rhodamine-labelled tubulin for 1 h, overlayed with DAPI-containing fixative and imaged. <b><i>A</i></b><i>.</i> Two representative examples of “strong” spindles formed in such extract, with a dense microtubule network and well-resolved, mitotically condensed chromatin. <b><i>B</i></b><i>.</i> Two representative examples of “weak” spindles formed in CSF extract with low microtubule content and abnormally compacted chromatin. <b><i>C</i></b><i>.</i> Typical morphology of chromatin classified here as normal (left panel), rod-like (middle panel) and compacted (right panel) used for the quantification shown in D-F. <b><i>D</i></b><i>.</i> Proportion of phenotypically “weak” spindles formed around chromatin with normal, rod-like and compacted morphology was determined in three different preparations from the same extract. Between 62 and 85 spindles per slide were counted. <b><i>E</i></b><i>.</i> Quantification of chromatin morphology by categories depicted in C, after incubation in untreated (control), mock- or APC-depleted extracts for 1 h. 80–165 chromatin figures were scored for each condition. <b><i>F</i></b><i>.</i> Quantification of different chromatin morphologies according to categories depicted in C, after incubation in mock- or APC-depleted extracts for 1 h, followed by exposure to +12°C. 70–90 chromatin figures were scored for each condition. <b><i>G</i></b><i>.</i> Level of APC depletion in CSF extract visualized by immunoblotting mock- and APC-depleted extracts with anti-APC antibody. Tubulin was used as a loading control. <b><i>H</i></b><i>.</i> Representative images of demembranated sperm chromatin after incubation in ULSS extract at 23.5°C for 50 min, classified as normal, rod-like and compacted for quantification in I. Sum intensity projections of deconvolved z-stack images capturing the total chromatin fluorescence are shown. Size bar 15 µm. <b><i>I</i></b><i>.</i> Percent of normal, rod-like and compacted chromatin (as depicted in H) detected after incubation in mock- or APC-depleted ULSS extract after 40 (ULSS 1) or 50 (ULSS 2) min. Data from two independent experiments are shown. 74–133 chromatin figures were scored for each condition.