Figure 2.

A centrosome remains attached to chromatin despite the absence of actin, microtubules, the lamina and the nuclear envelope. A–C. α-Tubulin (centriole and microtubule component), lamin B (nuclear membrane marker), centrin (centrosome marker), histone (chromatin marker), and γ-tubulin (PCM and γ-tubulin nuclear boundary marker) were immunofluorescence stained in demembranated Xenopus laevis sperm that were isolated in the presence of cytochalasin B (depolymerized actin; A and B) or both cytochalasin B and colcemid (depolymerized actin and microtubules; C) and were subsequently pelleted on a cushion containing glycerol to remove debris, and then immunostained as previously described [20]. Chromatin was stained with DAPI. (A) To study the nucleation of microtubules on demembranated sperm, egg extracts were incubated with such sperm before fixation [20]. Immnunostaining with an anti-α-tubulin antibody showed that addition of egg extracts (A) to demembranated sperm (B) triggered the assembly of microtubules on chromatin (A and B). (C) Isolation of demenbranated X. laevis sperm in the presence of colcemid and subsequent fixation and immunostaining with the indicated antibodies showed that colcemid treatment removed centrioles (negative α-tubulin staining) from the PCM, but the PCM still remained attached to the chromatin. (A–C) Arrows and arrowheads indicate the location of PCM and the γ-tubulin nuclear boundary, respectively. Scale bars: 10 μm. This data is from unpublished works by Alvarado-Kristensson et al.