Fig. 5.

Nuclear actin and 4.1. (A) Acquisition of actin relative to 4.1 SABD epitopes during nuclear assembly in vitro. Nuclei (DNA, blue) assembled in Xenopus egg extract spiked with fluorescent actin (red) were fixed and probed with anti-4.1 SABD (green). Data presented, from one of three independent experiments, used the same extract as described for Fig. 4. Diffuse actin and SABD epitopes detected at 30 min in the merge were largely coincident (yellow). At later times, coincidence of actin with 4.1 foci was apparent along with some areas of noncoincidence. (B) Latrunculin A (Lat A) inhibition of nuclear assembly in vitro (Right). Nuclei assembled in Xenopus egg extracts containing fluorescent actin (red) and 0.1 mM latrunculin A had highly aberrant chromatin morphology (94%; DNA, blue), and actin was not detected by fluorescence microscopy. (Left) A control nucleus. An equal concentration of DMSO (latrunculin vehicle) had no effect on nuclear assembly. (C) By Western blot analysis of equivalent numbers of nuclei from reactions with 0.1 mM latrunculin A or dominant negative 4.1 SABD peptides, actin was not detected, whereas it was readily detected in controls (Left). Nuclei perturbed by 0.1 mM latrunculin A also had no detectable 4.1 epitopes when probed with anti-4.1 SABD or anti-4.1 C terminus (αCTD) (Right).