Figure 5

Actin polymerisation around isolated, differentiated, intact nuclei in vitro. (A) Isolated rat liver nuclei were incubated for 1 h at 37°C with 500 nM actin Alexa488 under polymerising conditions and then imaged by fluorescence microscopy. (a): Fluorescent actin-Alexa488 assembled around isolated whole intact nuclei (left panel). The nuclei were identified by DIC imaging (right panel). (b) Shows representative examples of nuclei that were pre-incubated with mAb414 (see methods) and subsequently subjected to the actin polymerisation assay. After this treatment, the nuclei did not polymerise actin at their cytoplasmic surface (fluorescent image (left panel) has been contrast enhanced in order to reveal background staining of nuclei as well as dim residual actin filaments) (left panel: actin Alexa488 image and DIC on the right panel). (B) The effect of various antibodies on the polymerisation of actin around isolated nuclei. Note how both mAb414, and antibodies against Nuance or the non-specific nuclear transport inhibitor wheatgerm-agglutinin WGA significantly reduced actin polymerisation. By contrast anti-Emerin had no significant effect compared with anti-c-Myc (three independent experiments 50 nuclei/condition).