Investigations of the pathway of incorporation and function of lamin A in the nuclear lamina.

Cell-free extracts of Xenopus eggs were used to investigate how differing lamin sub-types are incorporated into a lamina and how lamina composition influences DNA replication. Purified recombinant human lamin A (HlaminA) was inoculated into egg extracts, which support in vitro nuclear assembly. The route of incorporation of Hlamin A into the lamina was compared to Xenopus lamin B3 (XlaminB3), the major endogenous lamin sub-type in egg extracts. While Xlamin B3 was incorporated into the lamina directly, HlaminA first accumulated at nucleoplasmic foci, before entering the lamina. When HlaminA was inoculated into extracts depleted of XlaminB3 it entered nuclei efficiently and was incorporated into nucleoplasmic foci. However, in the absence of XlaminB3, HlaminA remained in the foci and did not enter the nuclear envelope. When HlaminA entered the nuclear envelope, it did not influence DNA replication. Nuclei containing HlaminA initiated DNA replication on queue and in addition the spatial distribution of replication centres in these nuclei was identical to controls. Taken together, these results suggest that the incorporation of A-type lamins into the nuclear envelope is dependent upon the presence of B-type lamins. However, the presence of A-type lamins alone is not sufficient to influence the spatial and temporal order of DNA replication.