In eukaryotes, chromosomes are wrapped in an endoplasmic reticulum (ER)-derived envelope to form the nucleus. Whether any DNA, or only chromosomes, can be enveloped in this way is unclear. Live-cell imaging revealed that DNA transfected into mammalian cells was either captured directly in the cytoplasm, or if it entered the nucleus was soon expelled from it. In the cytoplasm, plasmid DNA was rapidly surrounded by an ER-derived double membrane and frequently colocalized with extra-chromosomal DNA of telomeric origin expelled from the nucleus. Therefore, this structure was termed exclusome. Exclusome membranes contain the inner-nuclear membrane proteins Lap2β and Emerin but differ from the nuclear envelope by the absence of the Lamin B Receptor, nuclear pore complexes (NPCs) and by the presence of fenestrations. Strikingly, Emerin was strongly enriched at exclusomes and overexpression of its LAP2, Emerin, MAN1 (LEM)-domain reduced cells with exclusomes. Together, cells wrap chromosomes and two types of extra-chromosomal DNA into similar yet distinct envelopes. Thereby, they distinguish, sort, cluster, package, and keep chromosomal and extra-chromosomal DNA apart in the nucleus and the exclusome, respectively. We suggest that while all DNA molecules are enveloped through virtually identical mechanisms, only chromosomes somehow promote NPC assembly to form a nuclear envelope.

Expression from transfected plasmid DNA is generally transient, but it is unclear what process terminates it. We show that DNA entering mammalian cells is rapidly surrounded by a double membrane in the cytoplasm, in some cases after leaving the nucleus. This cytoplasmic container, termed exclusome, frequently also contains extra-chromosomal telomeric DNA, and is maintained by the cell over several division cycles. The exclusome envelope contains endoplasmic reticulum proteins and the inner nuclear membrane proteins Lap2β and Emerin, but differs from the nuclear envelope by its fenestrations and the absence of the Lamin B Receptor and nuclear pore complexes (NPCs). Reduction of exclusome frequency upon over-expressing Emerin's LEM-domain suggests a role for Emerin in plasmid DNA compartmentalization. Thus, cells distinguish extra-chromosomal DNA and chromosomes and wrap them into similar yet distinct envelopes keeping the former in the exclusome but the latter in the nucleus, where transcription occurs.