A dedicated cytoplasmic container collects extrachromosomal DNA away from the mammalian nucleus

Abstract

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.

The intracellular visualization of exogenous DNA in fluorescence microscopy

“…The role of these BAF/DNA clusters in the transfection process is not well understood, and they might support but concurrently also impede the nuclear uptake of exogenous DNA. Importantly, the BAF/DNA clusters form dominant structures inside the cell, which seem to contain most or all the transfected DNA [ 45 – 47 ]. However, with the current DNA visualization methods, the detection of weaker signals, e.g.…”

“…Even though many studies assert to detect the signal of labeled DNA inside the nucleus with a simple co-staining of the nuclear chromatin, we believe that the correct identification of exogenous DNA inside the nucleus requires additional evidence. The labeled DNA clusters might also be enclosed in NE-like membranes in close vicinity to the nucleus [ 47 ], and/or entrapped in nuclear invaginations [ 121 , 122 ]. These membranous invaginations of the NE are channels with a diameter of 100–1000 nm that span the nucleus, and are common in cancer cells [ 123 , 124 ].…”

“…deriving from single DNA molecules. The method is thus mainly applied to visualize large cytoplasmic DNA aggregates [ 19 , 47 , 119 , 133 ].…”

“…This includes the visualization of transfected DNA (Fig. 6 ) [ 19 , 47 , 152 ], but also lacO sequences artificially inserted into the genome, e.g. for the examination of chromatin dynamics [ 129 , 130 ], the assessment of genetic loci [ 145 ], characterization of bacterial chromosome arrangements [ 153 ], and for studying transcription dynamics in mammalian cells [ 154 ].…”

“…More recently, the lacO/lacI-FP-based tracking system was used in several studies to analyze the cellular fate of exogenous DNA after non-viral gene delivery [ 19 , 47 , 119 ]. Bright lacI-FP clusters were consistently observed in the cytoplasmic region of the cell, independent from the used transfection system.…”