A special envelope separates extra-chromosomal from mammalian chromosomal DNA in the cytoplasm

Abstract

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.

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 [9,36,37]. However, with the current DNA visualization methods, the detection of weaker signals, e.g.…”

“…The placO/lacI-FP system has often been applied to track the fate of various DNA sequences inside the cell. This includes the visualization of transfected DNA [9,37,142], but also lacO sequences artificially inserted into the genome, e.g. for the examination of chromatin dynamics [119,120], the assessment of genetic loci [134], characterization of bacterial chromosome arrangements [143], and for studying transcription dynamics in mammalian cells [144].…”

“…Between the steps, the sample is washed extensively to remove the applied chemicals and the unbound probes. Protocols for sample preparation are found in several publications [37,[159][160][161].…”

“…Therefore, the presence of placO was demonstrated both in living cells by lacI-FP staining as well as with the FISH method. In another study performed by this group, FISH probes were employed to show that cytoplasmic DNA clusters formed after DNA delivery also contain telomeric DNA [37]. However, in some cases the harsh manipulation for FISH staining prevents the combination with co-stainings.…”

“…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 [37], and/or entrapped in nuclear invaginations [111,112]. 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 [113,114].…”