Asymmetric partitioning of transfected DNA during mammalian cell division

Significance

What happens to foreign DNA in mammalian cells during divisions? We provide evidence that the mitotic machinery helps cope with cytoplasmic clusters of plasmid DNA, a prototype of foreign DNA. The endoplasmic reticulum embraces these clusters, probably restricting their mobility and preventing them from interfering with chromosomal DNA. Subsequently, these clusters predominantly partition into the daughter cell with the young centrosome and with successive divisions end up in a decreasing fraction of cells within the growing population. This process is similar to the partition of noncentromeric DNA in budding yeast and cell fates in stem cells. The behavior difference of the two centrosomes documented here might also facilitate asymmetric partition of other cellular components.

Abstract

Foreign DNA molecules and chromosomal fragments are generally eliminated from proliferating cells, but we know little about how mammalian cells prevent their propagation. Here, we show that dividing human and canine cells partition transfected plasmid DNA asymmetrically, preferentially into the daughter cell harboring the young centrosome. Independently of how they entered the cell, most plasmids clustered in the cytoplasm. Unlike polystyrene beads of similar size, these clusters remained relatively immobile and physically associated to endoplasmic reticulum-derived membranes, as revealed by live cell and electron microscopy imaging. At entry of mitosis, most clusters localized near the centrosomes. As the two centrosomes split to assemble the bipolar spindle, predominantly the old centrosome migrated away, biasing the partition of the plasmid cluster toward the young centrosome. Down-regulation of the centrosomal proteins Ninein and adenomatous polyposis coli abolished this bias. Thus, we suggest that DNA clustering, cluster immobilization through association to the endoplasmic reticulum membrane, initial proximity between the cluster and centrosomes, and subsequent differential behavior of the two centrosomes together bias the partition of plasmid DNA during mitosis. This process leads to their progressive elimination from the proliferating population and might apply to any kind of foreign DNA molecule in mammalian cells. Furthermore, the functional difference of the centrosomes might also promote the asymmetric partitioning of other cellular components in other mammalian and possibly stem cells.

Generally, noncentromeric DNA molecules are mitotically instable in eukaryotes. This results in their apparent disappearance from an ever-increasing proportion of the progeny of an affected cell (e.g., 1–3). Endogenous sources of such DNA are recombination byproducts [double minutes, extrachromosomal ribosomal (r)DNA circles (ERCs) and other DNA circles (3–6)] or mitotic defects generating noncentromeric chromosomal fragments and cytoplasmic micronuclei (1, 7). Exogenous sources are DNA of pathogens or DNA, typically plasmids, artificially introduced into cells. For the latter, decades of work established that plasmid-born protein expression is transient, persisting only for a few cell cycles (8). This finding is consistent with plasmid DNA being somehow eliminated through divisions. Thus, some mechanisms seem to prevent the propagation of foreign DNA and extrachromosomal DNA in proliferating eukaryotic cells. However, how this is achieved is unclear.

In animal cells, DNA sensors mediate the early detection of exogenous DNA, such as DNA of invading pathogens and artificially introduced DNA (9–11). Both in leukocytes and nonprofessional immune cells, these can trigger innate immune responses, such as cytokine production, autophagy, and apoptosis (9). However, what happens to the DNA molecules themselves over time is unclear. When microinjected into the nucleus, plasmid DNA clusters and is expelled into the cytoplasm at mitosis (12). In the cytoplasm, the amount of DNA decreased within a few hours without completely disappearing, suggesting a rapid degradation of a major fraction and the persistence of a minor fraction of the molecules (13). Within a few hours after introducing DNA into the cytoplasm, tubular membranes and Emerin, a protein synthesized in the endoplasmic reticulum (ER) and subsequently predominantly present in the inner nuclear membrane, appear in the cytoplasm (10, 11). However, the functional relevance of these observations is not clear. Furthermore, the destiny of the DNA molecules, especially during subsequent mitoses, is elusive.

To better understand these phenomena and their functional relevance, we analyzed the fate of transfected plasmid DNA in dividing mammalian tissue culture cells.

Results

Transfected Plasmids Localize in a Few Clusters, Which Are Predominantly in the Cytoplasm.

To image the subcellular localization of plasmid DNA upon transfection into cells, we used two fluorescence-based approaches. In the first one, rhodamine was covalently coupled to a plasmid (Rho-plasmid) encoding Histone2B fused to enhanced (e)GFP (H2B-eGFP), whereas in the second we introduced a plasmid containing Lac operon repeats (pLacO) into cells expressing the Lac inhibitor (LacI) fused to a fluorescent protein [eGFP, monomeric (m)Cherry]. Because of its high affinity to the LacO sequence, the LacI fusion protein efficiently decorated the plasmid.

Twenty-four hours after transfection of the Rho-plasmid into HeLa cells using lipofection, we detected rhodamine fluorescence either in cells with a few bright foci only in the cytoplasm (61%, filled circle, Fig. 1B) or in cells with foci in the cytoplasm and foci in the nucleus (39%, open circle, Fig. 1B) (Fig. 1 A and B and SI Appendix, Fig. S1A). No cell with only nuclear foci was found. Foci intensities varied over four orders-of-magnitude (Fig. 1B), indicating that plasmids aggregated into clusters containing possibly up to tens of thousands of molecules. Moreover, the cytoplasmic foci were hundreds to thousands fold more intense than the nuclear ones (Fig.1B). Despite covalent labeling, the plasmids were successfully transcribed because eGFP-H2B was detected in 69% of the analyzed cells (SI Appendix, Fig. S1B). Interestingly, eGFP-H2B was detectable in all cells containing nuclear foci, but only in a fraction of cells containing no or only cytoplasmic foci (SI Appendix, Fig. S1C). We rationalized that the cells without nuclear foci might have lost such foci after transgene expression. Taken together, our data are consistent with the notion that nuclear localization is important for successful transcription.

Fig. 1.

The majority of transfected plasmid DNA localizes in the cytoplasm. (A and B) Cells 24 h after lipofection of rhodamine-labeled plasmid (Rho-plasmid). (A) Cell images with Rho-plasmid foci. LaminB1 immunostaining to visualize nuclei. (Left, one cytoplasmic focus; Right, two cytoplasmic and one nuclear (arrowhead) foci with z-scan along white dashed line y*; gray line, cell outlines; yellow Inset, enlarged nuclear focus) (B) Fluorescence intensities of cytoplasmic (red, blue) and nuclear (black) Rho-plasmid foci were measured in cells with only cytoplasmic (solid circle) and with cytoplasmic and nuclear foci (open circle). Percentage of cells in each pattern (n, pooled cell number of three experiments; 17–27 cells per experiment; median values indicated). (C and D) Cells stably expressing LacI-eGFP lipofected with pLacO. (C) Images after no plasmid, pControl (as pLacO but LacO repeats were replaced by ORF for mCherry) and pLacO transfection (arrowheads, pLacO clusters). (D) DNA dose dependency of the number of cytoplasmic plasmid clusters per cell (mean with SEM of three experiments, n > 50 per experiment). (A and C) Maximal intensity-projected image stacks. [Scale bars (unlabeled), 10 µm.]

To avoid possible effects of the covalent labeling and the uncertainty in distinguishing intracellular from extracellular plasmids, we turned to the LacO/LacI system. The LacI-eGFP and LacI-mCherry proteins contained a nuclear localization signal (NLS) to reduce the cytoplasmic background and a point mutation to avoid tetramerization (14). Twenty-four hours after pLacO transfection into HeLa stably expressing LacI-eGFP, bright LacI fluorescence was observed in one or a few cytoplasmic foci of various sizes (Fig. 1 C and D). Confirming that these foci contained pLacO, a control plasmid without LacO sequence did not induce such foci (Fig. 1C). Furthermore, the LacI-eGFP foci nearly perfectly colocalized with pLacO DNA, as detected by fluorescence in situ hybridization (SI Appendix, Fig. S1 D and E). Similar foci were also observed after pLacO electroporation (SI Appendix, Fig. S1 F and G), indicating that plasmid clustering was not caused by the lipofection method. Using both methods, the cells containing only one cluster remained predominant even after transfection of increasing DNA concentrations (Fig. 1D and SI Appendix, Fig. S1G). We rarely observed nuclear foci in these cells, possibly because they were drowned in the nuclear signals of the LacI fusion proteins.

We concluded that independently of transfection and detection methods the transfected plasmid is essentially deposited in one main cytoplasmic cluster that contains thousands of molecules.

During Mitosis, Plasmid Clusters Are Preserved and Partition Predominantly with the Young Centrosome.

Next, we imaged the cytoplasmic pLacO clusters in dividing cells. The clusters did not disassemble during mitosis and remained in the cytoplasm after mitosis (n >100 clusters) (Figs. 2A and 3D). Remarkably, four of eight initial cells with two or three pLacO clusters copartitioned all clusters to one of the two daughter cells in three consecutive mitoses, resulting in a majority of progeny cells being free of plasmids (Fig. 2A and Movie S1). Analysis of fixed telophase cells showed that in cells with two or three clusters asymmetric partition patterns were significantly higher than random [65% vs. 50% expected for 2:0 (n = 84 cells) and 41% vs. 25% expected for 3:0 (n = 29 cells)] (Fig. 2B).

Fig. 2.

Plasmid clusters are partitioned to the daughter cell harboring the young centrosome in a controlled manner. (A) Time-lapse images of the partition of three pLacO clusters (arrowheads) in three consecutive mitoses of a cell (dashed line, outline of mitotic cell). (B) Quantification of cells with x:0 partition patterns in fixed telophase cells with x clusters (x = 2 or 3 clusters) (dashed bar, theoretical random expectation). (C–F) Correlation analysis between the pLacO clusters and the centrosome type in dividing cells. (C) Representative image of a telophase cell, expressing Centrin1-eGFP and LacI-mCherry, with one pLacO cluster (yellow arrowhead) anti–ODF2-immunostained. (Insets) Enlarged centrosome areas. (D–F) Correlations between pLacO clusters and indicated centrosome types in late mitotic cells with x: 0 partition patterns (x = 1, 2, and 3 clusters or beads). (D and E) Cells without pretreatment. (E) Cells with both beads and pLacO clusters. (F) Cells pretreated with siRNA oligos before pLacO transfection. (A and C) Maximal intensity-projected image stacks; (B and D–F) n, pooled cell number of ≥3 experiments [cell number range per experiment: 14–38 (B, two clusters); 4–19 (B, three clusters); 30–59 (D, HeLa); 17–22 (D, MDCK); 16–18 (E), 26–75 (F, siControl, Left graph); 10–34 (F, siNinein), 13–37 (F, siControl, Right graph); 13–24 (F, siODF2); 10–22 (F, siAPC)]; statistics: (B) Binomial test, (D–F) Binomial test to compare experimental to random frequency, χ² test to compare pLacO with bead, siRNA with siControl, and siODF2 with siAPC. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. (Scale bars, 10 µm.)

Fig. 3.

Predominantly the old centrosome moves away from a stable plasmid cluster and the young centrosome during centrosome splitting. (A) Representative images of a cell with four beads (blue, b1–b4) and one pLacO cluster (red) at indicated times (gray lines, reference at t = 0 s. (B) Trajectories (0–400 s) of beads and the pLacO cluster of one cell. (C) Mean square displacements (MSD) of beads and pLacO clusters in 16 cells with both beads (one to four beads per cell) and pLacO clusters (one to two clusters per cell) (mean with SEM; dT, interval time). (D) Representative time-lapse images of a cell with one pLacO cluster during mitosis (white and gray arrowheads, old and young centrosomes, respectively; dashed circle, original location of centrosome pair at 0 min). (E) Difference of the distances of pLacO cluster to the old (OC) and young centrosome (YC), respectively, in individual cells (OC-YC). Results at the times: before split, maximal split, metaphase, and the second frame at anaphase. Percentage of cells with OC > YC indicated above. [3D distances; red, blue lines, clusters associated with young (red) and old centrosomes (blue) at anaphase] (F) Schematic behavior summary of cluster and centrosome dynamics in 60 cells containing one plasmid cluster. (G) Displacements of the centrosomes and pLacO clusters during splitting in cells with one plasmid cluster (mean with SD; **P < 0.01; ****P < 0.0001). (H) As in G, but for cells without plasmid clusters. (G and H) Displacements are 3D distances to the cluster’s or centrosomes’ start locations 5 min before split; (C) n, cluster or bead number from multipositions of one reproduced experiment; (E–H) n, pooled cluster (E) or cell (F–H); number of > 3 experiments, number range per experiment: 16–31 (E), 6–19 (F and G), 11–24 (H); statistics, Dunn's multiple comparisons test (G) or Wilcoxon matched-pairs signed rank test (H) was performed for each time point separately; only significant differences were indicated. (A and D) Maximal intensity-projected image stacks. (Scale bars, 10 µm.)

Every mitosis is asymmetric, at least for what concerns centrosomes. Indeed, the centrosomes at the poles of the bipolar spindle and inherited separately by the two resulting daughter cells have different ages (15). Although the relevance of this asymmetry is unclear, the inheritance of the old and young centrosomes correlates with specific cell fates in many stem cells (16–18). We investigated whether the partition of the plasmid clusters correlated with centrosome age. To this end, the centrosomes were labeled with stably expressed Centrin1-eGFP, whose abundance correlates with centriole age (19), or with an anti-outer dense fiber protein 2 (ODF2) antibody. ODF2 (Cenexin) is an old centrosome reporter (20). In late mitotic cells, both proteins decorated most strongly the old centrosome (Fig. 2C and SI Appendix, Fig. S2). Remarkably, in HeLa and Madin-Darby canine kidney (MDCK) cells the DNA clusters copartitioned with the young centrosome in 64.9% and 66.9% of the divisions, respectively (x: 0 partition patterns, x = 1, 2, or 3 clusters) (Fig. 2D). The same effect was observed with a linear fragment (SI Appendix, Fig. S3 A and B). These results were significantly different from random, showing that in distinct mammalian cells the partition of plasmid clusters was biased toward the young centrosome. To analyze whether this fate is somewhat specific for DNA or observed for any other particle of similar size (SI Appendix, Fig. S8D), we investigated how negatively charged polystyrene beads of 1 µm diameter partition at mitosis when introduced into HeLa cells. Unlike pLacO clusters, the beads did not preferentially copartition with either centrosome independently of whether they were transfected alone or together with pLacO (Fig. 2E and SI Appendix, Fig. S3 C–F). Thus, the biased partition of the DNA clusters was driven by neither their charge nor their size.

Ninein, a component of mature centrioles involved in microtubule anchoring, is thought to mediate the functional differences between old and young centrosomes (18, 21). Furthermore, like ODF2, adenomatous polyposis coli (APC) localizes to mature centrioles (20, 22). Thus, we tested whether down-regulation of any of these proteins (validated in SI Appendix, Figs. S5 A and B and S6) interfered with the biased partition of the plasmid in HeLa cells (Fig. 2F). Strikingly, upon Ninein and APC down-regulation, the results were no longer significantly different from random (HeLa siNinein: 44.9%; siAPC 46.2%) (Fig. 2F), suggesting that the partition bias was lost in both cases. Ninein down-regulation had a similar effect in canine cells (43.4% MDCK) (SI Appendix, Fig. S5C). As the fraction of cells with only cytoplasmic, nuclear and cytoplasmic, or only nuclear clusters was not changed between siControl and siNinein-treated cells, it is unlikely that a change in the localization of the clusters contributed to the loss of the partition bias (SI Appendix, Fig. S9H). Strikingly, neither Ninein overexpression nor ODF2 down-regulation affected the biased correlation of pLacO clusters with the young centrosome, indicating that the reported link between Ninein and ODF2 is irrelevant for Ninein’s effect on the partition of DNA clusters during mitosis (Fig. 2F and SI Appendix, Fig. S5 D and E) (23). We conclude that the biased partition of plasmid clusters toward the young centrosome is the result of a process controlled at the centrosome level and might be conserved across mammalian cells.

Plasmid Immobility, Initial Vicinity to Centrosomes, and High Mobility of the Old Centrosome Bias Plasmid Partition.

Next we wondered how cells biased the inheritance of the plasmid clusters toward the young centrosome. We first noticed that despite their similar size the pLacO clusters exhibited a very limited motion in interphase cells compared with the cotransfected beads, indicative that they interacted differently with cellular structures (Fig. 3 A–C, SI Appendix, Figs. S4 and S8D, and Movie S2). Cluster motion was also very limited during mitosis in comparison with chromosome and centrosome dynamics (Fig. 3D, SI Appendix, Figs. S7A and S8B, and Movie S3). Thus, their biased partition was not driven by an active movement of the cluster toward the young centrosome, but rather by a biased movement of the centrosomes at some point during mitosis. Because a substantial fraction of HeLa cells elongate their spindle asymmetrically (24), we next asked whether this drove the segregation of the young centrosome with the clusters. Quantitative analysis indicated that all spindles elongated asymmetrically but to different extents (SI Appendix, Fig. S7 B and C). The plasmid clusters remained in the nonelongating half of the cells, provided that they were at a relative center position in metaphase (n = 10 cells, SI Appendix, Fig. S7A). However, the elongating half of the cell contained the old centrosome nearly as frequently as the young centrosome, irrespectively of whether the cells had pLacO clusters or not (SI Appendix, Fig. S7C). Therefore, anaphase elongation is not biased to a centrosome type and thus is not biasing plasmid partition toward any centrosome.

Consequently, we analyzed the impact of earlier mitotic events by live-cell imaging (Fig. 3 D–H). The two centrosomes start to separate shortly before the nuclear envelope breaks down. As previously reported (25), the two centrosomes reached their maximal distance to build the spindle either right before (30%) or shortly after (70%) nuclear envelope breakdown (prophase and prometaphase pathways) (n = 75). In movies of both centrosome-splitting types, we measured the 3D distances of individual pLacO clusters (C) to the old (O) and the young (Y) centrosome (OC and YC distances, respectively). Remarkably, 5 min after they split the old centrosome was on average further away from the plasmid cluster than the young centrosome (SI Appendix, Fig. S8A). To characterize this behavior in individual cells, we calculated the differences between OC and YC (OC-YC) at four stages of mitosis: the last movie-frame before the centrosomes split (before split), when the centrosomes were maximally distant from each other before metaphase (maximal split), during metaphase, and at the second frame of anaphase (Fig. 3E). The distance of the cluster to the old centrosome was larger than to the young centrosome in 69% of the analyzed cells at the maximal split stage; the fraction increased slightly in metaphase (74%) and came back to 69% in anaphase, consistently with the analysis of fixed cells (Fig. 2D). Remarkably, 77% of the clusters that were closer to the young (red lines) or old centrosome (blue lines) in anaphase were also closer to the same centrosome at the maximal split stage (Fig. 3E). Therefore, the spatial association of the cluster with the young centrosome emerged as soon as the two centrosomes split and was generally maintained afterward.

Strikingly, we noticed that before centrosome splitting (−10, 0 min) (Fig. 3D), the majority of plasmid clusters were in the vicinity of the centrosome pair: In 73% of the cases (44 of 60 cells), the distance from the cluster to the centrosomes was shorter than the short diameter of the nucleus (average 17.5 μm), the biggest obstacle in the cell (Fig. 3F and SI Appendix, Fig. S8C). Furthermore, we noticed that in the majority of the analyzed movies the old centrosome moved further than the young one during splitting, leaving the young centrosome closer to the pLacO cluster [Fig. 3D (0–10 min), Fig. 3 E–G, and Movie S3]. Indeed, the displacement of the old centrosome between the time points “before split” and “maximal split” was in 70% of the cases bigger than that of the young centrosome [displacement ratio (old/young centrosome) > 1] (SI Appendix, Fig. S8E, plus cluster). Among the 44 cells in which the cluster was within 17.5 μm of the centrosomes before split, 34 old centrosomes migrated further than their younger counterpart, resulting in 82% of these anaphases in a copartition of the young centrosome with the cluster (28 of 34) (Fig. 3F). In the six remaining cells, partition of the cluster with the old centrosome seemed to be caused by the cells being very small (two cells), by spindle rotation (three cells), and by the cluster moving (one cell). In the 10 cells where the cluster was close to the centrosome pair at the time of splitting and the young centrosome moved further than the old one, the clusters did not preferentially copartition with either centrosome (Fig. 3F). Detailed measurements of displacement and speed during splitting confirmed the relative immobility of the plasmid cluster, and, on average, the bigger displacement of the old centrosome compared with the young one (Fig. 3G and SI Appendix, Fig. S8 B and E). The differential displacement of the two centrosomes in control cells without pLacO clusters was not significantly different compared with cells with clusters (Fig. 3H and SI Appendix, Fig. S8E). Finally, the LacI-GFP intensities and approximated cluster sizes were independent of whether they were close or far from the centrosome pair (SI Appendix, Fig. S8D). Thus, our data indicate that the two centrosomes behave intrinsically differently during spindle formation and that the plasmid cluster does not induce this difference.

We performed this analysis also upon Ninein knockdown to determine why this affected the partition bias (SI Appendix, Fig. S9). The distance between cluster and centrosomes before centrosome split and the centrosome displacements early in mitosis revealed no major differences between siControl and siNinein-treated cells (SI Appendix, Fig. S9 B, C, and E). However, upon Ninein down-regulation, the mitotic spindles containing aligned centrosomes moved more frequently compared with siControl cells (40% SiNinein, n = 15 cells; 5% siControl, n = 19 cells), randomizing the position of the centrosomes relative to the cluster (SI Appendix, Fig. S9F). Thus, Ninein down-regulation did not abolish the establishment but the maintenance of the spatial association between the cluster and the young centrosome. In these cells, the microtubules emanating from the centrosomes might no longer establish a stable connection to the cell cortex, as already proposed (26).

In summary, the three requirements driving the partition of plasmid clusters toward the young centrosome are (i) that the plasmid cluster must be relatively immobile and (ii) in proximity of the centrosomes before splitting. (iii) During spindle formation, the old centrosome migrates away from the young centrosome and plasmid cluster, leaving the latter two together. The maintenance of this association toward the end of mitosis requires the orientation of the mitotic spindle to remain stable.

Mitotic Plasmid Cluster Appears to Be Physically Linked to the ER.

One of the intriguing prerequisites for the biased inheritance of the plasmid cluster is its immobility. Because beads of similar electrostatic nature and size do not show such immobility (Fig. 3 A–C and SI Appendix, Fig. S4), we reasoned that DNA clusters might be anchored to some structure. Supporting this idea, most of the plasmid clusters were very close to the cell cortex during mitosis (Fig. 3D). The intermediate filament protein Vimentin can cage damaged proteins and partitions asymmetrically in mammalian cells (27, 28). It is also well established that the ER is close to the cortex, away from the mitotic spindle (29). We therefore probed if the DNA cluster contacted cytoskeletal elements or membrane structures during mitosis.

None of YFP-αTubulin, Vimentin, and actin showed any evident association with the plasmid clusters in metaphase cells (SI Appendix, Fig. S10A). However, the ER membrane reporter Sec61-mCherry always surrounded the plasmid clusters and was sometimes (10%, n = 21 mitotic cells) even enriched at that position (Fig. 4A). A reporter of ER lumen, eGFP–Lys-Asp-Glu-Leu (KDEL), and the nuclear membrane proteins Emerin and Lap2β, which localize in the ER during mitosis, also closely surrounded the mitotic plasmid clusters (SI Appendix, Fig. S10B). The tight association of the DNA cluster, but not of the beads, with the ER was also observed in all analyzed interphase cells (Fig. 4A).

Fig. 4.

Plasmid clusters connect to ER in mitotic cells. (A) PLacO clusters in mitotic (Left) and interphase (Right, panel 1) cells expressing LacI-eGFP and Sec61-mCherry and beads in interphase cells expressing Sec61-eGFP (Right, panel 2). (Insets, areas of clusters and beads; Left, panel 1: arrowhead, pLacO cluster; panels 1 and 2: quantification of 21 metaphase cells (pool of three experiments); Right, panel 1: representative of 70 cells (pool of three experiments); Right, panel 2: representative of 27 cells (pool of two experiments, reproduced also with other membrane reporters), (B) Correlative fluorescence with electron microscopy (EM) of a metaphase cell containing one pLacO cluster. The EM image corresponds to the confocal image at a perpendicular view (xz). Boxed areas show enlarged plasmid cluster (blue) and chromosomes (gray) (yellow arrowhead, tubular membrane; red arrowhead, connecting junction between the pLacO cluster and membrane; z1, z2, and z3, the blue-boxed area at three different z levels). (Single-z-focus images) [Scale bars (unlabeled), 10 μm.]

To visualize the relationships of the pLacO cluster and membranes at high resolution, we performed correlative light with focused ion beam scanning electron microscopy. Tubular membranes, reminiscent of ER tubules, were in close proximity to the plasmid location (Fig. 4B and SI Appendix, Fig. S10C). Most remarkably, in several distinct regions, electron-dense bridges were found between the plasmid cluster and the surrounding tubular structures (Fig. 4B, red arrowheads, and SI Appendix, Fig. S10C), suggesting physical connections. No such structures were observed around individual metaphase chromosomes (Fig. 4B, gray box). Furthermore, the plasmid clusters were of equal or higher electron density compared with the chromosomal DNA, indicating a tight packing or high DNA concentration (Fig. 4B and SI Appendix, Fig. S10C). Together, our data suggest that plasmid clusters are physically linked to ER membranes. This confinement separates the plasmid from the mitotic spindle area and probably restricts the mobility of the plasmid.

Discussion

Here, we show that 24 or more hours after transfection, the majority of transfected plasmid is mostly in one cluster in the cytoplasm, and only a very small fraction is present in the nucleus. Moreover, we show that during mitosis, plasmid clusters are outside of the spindle region and appear to be physically associated to the ER, even in mitosis. Sting (stimulator of IFN genes), an ER membrane protein involved in the transmission of signals from DNA sensors to the native immune system (9), might be implicated therein. Plasmid DNA microinjected into the nucleus is expelled into the cytoplasm after mitosis (12). These results support the notion that plasmid DNA, as a model for foreign DNA, is actively separated from chromosomal DNA in interphase and during the open mitosis of mammalian cells.

We also show that the plasmid clusters preferentially partition to the daughter cell with the young centrosome. This asymmetric partition is the result of a combination of events. First, clustering of the DNA molecules promotes the fidelity of partition by reducing the number of entities to be partitioned. However, although clustering is observed in yeast cells as well (3), its mechanisms are currently unknown. Second, a close proximity of the plasmid cluster with the centrosomes at the start of mitosis is required for biasing plasmid partition. Future work will determine how this proximity is established in the first place. Third, the two centrosomes move differentially during early mitosis. During centrosome splitting, which prepares the formation of the bipolar spindle, the old centrosome moves further away from its original position compared with the young centrosome. Importantly, this differential behavior already exists in HeLa cells without plasmid DNA. This might be due to the old centriole nucleating more astral microtubules (19) and the amount of a specific subgroup of astral microtubules required for asymmetric stem cell divisions (18, 30). Last but most importantly, we found the plasmid cluster to be relatively immobile during mitosis, which is a prerequisite for differential centrosome movement to bias the partition of the DNA clusters together with the young centrosome. We suggest that physical links between the plasmid cluster and the ER membrane anchor the plasmid DNA and thus restrict its mobility.

In budding yeast, noncentromeric plasmid DNA and endogenous circular DNA popping out from the rDNA locus act as aging factors (31). They are inherited by the yeast mother cell, reducing her division potential with each subsequent division, compared with newborn daughter cells (3, 32). The yeast mother cell also inherits the young spindle pole, the functional homolog of the young centrosome in mammalian cells (33). Therefore, we speculate that the asymmetric partition of the plasmid cluster promotes the maintenance of a high division potential in the majority of the progeny, namely, in those cells that are thereby cleared of foreign DNA. In reverse, cells containing foreign DNA might be more prone to aging. In support of that notion, we found that the cell cycle duration of the daughter cell with plasmid cluster is on average 0.7 h longer than that of its sibling (SI Appendix, Fig. S11), similar to the effect of protein aggregates on the proliferation of neural stem cells (28). Germ and renewing stem cells often retain the old centrosome upon division. Therefore, our data predict that these cells are cleared of any entering foreign DNA. Future studies will determine whether this is the case and how relevant this is for the biology of these cells.

To conclude, we identified a mechanism facilitating the biased partition of plasmid DNA to the daughter cell inheriting the young centrosome during mitosis. By this, the fraction of cells harboring DNA clusters in a population is expected to decrease with continued divisions, resulting in their apparent elimination. Further, we suggest that this mode of asymmetric partition of plasmid DNA acts in many mammalian cell types and protects the chromosomes from foreign DNA. We postulate that this is especially important for cell lineages with high division potential, such as germ and stem cells. Last but not least, we expect that the differential behavior of the two centrosomes identified will prove relevant for the asymmetric partition of other cellular components, possibly in many mammalian cell types.

Materials and Methods

Detailed materials and methods are provided in SI Appendix. Presented experiments are approved by the Swiss Federal Office of Public Health.

LacO Plasmid.

PLacO (13.5 kb) with 256 x LacO spacer (aattgtgagcggataacaattt-gtggccacatgtgg) is a gift from Susan M. Gasser, Friedrich Miescher Institute, Basel, Switzerland (34).

Centrosome Classification.

The method to distinguish old and young centrosomes is described in SI Appendix, Fig. S2A. To validate the classification of the centrosome age in HeLa cells stably expressing Centrin1-eGFP, the fluorescence intensity of Centrin1-eGFP was compared with that of immunostained ODF2 at centrosomes (SI Appendix, Fig. S2 B and C).