HI-NESS distribution in wild-type Escherichia coli and E. coli Δhns (Confocal microscopy, single Z-plane). (A) HI-NESS (green) labels the nucleoid in wild-type E. coli where its distribution correlates—albeit poorly—with the DAPI signal (magenta). HI-NESS also distributes in the cytoplasm of these cells, decreasing the signal-to-noise ratio and the applicability of HI-NESS as a DNA label in wild-type E. coli. (B) In E. coli Δhns, HI-NESS localises in the nucleoid. Line scans across E. coli and E. coli Δhns cells are marked in red with start and end positions indicated with S and E, respectively. A white signal in the Overlay images represents colocalization.

HI-NESS labels chromosomes in HeLa cells in culture (Confocal microscopy, single Z-plane). Line scans (marked in red with start and end positions indicated with S and E, respectively) across nuclei of HeLa cells co-stained with HI-NESS (green) and SiR-Hoechst (magenta) highlight the overlap between the distributions of the two DNA labels. However, HI-NESS also accumulates in nucleoli due to high levels of the protein in the nucleus and its affinity for RNA as detected in in vitro studies (Supplementary Figure S4; Supplementary Table S1).

The overlap between H2A-mTurquoise2 (green) and SiR-Hoechst (magenta) signals in HeLa cells (Confocal microscopy, single Z-plane). Nuclei 1 and 2: Extensive nucleolar accumulation of H2A-mTurquoise2 (H2A-mTq2) drowns its fluorescent signal over the rest of the nucleus. Line scans (marked in red with start and end positions indicated with S and E, respectively) across such nuclei show that the H2A-mTq2 signal only recapitulates that of SiR-Hoechst when the line scan does not cross a nucleolus. Nuclei 3, 4, and 5: in cells expressing low levels of H2A-mTq2 (nuclei 3 and 4), and in nuclei with no visible nucleoli (nucleus 5) the distribution of the SiR-Hoechst and H2A-mTq2 signals are comparable.

The distribution of mEos3.2-tagged HI-NESS (green) and H2A-mTq2 (magenta) in the nuclei of HeLa cells in culture (Spinning disk microscopy, single Z-plane). In HeLa cells co-expressing mEos3.2-tagged HI-NESS and H2A-mTq2, the fluorescently-labelled histone exhibits extensive nucleolar retention, and consequently, stains the chromosomes with a low signal-to-noise ratio. The decreased nucleolar accumulation of HI-NESS allows chromosomes to be visualised with a higher signal-to-noise ratio. Line scans (marked in red with start and end positions indicated with S and E, respectively) across these nuclei highlight the effect of nucleolar retention on the signal over the rest of the nucleus.

The distribution of mScarlet-I-HI-NESS (magenta) and H2A-mTq2 (green) in the nuclei of HeLa cells expressing lower levels of the fluorescent proteins (Spinning disk microscopy, single Z-plane). The distribution of the mScarlet-I-HI-NESS signal (left) in the nuclei recapitulates that of earlier experiments (Figures ​(Figures22 and 4), with the label showing some nucleolar accumulation, and the presence of dense foci. The distribution of H2A-mTq2 (middle), on the other hand, differs (Figures ​(Figures33 and 4). H2A-mTq2 does not accumulate in the nucleoli of cells expressing lower levels of the protein. H2A-mTq2 also exhibits a visibly different distribution compared to that of mScarlet-I-HI-NESS, with a relatively homogeneous signal over the nucleus and the apparent lack of the dense foci observed with HI-NESS. The discrepancy may arise from the homogeneous binding of H2A-mTq2 along the chromosome, in contrast to the preferential binding of Hoechst and HI-NESS to AT-rich sequences (Supplementary Figure S4; Supplementary Table S1) (4–6).

HI-NESS does not induce increased rates of DNA damage. The induction of γH2AX—a DNA damage biomarker—in U2OS (FRT) cells stably expressing mEGFP-HI-NESS is comparable to cells expressing mEGFP-NLS as visualised by microscopy (Wide field microscopy, single Z-plane) (Panel A, left). On average, 7.4 ± 3.0 γH2AX foci were detected in U2OS cells expressing mEGFP-HI-NESS cells, compared to 9.3 ± 2.9 foci with mEGFP-NLS expression (Panel B, green). γH2AX is induced when the cells are treated with 3 Gy ionizing radiation (IR) (Panel A, right), 51.0 ± 6.4 γH2AX foci appear in U2OS (FRT) mEGFP-HI-NESS cells, and 57.9 ± 2.6 in U2OS (FRT) mEGFP-NLS cells (Panel B, magenta). Panel C: Western blot shows that γH2AX induction is unaffected by the expression of HI-NESS. Furthermore, γH2AX induction and hence, the DNA damage response, following IR treatment is not affected by HI-NESS.

HI-NESS does not interfere with cell cycle progression. Panel A: U2OS (FRT) cells in the S-phase were visualised (Wide field microscopy, single Z-plane) by pulse-labelling the cells with EdU and ‘clicking’ the molecule to 5/6-FAM. Panel B: 56.6 ± 2.9% of U2OS (FRT) cells expressing mEGFP-HI-NESS (U2OS-HI-NESS) occur in the S-phase of the cell cycle compared to 65 ± 7.2% of cells expressing mEGFP-NLS (U2OS-mEGFP). Flow cytometry of propidium iodide-labelled cells (Panels C–E) detects 37% of U2OS-HI-NESS cells in the G1 phase of the cell cycle, 33% in the S phase and 30% in the G2/M phase (Panels C and E). Of the U2OS-mEGFP cells, 38% occur in the G1 phase, 32% in the S phase and 30% in the G2/M phase (Panels D and E).

HI-NESS labels chromosomes in zebrafish larvae (confocal microscopy, single Z-plane). The distribution of the mEos3.2 signal (green) in nuclei of zebrafish larvae overlapped with that of DAPI (magenta). Nucleolar accumulation of HI-NESS was not observed. (A) Striated muscle cells (Trunk); (B) from left to right: trunk, blood vessel, yolk extension. The HI-NESS labelled cell is within a blood vessel. The approximate locations in the zebrafish embryo at which (A) and (B) were imaged is provided in (C).

HI-NESS can be used to visualise chromosome dynamics during the cell cycle (wide field microscopy, single Z-plane). A time-lapse of a dividing HeLa cell (Movie S8) shows that HI-NESS remains bound to the chromosome during mitosis. From left to right: Phase contrast image, Lck-mTurquoise2 (membrane label), mScarlet-I-HI-NESS (DNA label).