MORC3 Forms Nuclear Condensates through Phase Separation

Abstract

Phase separation can produce local structures with specific functionality in the cell, and in the nucleus, this can lead to chromatin reorganization. Microrchidia 3 (MORC3) is a human ATPase that has been implicated in autoimmune disorders and cancer. Here, we show that MORC3 forms phase-separated condensates with liquid-like properties in the cell nucleus. Fluorescence live-cell imaging reveals that the MORC3 condensates are heterogeneous and undergo dynamic morphological changes during the cell cycle. The ATPase activity of MORC3 drives its phase separation in vitro and requires DNA binding and releasing the MORC3 CW domain-dependent autoinhibition through association with histone H3. Our findings suggest a mechanism by which the ATPase function of MORC3 mediates MORC3 nuclear compartmentalization.

Keywords: Biological Sciences; Biophysical Chemistry; Molecular Biology.

Graphical abstract

Figure 1

MORC3 Phase Separates to Condensates in Cells (A–C) Representative fluorescence microscopy images of live HeLa cells expressing H2B-GFP (green) and MORC3 labeled with mCherry (red). Scale bar, 5 μm. (D) Representative FRAP images of mCherry-MORC3 expressed in HeLa cells. The images were taken before (left) and after (middle and right) photobleaching. The condensate that was bleached is indicated by a white arrow. Scale bar, 2 μm. (E) FRAP curve of mCherry-MORC3 NBs in HeLa cells. The FRAP curve was obtained from averaging data from 10 cells. Error bars represent SEM. (F) Representative images of MORC3-NBs before and after treatment with 10% 1,6-hexanediol. Scale bar, 2 μm. See also Figure S1 and Video S1.

Figure 2

The Number of MORC3-NBs Changes during Cell Division (A) A representative cell division with chromatin labeled with H2B-GFP (green) and MORC3 labeled with mCherry (red) visualized by confocal time-lapse microscopy in live cells. Foci numbers decrease in metaphase but recover to initial levels after cell division is complete. Scale bar, 6 μm. (B) A plot of the changes of H2B volume (green) and MORC3 foci number (red) during cell division. (C) Statistics and cartoon plots of the dynamic morphological change of MORC3-NBs after aligning cells to the entry point of mitosis. (D) A representative cell division with chromatin labeled with H2B-GFP (green) and MORC3 labeled with mCherry (red) visualized by confocal time-lapse microscopy in live cells. Scale bar, 6 μm. See also Figure S1 and Video S2.

Figure 3

MORC3-ATPase Phase Separation Requires DNA (A) Phase separation of 13.3 μM MORC3 ATPase with (right) or without (left) 6.7 μM 37-bp double-stranded (ds) DNA. (B) A representative image of a sample containing 20 μM MORC3 ATPase and 10 μM 37-bp dsDNA on the surface of cover slides. Scale bar, 20 μm. (C) Quantification of droplets counted in images acquired for indicated MORC3-ATPase concentrations. (D) Quantification of MORC3-ATPase droplets in samples containing 13.3 μM MORC3 ATPase protein and 6.7 μM 15-bp dsDNA (black), 6.7 μM 37-bp dsDNA (orange), and 1.4 μM 601 DNA (red) without or with 2 mM ATP. (E) Quantification of MORC3-ATPase droplets in samples containing 6.7 μM ATPase protein and 3.3 μM 37-bp dsDNA in the absence and presence of 2 mM AMPPNP and ADP. (F) Representative images of 13.3 μM MORC3 ATPase with indicated amount of 37-bp dsDNA on the surface of cover slides. Scale bar, 10 μm. (G) Quantification of (F). (H) Representative confocal images of phase-separated MORC3-ATPase (13.3 μM) condensates (left and middle) or buffer with FAM-labeled DNA (6.7 μM) (right). Scale bar, 20 μm (left) and 10 μm (middle and right). Number of droplets were counted in a 50 × 50-μm square region (C, D, E, and G). For each sample, six square regions were counted. Error bars represent SEM. See also Figures S2 and S3.

Figure 4

Phase Separation of MORC3 Is Impeded by the CW Domain (A and B) Representative images of MORC3 ATPase-CW (20 μM) with or without 37-bp double-stranded DNA (5 μM) and histone H3K4me0 (1–12) and H3K4me3 (1–12) peptides (200 μM) on the surface of cover slides. Scale bar, 10 μm. As the MORC3 ATPase-CW condensates settled on the glass slide, droplets with irregular shapes gradually became spherical while small droplets fused together, as indicated by arrows in (B). (C) Quantification of (A). Number of droplets was counted in a 50 × 50-μm square region. For each sample, six square regions were counted. Error bars represent SEM. (D) Representative confocal images of phase-separated MORC3 ATPase-CW (20 μM) condensates in the presence of 6.7 μM FAM-labeled DNA and 200 μM H3K4me3 peptide (left). No condensates are detected without H3K4me3 peptide (right). Scale bar, 10 μm. See also Figure S2. (E) Representative images of MORC3 ATPase-CW (20 μM) incubated with reconstituted unmodified nucleosome (10 μM) on the surface of cover slides. Scale bar, 10 μm.