The F-Actin-Binding MPRIP Forms Phase-Separated Condensates and Associates with PI(4,5)P2 and Active RNA Polymerase II in the Cell Nucleus

Figures

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  Figure 1. Myosin phosphatase rho-interacting protein (MPRIP) localizes to the cytoplasm and cell nucleus: (A) confocal microscopy of an immunofluorescence (IF) experiment shows the localization of endogenous MPRIP in U2OS nucleus and cytoplasmic stress fibers. Upper and lower images represent different focal planes: nuclear and cytoplasmic, respectively. Arrowheads point to the MPRIP staining on stress fibers. Scale bars represent 10 µm; (B) Western blot (WB) of HeLa cells fractionated to nuclear and cytoplasmic fractions with detected MPRIP, Lamin B and GAPDH by a specific antibody (n = 3).

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  Figure 2. MPRIP specifically interacts with Phosphatidylinositol 4,5-bisphosphate (PIP2). Various PIP-coated agarose beads were used as a bait to pull-down interacting proteins from HeLa nuclear fraction. The membrane was WB analyzed with anti-MPRIP antibody. I—input 2%; C—control beads; 3—PI(3)P, 4—PI(4)P; 5—PI(5)P; 3,4—PI(3,4)P2; 3,5—PI(3,5)P2; 4,5—PI(4,5)P2; 3,4,5—PI(3,4,5)P3. n = 3.

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  Figure 3. Super-resolution microscopy revealed that Myosin phosphatase rho-interacting protein (MPRIP) localizes in close proximity to Phosphatidylinositol 4,5-bisphosphate (PIP2) at nuclear speckles and nuclear lipid islets (NLIs): (A) immunofluorescence labelling experiment showing the presence of endogenous MPRIP and PIP2 in U2OS nucleus. Scale bar represents 5 µm; (B–D) zoomed view, intensity profiles and statistical analysis of the NLIs. Scale bar corresponds to 1 µm. (E–G) Zoomed view, intensity profiles and statistical analysis of the speckle-associated PIP2 pool. Scale bar corresponds to 1 µm. Intensity profiles plotted correspond to the lines on the zoomed view. (D,G) The bar graph shows the statistical Mander’s and Spearman coefficients of the MPRIP and PIP2 signal. Mander’s A analysis: PIP2 over MPRIP channel, and Mander’s B analysis: MPRIP over PIP2 channel. Bars that are marked as Random were obtained by analyzing randomized images (see M.M.). Single asterisk * corresponds to a significance level of p ≤ 0.05 and the double asterisks ** to p ≤ 0.007.

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  Figure 4. The immunoprecipitation assay confirms the existence of a MPRIP/RNAPII/MYO1C nuclear complex. The MPRIP immunoprecipitation was performed with HeLa nuclear lysates and the corresponding proteins were detected by Anti-RNAP II CTD Phospho S5 and anti-MYO1C antibodies. I—input 2%; C—control non-specific rabbit Immunoglobulin G. n = 3.

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  Figure 5. The overexpression of GFP-MPRIP results in three different phenotypes. Confocal microscopy of U2OS cells over-expressing GFP-MPRIP. (A) diffuse; (B) condensed; and (C) fibrous structures, which constitute the three different phenotypes. DAPI in blue and MPRIP in green. Scale bars correspond to 5 µm. (D) The graph reflects the changes in population of cells showing different phenotypes over the time post transfection.

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  Figure 6. 1,6-Hexanediol treatment of the cells overexpressing GFP-MPRIP dissolves the condensates. Live cell imaging showing U2OS cells over-expressing GFP-MPRIP before and after 2 min of Hexanediol treatment: (A) the GFP-MPRIP condensates in a cell nucleus treated with 1,6-Hexanediol; (B) with 2,5-Hexanediol. Green is GFP-MPRIP. Scale bars correspond to 10 µm. n = 2.

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  Figure 7. MPRIP condensates show typical features of liquid-like structures. Live cell imaging showing the nucleus of an U2OS cell over-expressing GFP-MPRIP. (A) The GFP-MPRIP condensates were visualized for 15 min. Arrows show the structures that merge during the imaging. Green is GFP-MPRIP. Scale bar represents 3 µm. (B) The GFP-MPRIP expressing cell was visualized for 8 h and 10 min. Arrowheads show the spherical condensates that disappear and form fibers, and then revert back into condensates over time. Imaging started at the 12th hour after transfection. The scale bar represents 10 µm.

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  Figure 8. MRPIP nuclear condensates show dynamic liquid-like properties. (A) Representative images of Full-Fluorescence recovery after photobleaching (FRAP) experiments that were performed on GFP-MPRIP nuclear condensates. The lower row images correspond to a solidified structure. Imaging rate was 23 ms. Scale bars represent 0.5 µm. (C) Representative images of nucleoplasmic-, inner- and fiber-FRAP. r is the radius of the circular bleached area. Scale bars represent 3 µm, 0.5 µm and 0.25 µm from the top to the bottom, respectively. (B–D) The graphs represent the relative fluorescence values in time, starting with the first capture after bleaching. Each point corresponds to the corrected and normalized fluorescence values of the bleached areas of the FRAP experiments. The lines passing through the points are single exponential curves except for fiber-FRAP. The line for fiber-FRAP is a bi-exponential curve. The FRAP graph was created using one exemplary experiment for each FRAP experiment. “*” in subfigure B corresponds to a solidified structure.

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  Figure 9. Phalloidin staining of the GFP-MPRIP overexpressing U2OS cells. Fixed GFP-MPRIP transfected cells were stained with phalloidin and investigated by confocal microscopy. (A) GFP-MPRIP condensates localized on phalloidin labelled fibers in cell nucleus. Scale bar represents 5 µm (B) GFP-MPRIP fibers visualized by STED microscopy. Blue is DAPI, green is GFP-MPRIP and red is the phalloidin staining. The scale bar represents 1 µm.

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  Figure 10. Confocal microscopy revealed the sub-cellular localization of the overexpressed GFP-MPRIP fragments F1 and F2. Fragment F1 localizing to (A) stress fibers and (B) to nucleus. Fragment F2 shows (C) globular and fibrous structures in the cytoplasm but not in the (D) nucleus. Scale bars represent 10 µm.

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  Figure 11. Illustration of the hypothetical model of interplay between MPRIP phase separation and its interactors. RNAPII/NMI/MPRIP complex localizes in the proximity of nuclear PIP2-rich structures. The MPRIP protein contains two PH domains, an F-actin binding site and C-terminal IDR. MPRIP is able to form phase-separated condensates through IDR interactions. These condensates can form fibers when associated with nuclear F-actin. (*) This association is mediated either by intrinsic MPRIP F-actin-binding site or by other F-actin binding factors. (?) It remains unclear how MPRIP/RNAPII/NM1/PIP2 complex formation depends on the phase separation, actin polymerization or association with PIP2-rich nuclear structures. It also needs further clarification as to how these processes orchestrate the MPRIP-mediated regulation of RNAPII activity and the consequent transcriptional output of the cell.