Dataset Information

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.

ABSTRACT: Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.

SUBMITTER: Balaban C

PROVIDER: S-EPMC8068864 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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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.

Balaban Can C Sztacho Martin M Blažíková Michaela M Hozák Pavel P

Cells 20210408 4

Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F ...[more]

PMID: 33918018

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Project description:β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that β-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

Project description:Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) regulates activities of numerous ion channels including inwardly rectifying potassium (K(ir)) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K(V)) channels might be regulated by PI(4,5)P(2). Wide expression of K(V) channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K(V) channels by PI(4,5)P(2), we have coexpressed several of them in tsA-201 cells with a G protein-coupled receptor (M(1)R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P(2) with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P(2) at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K(V)7.1, K(V)7.2/7.3, and K(ir)2.1 channel current by 90-95%. Activation of M(1)R inhibited K(V)7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P(2) regulation of activity of K(V)1.1/K(V)β1.1, K(V)1.3, K(V)1.4, and K(V)1.5/K(V)β1.3, K(V)2.1, K(V)3.4, K(V)4.2, K(V)4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K(V)1.1/K(V)β1.1 and K(V)3.4, resulting in up-regulation of current density upon activation of M(1)R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P(2) at the plasma membrane by enzymes does not seem to influence activity of most tested K(V) channels, whereas it does strongly inhibit members of the K(V)7 and K(ir) families.

Dataset Information

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.

Publications

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.

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