Journal: Molecular Cell

Article Title: Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

doi: 10.1016/j.molcel.2015.01.013

Figure Lengend Snippet: Ddx4 Spontaneously Self-Assembles to Form Organelles in Live Cells (A) Evolutionary relationships between the disordered regions of Ddx4 homologs and their domain architectures. Disordered regions (green) and locations of DEAD-box helicase domains (brown) are indicated. (B) Schematic showing the DEAD-box helicase domain of Ddx4 replaced with YFP before being transfected into HeLa cells. Ddx4 YFP organelles appear over time. (C) Differential interference contrast (DIC) and corresponding extended focus fluorescence intensity images of a HeLa cell expressing Ddx4 YFP . Ddx4 YFP forms dense, spherical organelles in the nucleus. Cells were stained with antibodies to visualize nucleoli, PML bodies, nuclear speckles, and Cajal bodies as indicated, revealing that Ddx4 organelles are entirely distinct from these other bodies. (D) The variation in total droplet volume with time is explained by the Avrami equation for nucleated growth ( Section 5). The time is measured from the appearance of the first droplet.

Article Snippet: Genes for Ddx4 proteins were synthesized by GenScript and expressed recombinantly in E. coli .

Techniques: Transfection, Fluorescence, Expressing, Staining

Journal: Molecular Cell

Article Title: Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

doi: 10.1016/j.molcel.2015.01.013

Figure Lengend Snippet: Ddx4 YFP Organelles Are Internally Mobile and Respond Rapidly to Changes in Environmental Temperature and Tonicity (A) Fluorescence recovery after photobleaching (FRAP) of a Ddx4 YFP organelle in a live HeLa cell at 37°C. Sample bleaching is indicated with a gray bar. 50% of the fluorescence signal is recovered within approximately 2.5 s post-bleach, corresponding to a diffusion coefficient of 3 ± 1 × 10 −13 m 2 s −1 . (B) Cold shock induces condensation of sub-nuclear Ddx4 YFP droplets at low expression levels. Extended focus fluorescence intensity images showing the nucleus from a time series analysis of a HeLa cell expressing Ddx4 YFP undergoing cold shock. Images are shown at 2-min intervals. Prior to cold shock treatment, Ddx4 YFP had not reached the critical concentration for phase separation at 37°C and was diffuse in the nucleoplasm (first two frames). Rapid exchange of growth media at 37°C for media cooled on ice (time = 0) induced small Ddx4 YFP droplets to condense rapidly within the nucleus (purple line, number of droplets; blue line, total volume of droplets). Following cold shock, the number of Ddx4 YFP droplets decreased through a combination of coalescence and dissolution as the temperature rose. Scale bar, 5 μm (see ). (C) Extended focus fluorescence intensity image slices showing a section of the nucleus from a time series analysis of a HeLa cell containing Ddx4 YFP droplets undergoing osmotic shock. Images are shown at 2-min intervals. Axis labels, data colors, and scale as in (B). See and .

Article Snippet: Genes for Ddx4 proteins were synthesized by GenScript and expressed recombinantly in E. coli .

Techniques: Fluorescence, Diffusion-based Assay, Expressing, Concentration Assay, Dissolution

Journal: Molecular Cell

Article Title: Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

doi: 10.1016/j.molcel.2015.01.013

Figure Lengend Snippet: The N Terminus of Ddx4 Reversibly Forms Organelles In Vitro (A) Schematic showing the relationship between constructs of Ddx4 and the wild-type protein. Ddx4 N1 (residues 1–236) and Ddx4 N2 contain only the disordered N terminus. (B) DIC (left) and YFP fluorescence (right) images of (i) Ddx4 YFP organelles inside HeLa cells (scale bar, 2 μm) and (ii) 60:1 Ddx4 N1 :Ddx4 YFP organelles formed in vitro at 150 mM NaCl (scale bar, 10 μm). (C) FRAP curve of a 10 μm diameter droplet containing Ddx4 N1 and recombinant, purified Ddx4 YFP at a molar ratio of 60:1 in 150 mM NaCl buffer at 20°C. The bleach period is indicated with the gray bar. 50% of the fluorescence signal is recovered after approximately 1 min, corresponding to a diffusion coefficient of 4 ± 1 × 10 −13 m 2 s −1 . (D) Time series analysis of bright-field microscopy images of Ddx4 N1 (202 μM protein, 200 mM NaCl) with varying temperature, shown at 50 s intervals (scale bar, 50 μm). At 50°C, the sample was monophasic with low turbidity. Temperature was linearly decreased (4°C min −1 ) from 50°C to 22°C. At 36°C, the turbidity of the sample rapidly increased concomitant with the emergence of an incipient dense phase containing concentrated Ddx4 N1 . After holding at 22°C for 1 min, the sample was reheated to 50°C. At approximately 45°C during reheating, the condensed phase was completely dissolved and the turbidity of the solution returned to its initial turbidity. The thermal cycle was repeated with the same sample in situ (light green line), revealing that the changes in the droplet are fully reversible.

Article Snippet: Genes for Ddx4 proteins were synthesized by GenScript and expressed recombinantly in E. coli .

Techniques: In Vitro, Construct, Fluorescence, Recombinant, Purification, Diffusion-based Assay, Microscopy, In Situ

Journal: Molecular Cell

Article Title: Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

doi: 10.1016/j.molcel.2015.01.013

Figure Lengend Snippet: Quantitative Analysis and Interpretation of the Ddx4 N1 Phase Transition (A) The temperature at which the phase transition is observed, T P , was determined as a function of protein concentration and ionic strength at pH 8. At a given ionic strength, the Flory-Huggins model of polymer phase separation quantitatively describes each curve. This yields two fitting parameters, the enthalpy and entropy changes of the transition, which report on the microscopic interactions between molecules. (B) The interaction parameters varied in a predictable way with increasing salt. The enthalpic contribution to the interaction parameter (i) was found to decrease as a function of increasing NaCl. This is quantitatively explained by fitting the curve to a screened coulomb potential (light blue, Equation S19 ). The non-ionic component of the enthalpy is close to zero, −0.058 ± 0.137 kJ mol −1 , the relative permittivity within the condense phase was 45 ± 13, and the average spacing between opposite charges is 13 ± 2 Å. The entropic contribution to the interaction parameter (ii) decreases slightly with increasing salt, fitted to Equation S20 . The error bars represent the SE in the fitted parameters ( Figure 4 A). (C) The entropy and enthalpy values are correlated, suggesting that when the interactions are destabilized at higher salt, the chains in the interior of the droplet become more mobile. The error bars represent the SE in the fitted parameters ( Figure 4 A). (D) Schematic representation of dissolution of the Ddx4 condensed phase and expansion of the monomer in the disperse phase through increasing ionic strength or temperature. Ddx4 N1 protein chains depicted as green lines. Transition point (T p ) is indicated with a dashed gray line. The ionic interactions within the droplets are attenuated with increasing salt, as is the residual structure within the protein in the dispersed phase. Corresponding bright-field images are shown on the right. Scale bar, 10 μm.

Article Snippet: Genes for Ddx4 proteins were synthesized by GenScript and expressed recombinantly in E. coli .

Techniques: Sublimation, Protein Concentration, Polymer, Dissolution

Journal: Molecular Cell

Article Title: Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

doi: 10.1016/j.molcel.2015.01.013

Figure Lengend Snippet: Post-Translational Modification by Arginine Methylation Alters the Phase Transition of Ddx4 N1 (A) Sequence logo ( weblogo.berkeley.edu ) depicting the amino acid motifs surrounding arginine residues of Ddx4 N1 predominantly targeted by PRMT1. Arginine residues to be converted to aDMA are highlighted in dark red and with two small ellipses. The amino acid numbers of the modified arginine residues are shown within their respective sequence contexts. Asterisks highlight aDMA sites identified in Ddx4 N1 Me with 95% probability (Scaffold score) from a combination of trypsin and GluC digestion of recombinant, purified Ddx4 N1 Me. aDMA at sites 146 and 147 was identified at ∼65% probability (Scaffold score). (B) Schematic and mass reconstruction of +TOF MS spectra of Ddx4 N1 (green; 25.833 kDa) and Ddx4 N1 Me (dark red). In the latter, a series of peaks was observed between 1 and 20 methyl additions. The major peaks indicate complete aDMA modification at 5 and 6 sites, respectively. (C) A schematic of aDMA together with an insert showing the 1 H- 13 C HSQC NMR spectrum of the θ CH 3 of Ddx4 N1 Me. The chemical shifts of the methyl groups verify that the modification is aDMA (see Figure S5 ). (D) The phase-transition temperatures of Ddx4 N1 Me (dark red) are shifted compared to the unmodified form under the same conditions (light green). Modification with aDMA at a mixture of 5–6 aDMA sites reduces the transition temperature by 25°C, an effect on the phase transition comparable to increasing the ionic strength by 100 mM.

Article Snippet: Genes for Ddx4 proteins were synthesized by GenScript and expressed recombinantly in E. coli .

Techniques: Modification, Methylation, Sublimation, Sequencing, Recombinant, Purification

Journal: Molecular Cell

Article Title: Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

doi: 10.1016/j.molcel.2015.01.013

Figure Lengend Snippet: The Sequence Features that Enable Droplet Formation by Ddx4 and Their Distribution within the Human Genome (A) Sliding net charge (10 amino acid window, black) is shown for (i) Ddx4 N1 and (ii) a charge-scrambled mutant, Ddx4 N1 CS, obtained by swapping the positions of positive residues (blue bars) and negative residues (red bars) to minimize any persistence of blocks of charge. (iii) A mutant where nine phenylalanine residues, whose placement was highly conserved, were mutated to alanine (Ddx4 N1 FtoA, see Figure S6 ). The positions of the nine phenylalanine residues (yellow circles) mutated to alanine are indicated. (B) Representative fluorescence images from cell imaging experiments reveal that Ddx4 N1 CS and Ddx4 N1 FtoA do not form organelles in cells under physiological conditions. Residual HeLa nucleoli are still observed as fluorescence-depleted regions within the cell nucleus. (C) The human genome was surveyed for sequences with similar physical properties to the Ddx4 disordered termini. 1,556 sequences out of 14,198 were identified to have [F/R]G spacings in their sequence that are similar to the Ddx4 ortholog family. The top 10% of these are indicated (dotted line). A significant number of proteins associated with forming non-membrane organelles were present in this group. (D) Similar plots from the yeast (i) and E. coli (ii) genomes revealing a number of proteins closely associated with nucleic acid biochemistry.

Article Snippet: Genes for Ddx4 proteins were synthesized by GenScript and expressed recombinantly in E. coli .

Techniques: Sequencing, Mutagenesis, Fluorescence, Imaging, Membrane

Journal: Molecular Cell

Article Title: Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

doi: 10.1016/j.molcel.2015.01.013

Figure Lengend Snippet: Proteinaceous Organelles Differentially Solubilize Nucleic Acids (A) Ddx4 N1 organelles were allowed to form under near-physiological conditions at a total concentration of 162.5 μM. (i) Double- and (ii) single-stranded 32-nt DNAs (dsDNA and ssDNA, respectively) tagged with atto647N were added at a concentration of 1 μM. In the case of dsDNA, the majority of the material was excluded from the droplets. The reverse effect was observed for ssDNA. (B) The average and SD (error bar) confocal fluorescence emission intensities from both inside and outside the organelles were used to quantify the partition equilibrium coefficient and its corresponding free energy ( Equation 1 , Figure S7 ).

Article Snippet: Genes for Ddx4 proteins were synthesized by GenScript and expressed recombinantly in E. coli .

Techniques: Concentration Assay, Fluorescence

Journal: Drug Metabolism and Disposition

Article Title: Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine

doi: 10.1124/dmd.119.089656

Figure Lengend Snippet: Quantification of protein abundance and mRNA expression of drug transporters in the human intestine. In (A), scatter plots represent the protein abundance of relevant drug transporters from the ABC and SLC families, as well as the plasma membrane markers Na + /K + -ATPase and cadherin-17, in 16 human intestinal tissue samples from jejunum and ileum. Protein abundances are expressed as picomoles per milligram of total mucosal protein from intestinal tissue. In (B), scatter plots represent relative mRNA expression of selected drug transporters and the plasma membrane marker CDH-17 normalized to mRNA transcript levels of GAPDH in human intestinal tissue. RNA data were not collected for the gene ATP1A1 (corresponding to the protein Na + /K + -ATPase) and gene SLCO1A2 (protein OATP1A2). Proteins are denoted with their most common names, and transcripts are denoted by their corresponding gene names. The protein cadherin-17 corresponds to CDH-17, P-gp corresponds to ABCB1, MRP2 corresponds to ABCC2, BCRP corresponds to ABCG2, OST- α and OST- β correspond to SLC51A and SLC51B, respectively, and OATP2B1 corresponds to SLCO2B1. Bars in black represent the mean. RQ stands for relative quantification.

Article Snippet: Two different QconCATs previously designed to quantify human hepatic transporters (Concatemer of Standard Peptides from Human Hepatic Transporters; TransCAT) and human hepatic metabolizing enzymes (Concatemer of Standard Peptides from Human Drug Metabolizing Enzymes; MetCAT) were used to quantify the same transporters and DMEs from human intestinal tissues ( ; ).

Techniques: Quantitative Proteomics, Expressing, Clinical Proteomics, Membrane, Marker

Journal: Drug Metabolism and Disposition

Article Title: Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine

doi: 10.1124/dmd.119.089656

Figure Lengend Snippet: Expression levels of one plasma membrane marker (Na + /K + -ATPase), one cell adhesion protein (cadherin-17), and drug transporters with known involvement in drug clearance in human intestine Protein expression is represented by the mean, the S.D. of the mean, the %CV, and the range (min-max). Protein abundance is reported in picomoles per milligram of total mucosal protein.

Article Snippet: Two different QconCATs previously designed to quantify human hepatic transporters (Concatemer of Standard Peptides from Human Hepatic Transporters; TransCAT) and human hepatic metabolizing enzymes (Concatemer of Standard Peptides from Human Drug Metabolizing Enzymes; MetCAT) were used to quantify the same transporters and DMEs from human intestinal tissues ( ; ).

Techniques: Expressing, Clinical Proteomics, Membrane, Marker, Quantitative Proteomics

Journal: Drug Metabolism and Disposition

Article Title: Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine

doi: 10.1124/dmd.119.089656

Figure Lengend Snippet: Observed correlations between transporter protein abundance and relative mRNA expression levels. In (A), the x -axis represents mRNA expression levels of transporters normalized to mRNA expression levels of GAPDH, and the y -axis represents protein abundance. In (B), the x -axis represents mRNA expression levels of transporters normalized to mRNA expression levels of villin 1, and the y -axis represents protein abundance. Correlation analysis was assessed using Spearman rank-order correlation ( R s) test with t- distribution of the P value. Linear regression analysis was carried out to assess the linearity of relationships and scatter of the data ( R 2 ). Correlations were deemed strong when the R s values were greater than 0.60, P values were less than 0.05, and the data points demonstrated limited scatter ( R 2 > 0.30). R s values between 0.5 and 0.6 with a P value less than 0.05 were considered to represent moderate correlation. Clear circles with a green border correspond to ileal samples, and clear circles with a red border correspond to jejunal samples.

Article Snippet: Two different QconCATs previously designed to quantify human hepatic transporters (Concatemer of Standard Peptides from Human Hepatic Transporters; TransCAT) and human hepatic metabolizing enzymes (Concatemer of Standard Peptides from Human Drug Metabolizing Enzymes; MetCAT) were used to quantify the same transporters and DMEs from human intestinal tissues ( ; ).

Techniques: Quantitative Proteomics, Expressing

Journal: Mucosal immunology

Article Title: KLF6 contributes to myeloid cell plasticity in the pathogenesis of intestinal inflammation

doi: 10.1038/mi.2016.1

Figure Lengend Snippet: (A) RAW264.7 macrophages were transfected to overexpress KLF6 (pCl-neo-KLF6) or control plasmid (pCl-neo) together with a STAT3 luciferase reporter plasmid. Cells were treated with IL-10 or PBS (control), then assessed for the relative luciferase activity, which is expressed as a ratio of control-transfected cells. (B) RAW264.7 macrophages were transfected with siRNA specific to KLF6 or control (scrambled) RNA, together with a STAT3 luciferase reporter plasmid. Cells were treated with IL-10 or PBS (Ctrl) and then assessed for luciferase activity. (C) Wild-type PMs were treated with IL-10 or PBS (Ctrl) and ChIP assays were performed on the SOCS3 promoter (−313 to −319) following immunoprecipitation with anti-KLF6 or IgG (Ctrl). (D) PMs from indicated mice were treated with IL-10 and/or the STAT3 inhibitor NSC74859 as indicated. Gene expression of SOCS3 was determined by qPCR and is expressed as fold-change over PBS-treated, wild-type PMs. (E) RAW264.7 macrophages were transfected to overexpress KLF6 (pCl-neo-KLF6) or control plasmid (pCl-neo) together with an NFκB luciferase reporter plasmid. Cells were treated with IFNγ or PBS (control), then assessed for the relative luciferase activity. (F) RAW264.7 macrophages were transfected with siRNA specific to KLF6 or control (scrambled) RNA, together with an NFκB luciferase reporter plasmid. Cells were treated with IFNγ or PBS (Control) and then assessed for luciferase activity. (G) PMs were isolated from WT mice, treated with IFNγ or PBS (Ctrl), and ChIP assays were performed on the MCP-1 promoter (−1363 to −1378) following immunoprecipitation with anti-KLF6 or IgG (Ctrl. (H) RAW264.7 macrophages were transfected to overexpress the indicated plasmids, and then stimulated with IFNγ or PBS (Control). Relative gene expression of MCP-1 was determined by qPCR and is expressed relative to PBS-treated, mock-transfected cells. Graphs show mean ± SEM; * p ≤0.05; ** p ≤0.005, n=3.

Article Snippet: Luciferase reporter plasmids driven by the NFκB concatemer (Promega) or STAT3 (Affymetrix, Inc) were transfected alone or together with plasmids encoding KLF6 or siKLF6.

Techniques: Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Gene Expression, Isolation