Journal: Science Advances

Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

doi: 10.1126/sciadv.abm2094

Figure Lengend Snippet: Reticular, small-inclusion, and large-inclusion ER morphologies in CHO-K1 cells coexpressing mCherry-KDEL with ( A ) YFP-tagged M-α 1 -antitrypsin (YFP-M) or ( B ) YFP-Z. Intensity gamma manipulation of 0.5 was applied to enable visualization of both reticular ER and ER inclusions. ( C ) Proportion of transfected cells displaying ER morphologies. ( D ) Chemical structure of microviscosity-sensitive BODIPY-HaloLigand cartooned with HaloTag protein. ( E and F ) Example fluorescence decay curves (top) and residuals (bottom) of BODIPY-HaloTag ROVI probe localized to the ER of CHO-K1 cells expressing (E) M-α 1 -antitrypsin or (F) Z-α 1 -antitrypsin. ( G ) Fluorescence intensity images (left) and color-coded fluorescence lifetime images (right). ( H ) Fluorescence lifetime distribution histogram of CHO-K1 cells expressing HaloTag-KDEL labeled with BODIPY-HaloLigand with either M-α 1 -antitrypsin (top) or Z-α 1 -antitrypsin (bottom). ( I ) Mean ER fluorescence lifetime (corresponding to microviscosity) of CHO-K1 cells transiently transfected with HaloTag-KDEL together with either an empty vector control (Vector), M-α 1 -antitrypsin (M-A1AT), or Z-α 1 -antitrypsin (Z-A1AT) before labeling with BODIPY-HaloLigand and ER lumenal microviscosity quantitation by FLIM. Statistical significance was analyzed by analysis of variance (ANOVA) with Bonferroni’s multiple comparison test. A minimum of 19 cells were analyzed over three independent repeats. ns, not significant.

Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

Techniques: Transfection, Fluorescence, Expressing, Labeling, Plasmid Preparation, Quantitation Assay

Journal: Science Advances

Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

doi: 10.1126/sciadv.abm2094

Figure Lengend Snippet: CHO-K1 cells were transfected with YFP-M or YFP-Z and a HaloTagged ER protein 48 hours before FCS in large ER inclusions. ( A ) Schematic showing FCS detection volume (DV) within an ER inclusion being traversed by a fluorescent particle (FP). ( B and C ) Example FCS autocorrelation curves (top) and residuals (bottom) for HaloTag-KDEL in cells expressing (B) YFP-M or (C) YFP-Z. ( D ) Measured effective diffusion coefficients ( D eff ) for HaloTag-KDEL and ( E ) the corresponding anomalous parameter of diffusion (α). A minimum of 25 cells were analyzed, acquired over three independent experiments. ( F ) Domain organization of ER-AqLs-Sapphire; preprolactin signal sequence (PrSS), the Aquifex aeolicus lumazine synthase scaffold (AqLs), and Sapphire fluorescent protein (aA denotes number of amino acids). ( G to J ) HILO micrographs of ER in a CHO-K1 cell coexpressing (G) Sec61TA-HaloTag (JF646 labeled) and (H) AqLs-Sapphire, merged in (I). (J) Maximum intensity projection of AqLs intensity from 1000 frames acquired over 20 s. ( K to N ) ER inclusion containing (K) HaloTagged–M-α 1 -antitrypsin (Halo-M) or (M) HaloTagged–Z-α 1 -antitrypsin (Halo-Z) (JF646 labeled) and AqLs-Sapphire. Dashed lines denote ROIs used to generate fluorescence kymographs [(L) and (N), respectively], displaying a 20-s imaging period. Images are representative of three independent repeats.

Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

Techniques: Transfection, Expressing, Diffusion-based Assay, Sequencing, Labeling, Fluorescence, Imaging

Journal: Science Advances

Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

doi: 10.1126/sciadv.abm2094

Figure Lengend Snippet: ( A ) Schematic depicting photobleaching of fluorescent protein (green) within an ER inclusion that can exchange fluorescent protein with the ER network. Solid circle represents a bleached ROI, while the dashed-line circle represents a control ROI. Represented are two situations, showing a fluorescent protein with relatively high (top) or low (bottom) mobility. Cartooned graphs show the bleached ROI intensity (left) versus control ROI intensity minus bleached ROI intensity (right) as used in intensity differential (Δ I ) FRAP (ID-FRAP). ( B to D ) Representative examples of CHO-K1 cell large ER inclusions containing (B) mobile, (C) semi-mobile, and (D) immobile YFP-Z before and at 0 or 80 s after bleach. Δ I (Intensity control − Intensity bleached ) is shown as a percentage of the initial fluorescence intensity. ( E ) Distribution of YFP-M or YFP-Z mobilities (coexpressed with mCherry-KDEL) in CHO-K1 cell large ER inclusions determined by ID-FRAP, shown as a percentage of cells analyzed. ( F ) A representative example of large ER inclusions containing mobile M-α 1 -antitrypsin. ( G ) A representative example of large ER inclusions containing immobile YFP-Z coexpressed with mCherry-KDEL in a MEF. ( H ) A representative example of a large ER inclusion containing immobile mEmerald-tagged neuroserpin G392E in a CHO-K1 cell, coexpressed with mCherry-KDEL.

Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

Techniques: Fluorescence

Journal: Science Advances

Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

doi: 10.1126/sciadv.abm2094

Figure Lengend Snippet: ( A ) A structural model of a predicted late folding intermediate of Z-α 1 -antitrypsin, showing three Glcα1–3Manα1–2Manα1–2Man glycans at residues N46, N83, and N247. The C-terminal region is shown in blue. ( B ) An enlargement of the C terminus shows hydrophobic amino acid side chains in red (and in inset peptide sequence). ( C ) Whole-cell lysates of CHO-K1 cells transiently transfected to express untagged M- or Z-α 1 -antitrypsin (M- or Z-A1AT) with mCherry-KDEL and Z-A1AT with either HaloTag-calreticulin (Halo-CRT) or HaloTag-calreticulin Y92A,W244A (Halo-CRT Y92A,W244A ) were separated by native-PAGE, and Western blots were probed with the α 1 -antitrypsin polymer-specific mAb 2C1 . The same samples were separated by SDS-PAGE and blotted for total α 1 -antitrypsin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Quantitation was performed on ( D ) total α 1 -antitrypsin on SDS-PAGE and ( E ) total lane intensity of native-PAGE mAb 2C1 signal, from three independent experiments, with means and SEs shown. ( F ) mAb 2C1 signal intensity quantification on the blot shown in (C) was profiled from bottom to top of the gel. The X axis reflects increasing polymer size. ( G ) CHO-K1 cell lysates prepared as in (C) were centrifuged at 16,100 g before both supernatant and pellet fractions were separated by native-PAGE. Western blots were probed with polymer-specific mAb 2C1 antiserum. Representative gel of three experiments.

Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

Techniques: Sequencing, Transfection, Clear Native PAGE, Western Blot, SDS Page, Quantitation Assay

Journal: Science Advances

Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

doi: 10.1126/sciadv.abm2094

Figure Lengend Snippet: CHO-K1 cells expressing mEmerald-tagged Z-α 1 -antitrypsin (mEmerald-Z) and HaloTag-KDEL (Halo-KDEL) labeled with TMR HaloTag ligand were analyzed by ID-FRAP to assign α 1 -antitrypsin mobility phenotype. ( A ) Semi-mobile mEmerald-Z or ( B ) immobile mEmerald-Z inclusions were imaged before (top) and after (bottom) 5 min of treatment with hypotonic buffer, leading to ER swelling. Images are representative of three independent experiments. Example inclusions marked “i” and “ii” are expanded in ( C ), as three-dimensional renderings from Z -stacked confocal image series, 7 min after hypotonic shock. ( D ) CHO-K1 cells were transiently transfected with expression plasmids encoding mEmerald-Z and HaloTag-KDEL and were analyzed by ID-FRAP. Images show ER inclusions of immobile mEmerald-Z before (left) and immediately after photobleach (middle). Cells were subsequently treated with 4% (w/v) 1,6-hexanediol (1,6-Hex) for 20 min before ID-FRAP assessment of Z-α 1 -antitrypsin mobility in the same inclusion (right). Images are representative of all 14 cells analyzed over three experiments. ( E to G ) CHO cells expressing (E) mEmerald-M and Halo-KDEL, (F) mEmerald-Z and Halo-KDEL, and (G) mEmerald-Z and Halo-CRT were imaged before (top) and after (bottom) saponin treatment. ( H to J ) Lattice SIM images of mEmerald-Z puncta detergent-extracted from cells coexpressing AqLs-HaloTag labeled with JF646 ligand. Images were reconstructed using the Zeiss SIM 2 algorithm. The white line overlaid on the merged channel image (J) represents a linear ROI used to produce the histogram of fluorescence intensity gray values [kilogray (kGy)] along the length of the ROI (distance) shown in ( K ). ( L ) Apparent Young’s moduli of YFP-Z puncta extracted from cells expressing YFP-Z with either Halo-KDEL or Halo-CRT, assessed by atomic force microscopy (AFM) on a glass substrate. P value was assigned by Student’s t test.

Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

Techniques: Expressing, Labeling, Transfection, Fluorescence, Microscopy

Journal: Science Advances

Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

doi: 10.1126/sciadv.abm2094

Figure Lengend Snippet: COS7 cells were transfected with mEmerald-tagged α 1 -antitrypsin and HaloTag-KDEL labeled with PA-JF646 ligand. Shown are fluorescence intensity micrograph of [( A ), left] mEmerald-M or [( B ), left] mEmerald-Z alongside projections of single-particle tracks (SPTs) acquired over 15,000 frames (corresponding right panels). Tracks are color-coded by mean track velocity. Frequency distribution histograms show mean track velocity of HaloTag-KDEL molecules collated from ( C ) 38 cells expressing mEmerald-M and ( D ) 50 cells expressing mEmerald-Z, fitted with a bimodal Gaussian distribution model color-coded for the low velocity peak (blue), high velocity peak (red), and combined fit (black). ( E ) Mean effective diffusion coefficients ( D eff ) of HaloTag-KDEL particles in each cell analyzed in (C) and (D), with mean and SE shown. P values were assigned by Student’s t test.

Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

Techniques: Transfection, Labeling, Fluorescence, Single Particle, Expressing, Diffusion-based Assay

Journal: Science Advances

Article Title: Z-α 1 -antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state

doi: 10.1126/sciadv.abm2094

Figure Lengend Snippet: Single-particle tracking of PA-JF646–labeled HaloTag–Z-α 1 -antitrypsin (HaloTag–Z-A1AT) was performed in COS7 cells. ( A ) Spline fit of mean track velocity histograms of HaloTag–Z-A1AT in cells coexpressing mEmerald-KDEL or calreticulin tagged with GFP (CRT-GFP). ( B ) Spline fit of mean track velocity histograms of HaloTag–Z-A1AT coexpressed with mEmerald-KDEL, treated for 8 hours with either DMSO, 0.02 μM TG, or TM (2 μg/ml). PA-JF646 labeling of HaloTag–Z-A1AT was performed before addition of ER stress inducers, ensuring that labeled HaloTag–Z-A1AT glycosylation state would be unaffected by tunicamycin. ( C ) Spline fit of mean track velocity histograms of PA-JF646–labeled HaloTag-KDEL molecules in cells treated for 8 hours with either DMSO, 0.02 μM TG, or TM (2 μg/ml). HaloTag-KDEL was labeled immediately before imaging. Effective diffusion coefficients ( D eff ) for ( D ) HaloTag–Z-A1AT and ( E ) HaloTag-KDEL were extracted from the data presented in (B) and (C), respectively. Images showing representative cells were analyzed in (B) and (D), treated with ( F ) DMSO, ( G ) TG, or ( H ) TM. Left-hand images show mEmerald-KDEL ER marker, and right-hand images show tracks of PA-JF646–labeled HaloTag–Z-A1AT molecules color-coded by mean velocity. Minimum number of cells analyzed for each treatment was (A) 15, (B) 28, and (C) 24, over a minimum of three independent experiments. P values were assigned by Student’s t test.

Article Snippet: Antibodies used in this study were raised against total α 1 -antitrypsin (A0409, Sigma-Aldrich), glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), and the α 1 -antitrypsin polymer–specific mAb 2C1 (HM2289, Hycult Biotech).

Techniques: Single-particle Tracking, Labeling, Imaging, Diffusion-based Assay, Marker

Journal: Cell reports

Article Title: Human iPSC-hepatocyte modeling of alpha-1 antitrypsin heterozygosity reveals metabolic dysregulation and cellular heterogeneity

doi: 10.1016/j.celrep.2022.111775

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-AAT , Proteintech , 16382-1-AP; RRID: AB_10641185.

Techniques: Recombinant, Electron Microscopy, Lysis, Lactate Assay, Pyruvate Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Sequencing, Mutagenesis, Software, Gentle, Saline, Modification

Journal: Journal of Inequalities and Applications

Article Title: Hermite–Hadamard inequalities and their applications

doi: 10.1186/s13660-018-1895-4

Figure Lengend Snippet: Graph of the function \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\sin\frac{1}{x}$\end{document} sin 1 x

Article Snippet: These are the solutions of the non-homogeneous Bessel’s differential equation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ x^{2}\frac{\mathrm{d}^{2}y}{\mathrm{d}x^{2}}+x\frac{\mathrm {d}y}{\mathrm{d}x}+ \bigl(x^{2}-a^{2} \bigr)y=\frac{4 ( \frac{x}{2} )^{\alpha+1} }{\sqrt{\pi}\varGamma ( \alpha+\frac{1}{2} ) }, $$\end{document} x 2 d 2 y d x 2 + x d y d x + ( x 2 − a 2 ) y = 4 ( x 2 ) α + 1 π Γ ( α + 1 2 ) , introduced by Hermann Struve in 1882.

Techniques: