egfp alpha e catenin ptk 1 cells  (ATCC)

Journal: Nature Communications

Article Title: Morphological control enables nanometer-scale dissection of cell-cell signaling complexes

doi: 10.1038/s41467-022-35409-9

Figure Lengend Snippet: Micropatterns are patterned on the carbon film of EM grids and surrounded by a bio-passivated layer to prevent cell adhesion outside of the patterned area, coated with Oregon Green gelatin and then populated with PTK-1 cells expressing EGFP-alpha E catenin. a , c Phase contrast and b , d corresponding fluorescence images of patterned areas are shown in the top panels. e – g Representative cell doublet fully confined within the patterned area ( e - phase contrast, f - fluorescence: DAPI in blue; Oregon Green gelatin and EGFP-alpha E catenin in green, and g - overlay of phase contrast and fluorescence images). Scale bars, 60 µm ( a – d ) and 20 µm ( e – g ). All ECM patterns are 66.5 × 66.5 µm.

Article Snippet: PTK-1 cells (source: ATCC CRL-6493) in log phase were electroporated with pEGFP-C1-ACAT (mouse alpha catenin subcloned into pEGFP-C1, ClonTech; EGFP is fused to the carboxyl terminus of the target protein; construct was a generous gift from Nelson’s group).

Techniques: Expressing, Fluorescence

Journal: Nature Communications

Article Title: Morphological control enables nanometer-scale dissection of cell-cell signaling complexes

doi: 10.1038/s41467-022-35409-9

Figure Lengend Snippet: PTK-1 cells stably expressing EGFP-alpha E catenin were plated on micropatterned grids treated with Oregon Green gelatin. Upon adhesion, many cells form doublets confined into the pattern, as shown in this example. a Phase contrast, b fluorescence image of the cell pair of interest, displaying both the EGFP signal from the tagged alpha E catenin and the Oregon Green signal marking the pattern. Note that the pattern signal intensity is inherently much higher than that of the tagged protein, somewhat overwhelming the EGFP signal, which is quite robust in the absence of the pattern (see Supplementary Figs. and ). The numbers “1” and “2” point to two regions rich in cell–cell contacts and thin enough for direct EM imaging. The insets to the right show the two regions at higher magnification and increased contrast. c Overlay of the aligned fluorescence and cryo-EM views used for identifying the same two regions selected in b for investigation by cryo-ET. d , e 2-nm thick slices from tomographic reconstructions obtained from regions 1 and 2. Scale bars, 20 µm ( a – c ), and 200 nm ( d , e ). All ECM patterns are 66.5 × 66.5 µm.

Article Snippet: PTK-1 cells (source: ATCC CRL-6493) in log phase were electroporated with pEGFP-C1-ACAT (mouse alpha catenin subcloned into pEGFP-C1, ClonTech; EGFP is fused to the carboxyl terminus of the target protein; construct was a generous gift from Nelson’s group).

Techniques: Stable Transfection, Expressing, Fluorescence, Imaging, Cryo-EM Sample Prep, Tomography

Journal: Nature Communications

Article Title: Morphological control enables nanometer-scale dissection of cell-cell signaling complexes

doi: 10.1038/s41467-022-35409-9

Figure Lengend Snippet: a Top view of a cryo-FIB-milled lamella across a vitrified PTK-1 cell–cell contact site imaged using a cryo-transmission electron microscope. The milling patterns above and below the sample were adjusted to leave a central ~200 nm thin slice (lamella) supported by the surrounding unmilled, vitrified material. The cuts below and above the lamella are for releasing stress introduced by the milling. Cell–cell contact regions are identified visually using the shape of the two interacting cells in cryo-SEM images (Fig. illustrate shape-guided targeting of a region of interest). b Higher magnification of targeted region marked by the red box in ( a ) representing the area of tomogram acquisition. c 2-nm thick virtual slice through a tomogram of the lamella region marked by the red box in b , showing a clearly discernible cell–cell contact (marked by red box, P: plasma adjacent plasma membranes) between two adjacent cells. Other cellular features visible include dense packing of macromolecular complexes in the cytoplasm; R: ribosomes; ER: endoplasmic reticulum; M: mitochondrion; V: vesicle; LD: lipid droplet; N: the nuclear envelope separating the nucleoplasm from the cytoplasm and C: Chromatin within the nucleus. d 3D surface representations of segmented features. Plasma membranes in white, nuclear envelope in yellow, other membrane structures in orange, ribosomes in blue and actin filament assemblies in pink. The cell–cell contact is marked by a red box. A slice of the tomogram is shown in the background for reference. e 3D surface representation of adherens junctional punctum within cell–cell contact. Membrane is in white, connecting rods are in red. The inset shows an enlarged view of the boxed area in c . Scale bars in a = 5 μm; b = 500 nm, c = 250 nm.

Article Snippet: PTK-1 cells (source: ATCC CRL-6493) in log phase were electroporated with pEGFP-C1-ACAT (mouse alpha catenin subcloned into pEGFP-C1, ClonTech; EGFP is fused to the carboxyl terminus of the target protein; construct was a generous gift from Nelson’s group).

Techniques: Transmission Assay, Microscopy

Journal: bioRxiv

Article Title: Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

doi: 10.1101/657072

Figure Lengend Snippet: Epithelial cells are confined to ECM patterns on EM grids generated by maskless photopatterning. PtK1 cells were plated on rhodamine-fibronectin square (A-C, 24.8 μm wide; D-F, 32.4 μm wide) and circle (36.6 μm wide) patterns, allowed to adhere and spread for 12 hr. (A, D, G) Rhodamine-fibronectin; (B, E, H) phase contrast; (C, F, I) overlay. Scale bar, 10μm.

Article Snippet: Parental Potorous tridactylus Kidney 1 (PtK1) cells (ATCC CRL-6493) were cultured in the same type of media used for MDCKII cells, without selection antibiotic.

Techniques: Generated

Journal: Molecular oral microbiology

Article Title: Structure-function aspects of the Porphyromonas gingivalis tyrosine kinase Ptk1

doi: 10.1111/omi.12173

Figure Lengend Snippet: Domain architecture of the Ptk1 tyrosine kinase in P. gingivalis. Amino acid residues that were mutated are indicated in bold. TD: transmembrane domain, RK: arginine rich domain, YC: tyrosine rich C-terminal cluster.

Article Snippet: Wild type Porphyromonas gingivalis ATCC 33277 and isogenic mutants 33277 + pT-COW, Δ ptk1 , CΔ ptk1 ( Wright et al., 2014 ), and CMΔ ptk1 (this study), were cultured anaerobically in trypticase soy broth (TSB) supplemented with yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml).

Techniques:

Journal: Molecular oral microbiology

Article Title: Structure-function aspects of the Porphyromonas gingivalis tyrosine kinase Ptk1

doi: 10.1111/omi.12173

Figure Lengend Snippet: Auto (A) and substrate (B) phosphorylation by Ptk1 and its phosphotransfer domain mutants. For autophosphorylation, recombinant FPtk1 (Ptk1:541–821) or FPtk1 with mutations in the Walker A (KS/MC), Walker B (D/N), RK (ERR/A) and YC (YA) domains were dephosphorylated with alkaline phosphatase and incubated with or without ATP in the presence of a phosphatase inhibitor. For substrate phosphorylation, Fptk1 and mutant derivatives were incubated with recombinant EpsD (PGN_0224) in the presence of ATP and a phosphatase inhibitor. The phosphorylation level of proteins was determined by Western blotting with phosphotyrosine antibodies.

Article Snippet: Wild type Porphyromonas gingivalis ATCC 33277 and isogenic mutants 33277 + pT-COW, Δ ptk1 , CΔ ptk1 ( Wright et al., 2014 ), and CMΔ ptk1 (this study), were cultured anaerobically in trypticase soy broth (TSB) supplemented with yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml).

Techniques: Recombinant, Incubation, Mutagenesis, Western Blot

Journal: Molecular oral microbiology

Article Title: Structure-function aspects of the Porphyromonas gingivalis tyrosine kinase Ptk1

doi: 10.1111/omi.12173

Figure Lengend Snippet: Mass spectroscopy sequence coverage for FPtk1 (Ptk1:541–821). A. High confidence MS/MS data for 37 unique peptides comprising 87 exclusive unique and 394 total spectra were used to assign sequence coverage for 246 (blue upper case font) of 281 amino acids achieving 87.5% coverage. Non-detected residues are in lower case. MS/MS spectra for peptide in box is shown in (B). Phosphorylated residues are shown in red upper case, and bolded font. B. Example of MS/MS spectra used to assign site specific phosphorylation to MS/MS, in this case to tyrosine-775 (Y775) in the carbamidomethylated (c) tryptic peptide FTNMcLVLNDVSYSGSR.

Article Snippet: Wild type Porphyromonas gingivalis ATCC 33277 and isogenic mutants 33277 + pT-COW, Δ ptk1 , CΔ ptk1 ( Wright et al., 2014 ), and CMΔ ptk1 (this study), were cultured anaerobically in trypticase soy broth (TSB) supplemented with yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml).

Techniques: Mass Spectrometry, Sequencing, Tandem Mass Spectroscopy

Journal: Molecular oral microbiology

Article Title: Structure-function aspects of the Porphyromonas gingivalis tyrosine kinase Ptk1

doi: 10.1111/omi.12173

Figure Lengend Snippet: Phosphotransfer by Ptk1 is required for P. gingivalis community development with S. gordonii and for extracellular polysaccharide production. A. P. gingivalis 33277, Δptk1 (Dptk1), Δptk1 + pptk1 (CDptk1) and Δptk1 + pptk1 containing a K614M/S615C mutation in the Walker A domain (CMDptk1) were reacted with a substrate of S. gordonii for 18 h. Total biovolumes of P. gingivalis and S. gordonii in a 213 × 213 μm area were obtained with by confocal microscopy, analyzed by Volocity and data are expressed as ratio of P. gingivalis (Pg) to S. gordonii (Sg). B. P. gingivalis strains were stained for extracellular polysaccharide with FITC-labeled concanavalin A and wheat germ agglutinin. Bacterial cells were stained with Syto-17. Fluorescent images were collected by confocal microscopy and the ratio of lectin staining of EPS to whole cell staining determined using Volocity. Error bars are SD, n = 3. ***P < 0.001; ****P < 0.0001, ns P > 0.05.

Article Snippet: Wild type Porphyromonas gingivalis ATCC 33277 and isogenic mutants 33277 + pT-COW, Δ ptk1 , CΔ ptk1 ( Wright et al., 2014 ), and CMΔ ptk1 (this study), were cultured anaerobically in trypticase soy broth (TSB) supplemented with yeast extract (1 mg/ml), hemin (5 μg/ml) and menadione (1 μg/ml).

Techniques: Mutagenesis, Confocal Microscopy, Staining, Labeling