Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: The full-length model indicated super-tertiary domain interactions in the dysferlin model. The RoseTTAFold models that were used in this study were flexibly aligned using FATCAT . Inconsistencies in the 3D models that were generated as a result of the elastic alignment process were repaired using PyMod . Figures were rendered with PyMol and displayed as 180° views of the model. The various domains of dysferlin are shown as colored surfaces and similarly colored labels. The other ferlin full-length models can be found in the supplemental information (S1-S17 Figs and S1-S7 Tables in ).

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: Generated

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: Dysferlin C2A (green, 4IHB), otoferlin C2A (blue, 3L9B), myoferlin C2A (cyan, 6EEL), and Fer1L5 C2A (yellow). The gray spheres are the divalent cations from the crystal structures of dysferlin C2A (4IHB, chain E) and myoferlin C2A (6EEL). The amino acid boundaries from each respective C2 domain are listed with the domain assignment. The superposition of all four C2A domains is labeled as ‘Overlay’. The ferlins without a C2A domain are listed as Not Applicable (N/A).

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: Labeling

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: Primary sequence alignment of the FerI region of the six human ferlin proteins.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: A. C2-FerA domain schematic showings the secondary structure connectivity and the large insertion of the β 4–5 FerA subdomain. B. SDS-PAGE showing the purified dysferlin C2-FerA domain versus molecular weight size markers. C. Far-UV CD spectrum of purified dysferlin C2-FerA (red curve) and predicted C2-FerA spectrum derived from the model (blue curve). The inset table reports the secondary structure summary of the purified dysferlin C2-FerA domain (Experimental), and the dysferlin C2-FerA model (Predicted).

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: SDS Page, Purification, Molecular Weight, Derivative Assay

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: A. Superimposed β 6–7 subdomain of Dysferlin C2F (green) and RNA binding domain of Staufen (1STU) in cyan; RMSD = 2.7 Å. B. Superimposed β 6–7 subdomain of Dysferlin C2F (green) and dsRNA binding domain of TARBP2 (4WYQ) in magenta; RMSD = 0.86 Å.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques: RNA Binding Assay, Binding Assay

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: Predicted ferlin transmembrane span boundaries and extracellular residues.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Redefining the architecture of ferlin proteins: Insights into multi-domain protein structure and function

doi: 10.1371/journal.pone.0270188

Figure Lengend Snippet: A: Dysferlin C2A structure (4IHB) colored to highlighted the various insertions of subdomains. B: The schematic highlights the loops with embedded subdomains. The Type-2 C2 domain β -strand topology is shown as grey arrows. The colored loops: β 1–2 (green, C2C), β 2–3 (orange, C2-FerA), β 4–5 (blue, C2-FerA, C2G), β 6–7 (red, C2C, C2E, C2G, C2F), and β 7–8 (purple, C2C) show where conserved subdomains are present.

Article Snippet: A codon-optimized human dysferlin cDNA gene was purchased from Addgene (Plasmid 67878) [ ].

Techniques:

Journal: bioRxiv

Article Title: Visualizing endogenous RhoA activity with an improved localization-based, genetically encoded biosensor

doi: 10.1101/2021.02.08.430250

Figure Lengend Snippet: A ) Still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD RhoA sensor (upper panel) or CMVdel-dimericTomato-2xrGBD RhoA sensor (lower panel) and H1R (not shown) which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. B ) Change in cytosolic intensity for mNeonGreen-1xrGBD/ - 2xrGBD/ -3xrGBD, 3xmNeonGreen-1xrGBD and dimericTomato-1xrGBD/ -2xrGBD in Hela cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as vertical, black line and the grey dashed line indicates no change in cytosolic intensity. The number of samples per condition is: 3xmNG-1xrGBD=16, mNG-1xrGBD=28, dT-1xrGBD=15, mNG-2xrGBD=34, mNG-3xrGBD=14, dT-2xrGBD=14. C ) Time traces of the normalized cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in grey and for the dimericTomato-2xrGBD sensor in black. Abbreviations: mNG: mNeonGreen, dT: dimericTomato, rGBD: rhotekin G protein binding domain

Article Snippet: The following plasmids are available on addgene ( http://www.addgene.org/ ): # 129633: mNeongreen-aGBD(anillin), # 129634: mNeongreen-pGBD(PKN1 codon optimized), # 129624: mNeongreen-2xrGBD, # 129625: dTomato-2xrGBD.

Techniques: Expressing, Protein Binding

Journal: bioRxiv

Article Title: Visualizing endogenous RhoA activity with an improved localization-based, genetically encoded biosensor

doi: 10.1101/2021.02.08.430250

Figure Lengend Snippet: A ) Change in cytosolic intensity for CMVdel-mNeonGreen-1xpGBD/ -2xpGBD/ -3xpGBD, CMVdel-dimericTomato-2xpGBD coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as vertical, black line. The number of samples per condition is: dT-2xpGBD=16, mNG-1xpGBD=24, mNG-2xpGBD=28, mNG-3xpGBD=14. B ) Colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-2xpGBD in HeLa cells. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. Each dot represents an individual cell. The median of the data is shown as a vertical, black line. The number of samples per condition is:H2A-dT-2xpGBD=19, RhoA-dT2xpGBD=16. C ) Change in cytosolic intensity for CMVdel-mNeonGreen-1xaGBD/ -2xaGBD/ -3xaGBD, CMVdel-dimericTomato-1xaGBD coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as vertical, black line. The number of samples per condition is: dT-1xaGBD=14, eGFP-AHD+PH=27, mNG-1xaGBD=17, mNG-2xaGBD=7, mNG-3xaGBD=9. D ) Colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-1xaGBD and mScarlet-I-AHD+PH in HeLa cells. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. The median of the data is shown as a vertical, black line. The number of samples per condition is: H2A-aGBD=12, RhoA-aGBD=10, RhoA-AHD=9. E ) Amino acid sequence alignment for aGBD, rGBD and pGBD by MUSCLE depicted with the clustalX color code. F ) On the left a structural alignment of PKN1 and Anillin by their RhoA binding domains. On the right a structural alignment of PKN1 and anillin by RhoA, showing the two binding positions at the RhoA molecule. Anillin and the bound RhoA are depicted in dark and light yellow, respectively. PKN1 and the bound RhoA are depicted in light and dark blue, respectively. (PDB: Anillin = 4xOI, PKN1 = 1cxz).

Article Snippet: The following plasmids are available on addgene ( http://www.addgene.org/ ): # 129633: mNeongreen-aGBD(anillin), # 129634: mNeongreen-pGBD(PKN1 codon optimized), # 129624: mNeongreen-2xrGBD, # 129625: dTomato-2xrGBD.

Techniques: Binding Assay, Expressing, Sequencing