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Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging <t>using</t> <t>3D-SIM</t> showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.
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Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging <t>using</t> <t>3D-SIM</t> showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.
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Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging <t>using</t> <t>3D-SIM</t> showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.
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Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging <t>using</t> <t>3D-SIM</t> showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.
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Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging <t>using</t> <t>3D-SIM</t> showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.
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Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging <t>using</t> <t>3D-SIM</t> showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.
3d Sim Imaging Zeiss Lsm710 Elyra P1, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging using 3D-SIM showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.

Journal: Nucleic Acids Research

Article Title: Mutational analysis of the F plasmid partitioning protein ParA reveals residues required for oligomerization and plasmid maintenance

doi: 10.1093/nar/gkaf537

Figure Lengend Snippet: Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging using 3D-SIM showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.

Article Snippet: 3D structured illumination microscopy (3D-SIM) imaging was carried out using a DeltaVision OMX-SR Blaze microscope equipped with a PCO Edge 4.2 sCMOS camera, and a ×60, NA 1.42 oil-immersion objective lens was used to acquire raw images.

Techniques: Binding Assay, Membrane, Staining, Clinical Proteomics, Imaging, Expressing