sim 3d structured illumination microscopy images  (Nikon)

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

Journal: bioRxiv

Article Title: Dynein acts to cluster glutamate receptors and traffic the PIP5 kinase, Skittles, to regulate postsynaptic membrane organization at the neuromuscular junction

doi: 10.1101/2021.09.27.462070

Figure Lengend Snippet: A-A”) The punctate distribution of dynein, while somewhat broader, is similar to that of the glutamate receptor subunit GluRIIC at the NMJ. A single optical section through the NMJ is shown. In the inset, many GluRIIC positive punctae overlap with dynein punctae and appear yellow (A’’) . An example of the GluRIIC localization in a control animal (B-B’) and in a dynein depleted animal (C-C’) used for the analyses in (D) . HRP labels the presynaptic side of the NMJ, and single optical sections through the NMJ are shown. D) Boxplots of the radius of iGluR spheres in control animals, and those depleted of dynein by RNAi. n=number of NMJ analyzed. Unpaired, one-tailed t-test results: ***, P<0.0001, **, P<0.005, *, P<0.05. E-E’’) Structured illumination microscopy images of the GluRIIC subunit and Bruchpilot (BRP), a presynaptic active zone component, in control animals and in animals depleted of dynein within the muscle (F-F’’) . G) Quantification of the number of BRP, active zone punctae apposed to a postsynaptic glutamate receptor cluster for control animals and those depleted of muscle dynein. n=number of NMJ analyzed, each point on the graph is an individual NMJ. Unpaired, two-tailed t-test results: ***, P<0.0001. Boxed areas are shown as insets. Scale, 10μm.

Article Snippet: Reconstructed 3D Structural Illumination Microscopy (SIM) images were analyzed using Imaris 9.5.1 (Bitplane, Belfast, UK).

Techniques: Control, One-tailed Test, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Dynein acts to cluster glutamate receptors and traffic the PIP5 kinase, Skittles, to regulate postsynaptic membrane organization at the neuromuscular junction

doi: 10.1101/2021.09.27.462070

Figure Lengend Snippet: Compared to controls (A-A’) , depletion of postsynaptic Sktl by RNAi results in increased cytoplasmic GluRIIC staining in the muscle and decreased extra synaptic GluRIIC punctae (B-B’) . Arrows indicated some of the extra synaptic punctae in (A,B) . C) Depletion of postsynaptic Sktl results in increased GluRIIC at the membrane compared to controls. Structured illumination microscopy was used to image and analyze GluRIIC clusters and their apposition to presynaptic BRP punctae. GluRIIC receptor cluster size, and their apposition to presynaptic Bruchpilot (BRP) punctae appear to be similar in controls (D-D’) and Sktl depleted animals (E-E’) . F) Quantification of the number of BRP, active zone, punctae apposed to a postsynaptic glutamate receptor cluster for control animals and those depleted of postsynaptic Sktl in comparison to the data shown in for dynein depletion, replicated again here for a side-by-side comparison. G-H’) Structured illumination microscopy was used to image glutamate receptor clusters and dynein together at NMJ to better determine the relationship between the two components. Two examples of wild type synaptic terminals are shown. GluRIIC labels glutamate receptor clusters, and Dhc64c labels the dynein heavy chain. In the merged image insets dynein often localizes to the center of the glutamate clusters (G’,H’) . I) A model for how dynein functions on the postsynaptic side of the NMJ to transport Sktl for local production of PIP 2 and organization of the postsynaptic spectrin cytoskeleton, and stabilization/organization of glutamate receptor clusters. n=number of NMJ analyzed, each point on the graph is an individual NMJ. Unpaired, two-tailed t-test results: ***, P<0.0001, **, P<0.005, *, P<0.05. Boxed areas are shown as insets. Scale, 10μm.

Article Snippet: Reconstructed 3D Structural Illumination Microscopy (SIM) images were analyzed using Imaris 9.5.1 (Bitplane, Belfast, UK).

Techniques: Staining, Membrane, Microscopy, Control, Comparison, Two Tailed Test

Journal: Scientific Reports

Article Title: The autophagy inducer SMER28 attenuates microtubule dynamics mediating neuroprotection

doi: 10.1038/s41598-022-20563-3

Figure Lengend Snippet: SMER28 treatment stabilizes microtubules by α-tubulin acetylation. ( a ) Western Blot analysis of U-2 OS cells treated with 50 and 200 µM of SMER28, 150 nM epothilone B (EpoB), 150 nM paclitaxel (Pacl) or 300 nM rapamycin (Rapa), as indicated, for 4 h. Lysates were analyzed for the protein levels of acetylated (ac)-α-tubulin. GAPDH served as loading control. ( b ) Quantification of relative ratios of ac-α-tubulin to GAPDH as indicated. Data show means of protein levels derived from six independent experiments ± s.e.m. *P < 0.05, ***P < 0.001, Mann–Whitney rank sum test. ( c ) U-2 OS cells were treated with DMSO as vehicle control, 50 µM or 200 µM SMER28, 150 nM epothilone B, 150 nM paclitaxel, 300 nM rapamycin or HBSS, as indicated, for 4 h. Cells were stained for ac-α-tubulin and visualized by SIM. Images show maximum intensity projections. ( d ) 3D object measurement tool of NIS Elements (Nikon) was used to measure the ac-tubulin filament volume of U-2 OS cells treated as indicated. SIM image stacks were used for quantification. Data show means of ac-tubulin filament volume in µm 3 from at least ten cells ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001, one way ANOVA.

Article Snippet: For SIM (3D structured illumination microscopy) images shown in Figs. and , an N-SIM E (Nikon) was used, built on a Ti-Eclipse microscope (Nikon).

Techniques: Western Blot, Derivative Assay, MANN-WHITNEY, Staining