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: Nature Communications

Article Title: A distal centriolar protein network controls organelle maturation and asymmetry

doi: 10.1038/s41467-018-06286-y

Figure Lengend Snippet: Topography of distal and daughter centriolar proteins. a Growing RPE1 cells were stained with the indicated combinations of antibodies and visualized using structured illumination microscopy (SIM). Bottom left: diameter of the Talpid3/C2CD3/OFD1 complex, DCPs, and DA protein, CEP164 ( N = 20). Cumulative data from two independent experiments are shown. Bottom right: schematic representation of the centrosome illustrates the localization of Talpid3/C2CD3/OFD1 complex, DCPs, and DA protein, CEP164. b Localization of CEP120 and Centrobin was examined in control, Talpid3 −/− , C2CD3 −/− , and PCM1 −/− cells using SIM. Cells were serum-starved for 24 h and then visualized with indicated antibodies. Scale bars = 0.5 μm

Article Snippet: Super-resolution microscopy was performed using a structured-illumination microscopy (SIM) system (DeltaVision OMX 3D; Applied Precision).

Techniques: Staining, Microscopy, Control

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