Journal: Journal of Cell Science

Article Title: Parallel assembly of actin and tropomyosin, but not myosin II, during de novo actin filament formation in live mice

doi: 10.1242/jcs.212654

Figure Lengend Snippet: De novo actin and Tpm 3.1 filament polymerisation forms a scaffold around granules after fusion with the APM. (A–D) Intravital confocal imaging of actin filament assembly during secretory granule exocytosis in submandibular salivary gland acinar cells in the progeny of an mTomato and Lifeact–GFP mouse cross. (A,B) Snapshots of salivary acini in situ showing the membrane marker mTomato (red) and F-actin marker Lifeact–GFP (green). (A) Snapshot was taken at a depth of 12 µm from the surface of the gland. Three individual acinar epithelial cells (numbers 1–3) are part of one acinus (encircled by dashed line). Actin is highly enriched at the APM/canaliculi (white arrowheads). Enlarged split channel images displayed on the right show an APM/canaliculus cross-section (red arrowhead) enriched with F-actin (green arrowhead). (B) The same area as in A was imaged 5 min after subcutaneous injection of isoproterenol. Granules fused to APM are seen (white arrows) after stimulation with isoproterenol. Enlarged split channel images show actin recruitment (green arrow) onto the fused granule as seen by the appearance of the mTomato membrane marker (red arrow). Scale bar: 10 µm; the width of the insets is 5.25 µm. See also Fig. S1 and Movie 1. (C) Enlarged time-course images of a representative granule fusion event. The granule acquires an mTomato signal at t=0 s, marking the fusion event (red arrows), and Lifeact–GFP is first detected around the granule circumference at t=1 s (green arrows). Each panel is 5.25 µm wide and the granule diameter is ∼1.2 µm; temporal sampling is 943 ms per frame. (D) Recruitment profiles of mTomato and Lifeact–GFP during a granule fusion event are shown as normalised fluorescence intensity over time. Increase in mTomato (red line) signal from baseline indicates fusion between the granule membrane and the APM. Increase in Lifeact–GFP (green line) indicates actin filament assembly. The frame before the first detection of actin polymerisation was set at t=0 s for this and all subsequent graphs. (E) Detection of mTomato, Tpm3.1 and actin (phalloidin) on fused granules (arrows) in a fixed salivary gland section from an mTomato mouse at 10 min after isoproterenol injection. The contrast was adjusted for each channel separately to facilitate consistent visualisation of the granules in all figures. Scale bar: 10 µm.

Article Snippet: A 3 μm line was drawn across each granule in the raw images and the average (over two pixels) fluorescence intensities in the red and green channels were recorded along the line in Metamorph software.

Techniques: Imaging, In Situ, Membrane, Marker, Injection, Sampling, Fluorescence

Journal: Journal of Cell Science

Article Title: Parallel assembly of actin and tropomyosin, but not myosin II, during de novo actin filament formation in live mice

doi: 10.1242/jcs.212654

Figure Lengend Snippet: The initial actin and Tpm3.1 filament assembly exhibits a close temporal relationship, unlike what is seen with myosin IIA. (A) Live intravital confocal imaging of de novo cytoskeleton assembly after isoproterenol-stimulated secretory granule fusion in the progeny of a Tpm3.1–NG KI and Lifeact-RFP mouse cross. The snapshot (left) shows a fused secretory granule (white arrow) near the APM/canaliculus. The image sequence (right) shows the progression of Tpm3.1–NG (green arrows) and Lifeact–RFP (red arrows) localisation over time. See also Movie 2. (B) Snapshot of intravital confocal imaged salivary acini in situ in the progeny of a myosin IIA–GFP KI and Lifeact–RFP mouse cross (left) after isoproterenol injection. Time-lapse sequence (right) showing the progression of actomyosin scaffold assembly around the granule with F-actin (red arrows) and myosin IIA (green arrows) localisation over time. See also Movie 3. (C) Recruitment kinetics of Lifeact–RFP and Tpm3.1–NG acquired at 241 ms intervals shown as mean normalised fluorescence ±95% CI for each time point. An average of 16 fusion events from four mice were plotted. The first significant (P<0.05) increase in fluorescence intensity versus the zero time point was determined by one-way ANOVA and Dunnett's multiple comparison test for Lifeact–RFP (red asterisk, P=0.0025) and Tpm3.1–NG (green asterisk, P=0.0215). (D) Recruitment kinetics of Lifeact–RFP (red line) and myosin IIA (blue line) acquired at 241 ms intervals are shown as normalised mean fluorescence intensities±95% CI from 17 fusion events in four mice. The first significant (P<0.05) increase in fluorescence intensity versus the zero time point was determined, as in Fig. 4C, for Lifeact–RFP (red asterisk, P=0.0283) and myosin IIA (blue asterisk, P=0.0091). Scale bar: 5 µm; the width of the insets is 6.16 µm.

Article Snippet: A 3 μm line was drawn across each granule in the raw images and the average (over two pixels) fluorescence intensities in the red and green channels were recorded along the line in Metamorph software.

Techniques: Imaging, Sequencing, In Situ, Injection, Fluorescence, Comparison

Journal: Cell stem cell

Article Title: Multimerization of Zika Virus-NS5 causes ciliopathy and forces premature neurogenesis

doi: 10.1016/j.stem.2020.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Since microtubules constitute the core structure of primary cilia, the fluorescence average pixel intensity of tubulin polyglutamylation (Enzo Life Sciences) has been measured.

Techniques: Recombinant, Modification, Protease Inhibitor, Infection, Mutagenesis, Luciferase, Reporter Assay, Plasmid Preparation, Sequencing, Marker, Software, Imaging