Journal: Traffic (Copenhagen, Denmark)

Article Title: Dynamics of axonal β‐actin mRNA in live hippocampal neurons

doi: 10.1111/tra.12865

Figure Lengend Snippet: Single‐molecule fluorescence in situ hybridization (smFISH) targeting β‐actin mRNA in sparsely transfected neurons. (A) DIV11 neurons were transfected with CMV‐GFP‐UtrCH to label filamentous actin, and smFISH was performed on the following day. (B) To image axons over a broad area, grid imaging was performed prior to the stitching process. Axonal β‐actin mRNA molecules of the transfected neuron were identified by three‐dimensional (3D) axon segmentation and 3D particle detection. (C) Left, representative smFISH image. The dotted lines delineate an axon. Right, enlarged images of the areas within the blue and pink boxes in the left panel. The green arrows indicate β‐actin mRNA molecules within actin patches (APs), and the blue arrows indicate β‐actin mRNA molecules outside APs. Scale bars = 50 μm (Left panel), 5 μm (right panel). (D) Fractions of β‐actin mRNA molecules colocalized with APs ( n = 7 axons, 367 mRNAs, ** p < 0.01 by pairwise t ‐test). The error bars represent the SEM.

Article Snippet: Hippocampal neuron cultures at DIV11 were transfected with CMV‐GFP‐UtrCH (Addgene, #26737) by using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Fluorescence, In Situ Hybridization, Transfection, Imaging

Journal: eLife

Article Title: The recycling endosome protein Rab25 coordinates collective cell movements in the zebrafish surface epithelium

doi: 10.7554/eLife.66060

Figure Lengend Snippet: ( A–A” ) Confocal z-projections of stills from time-lapse of a WT EVL cell during mitosis expressing Gfp-Utrophin (green) and mRfp (magenta). Scale bar 20 μm. ( B ) Lateral views with apical to the top of stills from single-plane confocal time-lapses of WT EVL cells during mitosis expressing Gfp-Utrophin (green) and mRfp (magenta). Arrowheads denote cleavage furrow ingression from basal to apical. ( C ) Confocal z-projections of stills from time-lapse of MZ rab25b multipolar cleavage failure. F-actin labeled with Gfp-Utrophin. Scale bar 20 μm. ( C’ ) Confocal z-projections of time-lapse of MZ rab25b Tg (Myl1.1-Gfp) (Fire-LUT) during multipolar cytokinesis failure. White arrows indicate Myosin-Gfp foci. ( D ) Confocal z-projection of MZ rab25b embryo showing an array of EVL cells interconnected via cytokinetic bridges at 30% epiboly. Microtubules (green), nuclei (magenta), and plasma membrane (magenta), arrows and numbers denote connected cells and cytokinetic bridges. Scale bar 20 μm.

Article Snippet: Recombinant DNA reagent , pCS2+- Gfp-Utrophin , Addgene , RRID: Addgene_26737 , Gfp-Utrophin version of pCS2+.

Techniques: Expressing, Labeling

Journal: eLife

Article Title: The recycling endosome protein Rab25 coordinates collective cell movements in the zebrafish surface epithelium

doi: 10.7554/eLife.66060

Figure Lengend Snippet: ( A ) Confocal z-projections of stills from a time-lapse of a WT embryo expressing mGfp starting at 7hpf. Lateral view focused on the margin. Green shaded cells shrink the EVL-YC junction and intercalate into submarginal zones. Orange shaded cells establish new cell-cell contacts following intercalation events (denoted by red dotted line). Circle denotes shared vertex with underlying yolk cell. Scale bar 20 μm ( A’ ) Confocal z-projection time-lapse of WT embryo labeled for F-actin; (Fire-LUT) (Gfp-Utrophin) starting at 7hpf; lateral view; scale bar 20 μm. ( B ) Confocal z-projection of stills from a confocal time-lapse starting at 7hpf of an MZ rab25b embryo expressing membrane-Gfp. Purple shaded cells exit EVL marginal region. Scale bar 20 μm ( B’ ) Confocal z-projection of stills from time-lapse starting at 7hpf of an MZ rab25b embryo labeled for F-actin (Fire-LUT) (Gfp-Utrophin); scale bar 20 μm. Shaded cells denote an EVL circumferential stretching event. Scale bar 20 μm. ( C–D ) EVL-YC mean contact length or shortening rate over time in rearranging EVL marginal cells in WT (N = 5) and MZ rab25b embryos (N = 5). Mean:SEM. Each color indicates a separate trial of a single embryo. Each line represents the average of the contact length or junction shrink rate in each trial (n = 2–5). ( E ) Resolution times following formation of EVL-YC multicellular vertices. Mean: SEM. WT (n = 20,N = 4) and MZ rab25b (n = 12,N = 5), unresolved MZ rab25b vertices (red) (n = 6,N = 5). Mann-Whitney, *p<0.05.

Article Snippet: Recombinant DNA reagent , pCS2+- Gfp-Utrophin , Addgene , RRID: Addgene_26737 , Gfp-Utrophin version of pCS2+.

Techniques: Expressing, Labeling, MANN-WHITNEY

Journal: eLife

Article Title: The recycling endosome protein Rab25 coordinates collective cell movements in the zebrafish surface epithelium

doi: 10.7554/eLife.66060

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCS2+- Gfp-Utrophin , Addgene , RRID: Addgene_26737 , Gfp-Utrophin version of pCS2+.

Techniques: Sequencing, Ligation, Recombinant, Plasmid Preparation

Journal: mSphere

Article Title: CRISPR/Cas9-Based Knockout of GNAQ Reveals Differences in Host Cell Signaling Necessary for Egress of Apicomplexan Parasites

doi: 10.1128/mSphere.01001-20

Figure Lengend Snippet: P. berghei liver stage egress is independent of host cell GNAQ. (A) P. berghei parasites show comparable growth in WT and GNAQ-KO cells. WT and GNAQ-KO cells were infected with P. berghei parasites, and at 48 hpi, the average parasite size (area) was determined by density slicing using ImageJ from 50 to 100 parasites per experiment. Shown are means ± SD from five independent experiments. (B) The formation of detached cells is not impaired in GNAQ-deficient host cells. Detached cells in the supernatant were counted at 65 hpi and normalized to the number of infected cells at 48 hpi. Shown are means ± SD from five independent experiments, each performed in triplicate. (C to E) P. berghei liver stage PVM rupture is not affected by the absence of GNAQ. WT and GNAQ-KO cells were infected with sporozoites, and the percentage of merozoite-forming parasites that ruptured the PVM and the time difference between the successful formation of merozoites and PVM rupture were measured by quantitative live-cell imaging. The influx of GFP into the PV was used as a measure of PVM rupture. Imaging was started at ∼55 hpi and lasted for 12 h. (C) Time between the formation of merozoites and PVM rupture. Each line represents the time difference between successful merozoite formation (beginning of the line) and PVM rupture (end of the line) and corresponds to one analyzed parasite. Continuous lines indicate parasites that did not rupture the PVM at all, which were not considered for the determination of the average PVM rupture time in panel E. Shown are combined data from three independent imaging experiments per cell line. (D) Percentage of merozoite-forming parasites that ruptured the PVM. The percentage of PVM rupture was determined in each of the three independent imaging experiments, in which the number of parasites that successfully developed to merozoites within the first 6 h of imaging was set to 100% in each experiment. Based on these, the percentage of parasites that successfully ruptured the PVM was calculated. Shown are means ± SD. (E) Elapsed time from merozoite formation to PVM rupture determined for all parasites that ruptured the PVM. Displayed are means ± SD. For all statistical analyses between WT and GNAQ-KO cells, one-way ANOVA followed by a Holm-Sidak multiple-comparison test was performed. All statistically significant differences are indicated (*, P < 0.05). (F) Egress-associated breakdown of the host actin cytoskeleton is comparable in WT and GNAQ-deficient cells. Cells expressing the filamentous actin marker GFP-utrophin (green) were infected with P. berghei parasites constitutively expressing mCherry (red), and their egress was studied by live-cell time-lapse microscopy. Representative images before and after egress are shown. Bars, 10 μm. See also and in the supplemental material. (G) Comparison and GNAQ dependence of the egress strategies of T. gondii tachyzoites and P. falciparum blood stages versus P. berghei liver stages. The PVM is displayed in red.

Article Snippet: A total of 2 × 10 6 HeLa cells were transfected with 4 μg of pEGFP-N3 (Clontech) or GFP-utrophin ( ) (Addgene plasmid 26737) plasmid DNA using program T-28 in an Amaxa Nucleofector (Lonza).

Techniques: Infection, Live Cell Imaging, Imaging, Expressing, Marker, Time-lapse Microscopy