coding sequence of plya2 fused to mcherry  (GenScript corporation)

Journal: PLoS Biology

Article Title: Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis

doi: 10.1371/journal.pbio.3001599

Figure Lengend Snippet: (A–D) Live spermatocytes were incubated with purified PlyA2-mCherry (10 μg/ml) for 30 min followed by washes with M3 insect media (2×) and imaged using spinning disk confocal microscopy. (A and B) w 1118 spermatocytes, (C and D) cpes spermatocytes. (E–P) Live spermatocytes expressing endogenous EYFP tagged Rab proteins were incubated with PlyA2-mCherry for 1 h followed by washes with M3 insect media (2×) and imaged using spinning disk confocal microscopy. (E–G) EYFP-Rab4 spermatocytes, (H–J) EYFP-Rab6 spermatocytes, (K–M) EYFP-Rab7 spermatocytes, (N–P) EYFP-Rab11 spermatocytes. Arrowheads indicate colocalizing structures. (Q) Colocalization coefficient (Mander’s coefficient) measurements showing fraction of EYFP Rab proteins colocalize with internalized PlyA2-mCherry. Mander’s coefficient was determined using ImageJ plugin JACoP. Each dot in the graph represents a 2D image consisting of 8–16 spermatocytes, Rab4 ( n = 42), Rab6 ( n = 47), Rab7 ( n = 36), and Rab11 ( n = 81). Statistical significance was calculated using mean, SD, and N in Prism 9. The ordinary 1-way ANOVA multiple comparison was used to calculate P values where **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, and ns P >0.05. The data underlying the graph shown in this figure can be found in . CPE, ceramide phosphoethanolamine; SD, standard deviation.

Article Snippet: Briefly, the coding sequence of PlyA2 (accession number AB777517) fused to mCherry at the C-terminus was synthesized by GenScript.

Techniques: Incubation, Purification, Confocal Microscopy, Expressing, Comparison, Standard Deviation

Journal: PLoS Biology

Article Title: Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis

doi: 10.1371/journal.pbio.3001599

Figure Lengend Snippet: (A–F) The EYFP-Rab7 expressing spermatocytes are treated with PlyA2-mCherry and snapshots of spermatocytes undergoing meiosis I cytokinesis are shown. (A–C) Horizontal view of 1 z slice is shown. (D–F) Cross-sectional view of a spermatocyte undergoing cytokinesis, maximum intensity projection image was shown. (G) Quantification of endosomal volume localized to cleavage furrow in EYFP-Rab7 expressing and PlyA2-mCherry treated spermatocytes. Using surfaces tool in Imaris software, we have calculated the total endosomal volume and fraction of which localized to cleavage furrow in 3D images ( n = 6 spermatocytes from 3 independent experiments). (H–J) Localization of EYFP-Rab7 in intact cysts where spermatocytes are encapsulated by cyst cells. Horizontal view of a single z slice is shown. (K–P) EYFP-Rab11 expressing spermatocytes were treated with PlyA2-mCherry and meiosis I cytokinesis was imaged. (K–M) Horizontal view of single z slice is shown. (N–P) Cross-sectional view, maximum intensity projection image is shown. White arrows indicate colocalization and white arrowheads indicate lack of colocalization. (Q) Quantification of a fraction of endosomal volume localized to cleavage furrow in EYP-Rab11 expressing, PlyA2-mCherry treated spermatocytes. Endosomal volume in 3D images were calculated using surfaces tool in Imaris software ( n = 9 spermatocytes from 3 independent experiments). The data underlying the graphs shown in this figure can be found in . CPE, ceramide phosphoethanolamine.

Article Snippet: Briefly, the coding sequence of PlyA2 (accession number AB777517) fused to mCherry at the C-terminus was synthesized by GenScript.

Techniques: Expressing, Software

Journal: PLoS Biology

Article Title: Delivery of ceramide phosphoethanolamine lipids to the cleavage furrow through the endocytic pathway is essential for male meiotic cytokinesis

doi: 10.1371/journal.pbio.3001599

Figure Lengend Snippet: (A) Combined transmitted and fluorescence image of dissected Drosophila cyst; dividing cells chosen for FIB-SEM imaging are boxed. Green, eYFP-Rab7; red, PlyA2-mCherry. Scale bar: 20 μm. (B) 2D section from FIB-SEM reconstruction correlated with “z slice” from confocal. Colors as in A, saturated for ease of visualization. Scale bar: 5 μm. (C) Area boxed in B; FIB-SEM image (top), Rab7 (middle), PlyA2 (bottom). Arrowheads indicate electron-dense endosomes. Scale bar: 2 μm. (D) Volumetric imaging of dividing spermatocytes, showing orthogonal imaging planes, and expected cross sections for LM and FIB-SEM. (E) Fluorescence “z slice” capturing a cleavage furrow in-plane. Scale bar: 4 μm. (F) FIB-SEM image capturing same furrow as in E cross-section, approximated by red lines. Scale bar: 2 μm. (G–I) Correlated fluorescence image, overlay, and rotated FIB-SEM section, respectively, of furrow in-plane. (J) Area boxed in I showing endosomes docked at the furrow (arrow). Scale bar: 1 μm. (J and K) Volume rendering of FIB-SEM 3D reconstruction, with furrow segmented and false colored red, undocked endosomes green, docked endosomes blue. (L) Close-up of K, with furrow segmented translucent and endosomes unsegmented. (M) Matching FIB-SEM section in the imaging plane. Arrows in K, L, M show same endosomes docked at furrow. Scale bar: 2 μm. (N) Independent CLEM/FIB-SEM experiment revealing at higher pixel sampling another example of docked vesicle. Scale bar: 1 μm. CPE, ceramide phosphoethanolamine; LM, light microscopy.

Article Snippet: Briefly, the coding sequence of PlyA2 (accession number AB777517) fused to mCherry at the C-terminus was synthesized by GenScript.

Techniques: Fluorescence, Imaging, Sampling, Light Microscopy