Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Protein-protein interaction network of LGN (GPSM2, green) showing several interactors (grey) of known and unknown mitotic functions. The green line highlights a novel interaction between LGN and ANXA1, which is further validated in this study. b Gene Ontology analysis of the LGN network presenting terms from Biological process (top) and Reactome pathway analysis (bottom) significantly enriched. P -values were derived from the hypergeometric test (two-sided Fisher’s Exact Test) with Bonferroni step-down correction from multiple tests. c ANXA1 and GFP-LGN co-immunoprecipitate from MCF-10A stably expressing GFP-LGN, arrested in metaphase. Lysates were subjected to affinity purification with GFP-Trap beads. The immunoprecipitates (IP) were analysed by western blotting (3 independent experiments). d LGN and ANXA1-mCherry co-immunoprecipitate from clonal MCF-10A cells stably expressing ANXA1-mCherry, arrested in metaphase. Lysates were subjected to affinity purification with RFP-Trap beads. The immunoprecipitates were analysed by western blotting (3 independent experiments). e Proximity ligation assay (PLA) performed using GFP and ANXA1 antibodies in GFP-LGN-expressing MCF-10A cells. Cells were counterstained with DAPI (DNA, magenta) (3 independent experiments). Yellow dotted lines indicate the cell contour. f Confocal images of representative MCF-10A cells stably expressing GFP-LGN (green) stained for ANXA1 (magenta) and counterstained with DAPI (DNA, blue) (3 independent experiments). g Average cortical fluorescence intensity profiles of GFP-LGN and ANXA1 in prometaphase cells (left) and metaphase cells (right) (3 independent experiments, prometaphase n = 33 cells; metaphase n = 36 cells). Data are presented as mean ± s.e.m. Positions along the cortex in the graphs correspond to coordinates as illustrated in the schematic. arb. units (arbitrary units). All scale bars, 10 µm. Source data are provided as a Source Data file.

Article Snippet: To clone pTK-ANXA1-mCherry, the ANXA1-mCherry sequence was synthesised (Eurofins) and inserted into pTK93-Lifeact-mCherry vector (Addgene, Plasmid #46357) where Lifeact-mCherry was removed by restriction digestion with NaeI and ApaI (New England Biolabs). pTK-mCherry was cloned as follows: The full-length mCherry sequence was amplified by PCR and inserted into pTK93-Lifeact-mCherry vector where the Lifeact-mCherry sequence was removed by restriction digestion with BamHI and SalI (New England Biolabs).

Techniques: Derivative Assay, Stable Transfection, Expressing, Affinity Purification, Western Blot, Proximity Ligation Assay, Staining, Fluorescence

Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Left: time-lapse images of representative MCF-10A cells stably expressing GFP-LGN (green), treated with si-Control or si-ANXA1#1. DNA is stained with Hoechst 33342 (magenta) 30 min before imaging. Right: illustration showing the observed cortical localisation of GFP-LGN. b Western blotting of extracts from siRNA-transfected cells. c Percentage of cortical localisation of GFP-LGN in siRNA-transfected cells (si-Control: n = 53 cells; si-ANXA1#1: n = 42 cells). Two-sided t -test, bilateral: **** P < 0.000001; unilateral: * P = 0.0475; central: **** P = 0.000004; circumferential: * P = 0.0233. d Confocal images of representative si-Control-, si-ANXA1#1- and si-ANXA1#2-transfected metaphase cells expressing or not ANXA1-mCherry and stained for LGN, NuMA or p150 Glued (green) and counterstained with DAPI (DNA, magenta). e Western blotting of extracts from siRNA-transfected cells expressing or not ANXA1-mCherry. f Percentage of cortical localisation of LGN, NuMA or p150 Glued in siRNA-transfected metaphase cells expressing or not ANXA1-mCherry: LGN (si-Control: n = 70 cells; si-ANXA1#1: n = 51 cells; si-ANXA1#2: n = 43 cells; si-Control + ANXA1-mCherry: n = 69 cells; si-ANXA1#2 + ANXA1-mCherry: n = 47 cells), NuMA (si-Control: n = 73; si-ANXA1#1: n = 62; si-ANXA1#2: n = 45; si-Control + ANXA1-mCherry: n = 41 cells; si-ANXA1#2 + ANXA1-mCherry: n = 50 cells), p150 Glued (si-Control n = 79; si-ANXA1#1 n = 101; si-ANXA1#2 n = 99; si-Control + ANXA1-mCherry: n = 33 cells; si-ANXA1#2 + ANXA1-mCherry: n = 34 cells). Two-way ANOVA with Dunnett’s multiple comparisons test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. g Confocal images of representative si-Control-, si-ANXA1#1- and si-ANXA1#2-treated metaphase cells stained for G αi1 (green) and counterstained with DAPI (DNA, magenta). h Average cortical fluorescence intensity profiles of G αi1 in siRNA-transfected metaphase cells (si-Control: n = 32; si-ANXA1#1: n = 30; si-ANXA1#2: n = 30). All data are presented as mean ± s.e.m. from 3 independent experiments. arb. units (arbitrary units). All scale bars, 10 µm. Source data are provided as a Source Data file.

Article Snippet: To clone pTK-ANXA1-mCherry, the ANXA1-mCherry sequence was synthesised (Eurofins) and inserted into pTK93-Lifeact-mCherry vector (Addgene, Plasmid #46357) where Lifeact-mCherry was removed by restriction digestion with NaeI and ApaI (New England Biolabs). pTK-mCherry was cloned as follows: The full-length mCherry sequence was amplified by PCR and inserted into pTK93-Lifeact-mCherry vector where the Lifeact-mCherry sequence was removed by restriction digestion with BamHI and SalI (New England Biolabs).

Techniques: Stable Transfection, Expressing, Staining, Imaging, Western Blot, Transfection, Fluorescence

Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Confocal images of representative MCF-10A cells treated with DMSO (Control) or 20 µM TG100-115 for 2 h, stained for ANXA1 (magenta) and S100A11 (green), and counterstained with DAPI (DNA, blue). b Average cortical and cytoplasmic fluorescence intensity profiles of ANXA1 and S100A11 from metaphase cells (Control: n = 30; TG100-115: n = 30). c Confocal images of representative MCF-10A cells treated with DMSO (Control) or 20 µM TG100-115 for 2 h, stained for LGN, NuMA or p150 Glued (green), and counterstained with DAPI (DNA, magenta). d Percentage of cortical localisation of LGN, NuMA or p150 Glued in Control and TG100-115-treated metaphase cells: LGN (Control: n = 39 cells; TG100-115: n = 37), NuMA (Control: n = 43 cells; TG100-115: n = 40), p150 Glued (Control: n = 31 cells; TG100-115: n = 32). Two-sided t test, LGN (bilateral: **** P = 0.00005; unilateral: *** P = 0.0008; central: ** P = 0.006; circumferential: ** P = 0.01); NuMA (bilateral: *** P = 0.001; unilateral: * P = 0.044; central: ** P = 0.005; circumferential: **** P = 0.00009; absent: P = 0.405); p150 Glued (bilateral: *** P = 0.0002; unilateral: P = 0.728; absent: ** P = 0.005). All data are presented as mean ± s.e.m. from 3 independent experiments. arb. units (arbitrary units). All scale bar, 10 µm. Source data are provided as a Source Data file.

Article Snippet: To clone pTK-ANXA1-mCherry, the ANXA1-mCherry sequence was synthesised (Eurofins) and inserted into pTK93-Lifeact-mCherry vector (Addgene, Plasmid #46357) where Lifeact-mCherry was removed by restriction digestion with NaeI and ApaI (New England Biolabs). pTK-mCherry was cloned as follows: The full-length mCherry sequence was amplified by PCR and inserted into pTK93-Lifeact-mCherry vector where the Lifeact-mCherry sequence was removed by restriction digestion with BamHI and SalI (New England Biolabs).

Techniques: Staining, Fluorescence

Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Time-lapse images of representative MCF-10A cells treated with si-Control or si-ANXA1#1. Microtubules are labelled with SiR-tubulin (green) and DNA with Hoechst 33342 (magenta), 2 h and 30 min before acquisition, respectively. Arrowheads indicate the phenotypes listed in ( e ). Scale bars, 5 µm. b Percentage of cells with abnormal mitosis (si-Control: n = 33; si-ANXA1#1: n = 44). Two-sided t -test, **** P < 0.0001. c Percentage of cells that completed mitosis (si-Control: n = 33; si-ANXA1#1: n = 44). Two-sided t -test, ** P = 0.0095. d Time from nuclear envelope breakdown (NEBD) to anaphase onset in siRNA-transfected cells (si-Control: n = 33; si-ANXA1#1: n = 25). Two-sided t -test, ** P = 0.006. e Percentage of chromosomes segregation and spindle assembly defects in siRNA-transfected cells (si-Control: n = 33; si-ANXA1#1: n = 44). Two-sided t test, normal segregation: **** P = 0.000033; chromosome bridges: ** P = 0.0055; abnormal bipolar spindle: ** P = 0.0043; multipolar spindle: * P = 0.032. f Dynamics of z orientation (α z ) during metaphase in siRNA-transfected cells (si-Control: n = 33; si-ANXA1#1: n = 39). Black lines represent the average spindle angles. g Oscillation index in siRNA-transfected cells, in the z-axis (si-Control: n = 33; si-ANXA1#1: n = 41). Two-sided t -test, * P = 0.0125. h Percentage of cells with spindle oscillations in the z axis (si-Control: n = 33; si-ANXA1#1: n = 44). Two-sided t -test, **** P < 0.0001. i Dynamics of xy orientation (α xy ) during metaphase in siRNA-transfected cells (si-Control: n = 33; si-ANXA1#1: n = 39). Black lines represent the average spindle angles. j Oscillation index in siRNA-transfected cells, in the xy axis (si-Control: n = 33; si-ANXA1#1: n = 41). Two-sided t -test, * P = 0.0153. k Percentage of cells with spindle oscillations in the xy axis (si-Control: n = 33; si-ANXA1#1: n = 44). Two-sided t test, * P = 0.0105. l Origin-aligned metaphase plate tracks in the xy plane of si-Control- (left) and si-ANXA1#1-transfected (right) cells (si-Control: n = 33; si-ANXA1#1: n = 43). m Displaceme n t length from the starting point to the position at the end of metaphase in siRNA-transfected cells (si-Control: n = 31; si-ANXA1#1: n = 30). Two-sided t -test, * P = 0.037. n Velocity of metaphase plate movements in si-RNA-transfected, calculated by dividing the track length by metaphase duration (si-Control: n = 31; si-ANXA1#1: n = 44). Two-sided t -test, P = 0.384. All data are presented as mean ± s.e.m. from 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: To clone pTK-ANXA1-mCherry, the ANXA1-mCherry sequence was synthesised (Eurofins) and inserted into pTK93-Lifeact-mCherry vector (Addgene, Plasmid #46357) where Lifeact-mCherry was removed by restriction digestion with NaeI and ApaI (New England Biolabs). pTK-mCherry was cloned as follows: The full-length mCherry sequence was amplified by PCR and inserted into pTK93-Lifeact-mCherry vector where the Lifeact-mCherry sequence was removed by restriction digestion with BamHI and SalI (New England Biolabs).

Techniques: Transfection

Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Confocal images of representative metaphase MCF-10A cells transfected with si-Control, si-ANXA1#1 or si-ANXA1#2 stained for α-tubulin (grey) and counterstained with DAPI (DNA, magenta). White arrowheads indicate elongated and buckled astral microtubules. Dashed lines outline the cell contour. b Confocal images of representative metaphase MCF-10A cells stably expressing EB3-GFP and transfected with si-Control, si-ANXA1#1 or si-ANXA1#2. Dashed lines outline the cell contour. c Ratio of astral microtubule and EB3-GFP comets length ( I ) in siRNA-transfected cells: astral microtubules (si-Control: n = 31; si-ANXA1#1: n = 43; si-ANXA1#2: n = 45); EB3-GFP comets (si-Control: n = 30; si-ANXA1#1: n = 34; si-ANXA1#2: n = 34). I 1 and l 2 of astral microtubules were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, astral microtubules: ** P = 0.007 and * P = 0.035; EB3-GFP: *** P = 0.001 and *** P = 0.0009. d Relative fluorescence intensities of astral microtubules ( I astral, rel ) in siRNA-transfected cells (si-Control: n = 31; si-ANXA1#1: n = 47; si-ANXA1#2: n = 47). Fluorescence intensities on the spindle ( I spindle ) and total cell ( I total ) were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, P = 0.467 and P = 0.676. e Ratio of pole-to-cortex distance ( d ) in siRNA-transfected cells (si-Control: n = 40; si-ANXA1#1: n = 48; si-ANXA1#2: n = 52). Pole-to-cortex distance d 1 and d 2 were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, ** P = 0.004 and * P = 0.032. f Confocal images of representative metaphase MCF-10A cells transfected with si-Control, si-ANXA1#1 or si-ANXA1#2 stained for F-actin (grey) and counterstained with DAPI (DNA, magenta). g Cortical to cytoplasmic ratio of actin fluorescence intensities ( I cortex / I cytoplasm ) in siRNA-transfected cells (si-Control: n = 30; si-ANXA1#1: n = 30; si-ANXA1#2 n = 30). Fluorescence intensities in the cytoplasm ( I cytoplasm ) and total cell ( I total ) were measured as depicted on the illustration. One-way ANOVA with Tukey’s test, *** P = 0.001 and *** P = 0.0003. h Confocal images of representative MCF-10A cells treated with DMSO (Control) or 1 µM Latrunculin A for 30 min, stained for F-actin (grey) and counterstained with DAPI (DNA, magenta). i Confocal images of representative MCF-10A cells treated with DMSO (Control) or 1 µM Latrunculin A for 30 min, stained for ANXA1, LGN, NuMA or p150 Glued (grey), and counterstained with DAPI (DNA, magenta). j Average cortical and cytoplasmic fluorescence intensity profiles of ANXA1, LGN, NuMA and p150 Glued from metaphase cells (Control: n = 30; Latrunculin A: n = 30). Asterisks indicate the spindle poles. All data are presented as mean ± s.e.m. from 3 independent experiments. n.s. (not significant). arb. units (arbitrary units). All scale bars, 5 µm. Source data are provided as a Source Data file.

Article Snippet: To clone pTK-ANXA1-mCherry, the ANXA1-mCherry sequence was synthesised (Eurofins) and inserted into pTK93-Lifeact-mCherry vector (Addgene, Plasmid #46357) where Lifeact-mCherry was removed by restriction digestion with NaeI and ApaI (New England Biolabs). pTK-mCherry was cloned as follows: The full-length mCherry sequence was amplified by PCR and inserted into pTK93-Lifeact-mCherry vector where the Lifeact-mCherry sequence was removed by restriction digestion with BamHI and SalI (New England Biolabs).

Techniques: Transfection, Staining, Stable Transfection, Expressing, Fluorescence

Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Confocal images of representative mouse mammary gland cryosections (50 µm-thick) stained for ANXA1 (green), K14 (grey) and E-cadherin (magenta). b Confocal images of representative mMEC acini stained for F-actin (green) and α-tubulin (grey) or Par6 (green) and counterstained with DAPI (DNA, magenta). c Spindle angle frequencies and mean angles ( m α) (3 independent experiments, 48 h: n = 42 acini; 72 h: n = 40 acini; 96 h: n = 39 acini). d Confocal images of representative acini stained for ANXA1 (green) and counterstained with DAPI (DNA, magenta). e Western blotting of extracts from shRNA-transduced acini. f Confocal images of representative shRNA-transduced acini (Venus, yellow) stained for F-actin (green) and α-tubulin (grey) and counterstained with DAPI (DNA, magenta). g Spindle angle frequencies and mean angles (mα) (3 independent experiments, sh-Control: n = 30 acini; sh-ANXA1#1: n = 30 acini; sh-ANXA1#2: n = 30 acini). Kolmogorov-Smirnov test, ** P < 0.01. h Confocal images of representative shRNA-transduced acini (Venus, yellow) stained for LGN (green) and counterstained with DAPI (DNA, magenta). i Confocal images of representative shRNA-transduced acini (Venus, yellow) stained for F-actin (green) and E-cadherin (grey) and counterstained with DAPI (DNA, magenta). j Percentage of acini with normal lumen (left) (4 independent experiments, sh-Control: n = 163 acini; sh-ANXA1#1: n = 142 acini; sh-ANXA1#2: n = 168 acini), acinus area (middle) and acinus roundness (right) (3 independent experiments, sh-Control: n = 30 acini; sh-ANXA1#1: n = 30 acini; sh-ANXA1#2: n = 30 acini). One-way ANOVA with Tukey’s test, left: *** P = 0.0008 and *** P = 0.0005; middle: P = 0.201 and * P = 0.067; right: ** P = 0.007 0.01 and ** P = 0.002. k Number of lumen per acinus (left), lumen area (middle) and lumen circularity (right) (3 independent experiments, sh-Control: n = 36; sh-ANXA1#1: n = 32; sh-ANXA1#2: n = 31). O n e-way ANOVA with Tukey’s test, left: * *** P = 0.00009 and **** P = 0.00002; middle: **** P = 0.0001 and **** P = 0.00007; right: **** P = 0.0001 and **** P = 0.00006. l Left: illustration showing quantification of cortical fluorescence intensity of F-actin (green) and E-cadherin (grey) in acini. Right: average cortical fluorescence intensity profiles of E-cadherin and F-actin (3 independent experiments, sh-Control: n = 30; sh-ANXA1#1: n = 30; sh-ANXA1#2: n = 30). All time-points of 3D culture are indicated in hours (h). All data are presented as mean ± s.e.m. n.s. (not significant). arb. units (arbitrary units). All scale bars, 10 µm. Source data are provided as a Source Data file.

Article Snippet: To clone pTK-ANXA1-mCherry, the ANXA1-mCherry sequence was synthesised (Eurofins) and inserted into pTK93-Lifeact-mCherry vector (Addgene, Plasmid #46357) where Lifeact-mCherry was removed by restriction digestion with NaeI and ApaI (New England Biolabs). pTK-mCherry was cloned as follows: The full-length mCherry sequence was amplified by PCR and inserted into pTK93-Lifeact-mCherry vector where the Lifeact-mCherry sequence was removed by restriction digestion with BamHI and SalI (New England Biolabs).

Techniques: Staining, Western Blot, shRNA, Fluorescence

Journal: Nature Communications

Article Title: Annexin A1 is a polarity cue that directs mitotic spindle orientation during mammalian epithelial morphogenesis

doi: 10.1038/s41467-023-35881-x

Figure Lengend Snippet: a Cortical ANXA1 interacts with LGN to instruct polarised accumulation of the LGN-NuMA complex at the lateral cortex. NuMA in turn, recruits Dynein-Dynactin generating pulling forces on the spindle poles that are balanced by ANXA1-dependent regulation of cortical F-actin and astral microtubule organisation, thereby ensuring planar alignment of the mitotic spindle. During 3D morphogenesis, ANXA1-dependent regulation of planar mitotic spindle orientation is crucial for single lumen formation and normal epithelial architecture. b Loss of ANXA1 results in a diffuse cortical distribution of LGN and NuMA, which in turn impairs the recruitment of Dynein-Dynactin to the cell cortex. This affects the length and dynamic instability of astral microtubules as well as cortical F-actin integrity, generating unbalanced forces that result in randomised mitotic spindle orientation. During 3D morphogenesis, mitotic spindle misorientation upon ANXA1 knockdown results in epithelial multi-layering, leading to multi-lumen formation and aberrant epithelial architecture.

Article Snippet: To clone pTK-ANXA1-mCherry, the ANXA1-mCherry sequence was synthesised (Eurofins) and inserted into pTK93-Lifeact-mCherry vector (Addgene, Plasmid #46357) where Lifeact-mCherry was removed by restriction digestion with NaeI and ApaI (New England Biolabs). pTK-mCherry was cloned as follows: The full-length mCherry sequence was amplified by PCR and inserted into pTK93-Lifeact-mCherry vector where the Lifeact-mCherry sequence was removed by restriction digestion with BamHI and SalI (New England Biolabs).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Nogo-B Receptor Directs Mitochondria-Associated Membranes to Regulate Vascular Smooth Muscle Cell Proliferation

doi: 10.3390/ijms20092319

Figure Lengend Snippet: NgBR KD cells show disrupted ER-mitochondria interaction. shControl- and shNgBR-transfected cells were harvested at 48 h post transfection. MAM formation was analyzed by evaluating the expression of MAM-related proteins in ( A ) total cell lysates (FACL-4 and IP 3 R3) and ( B ) crude mitochondrial extracts (FACL-4 and VDAC1); β-actin and COX IV were utilized as the loading controls, respectively. n = 3. ( C ) Cells were incubated with plasmid expressing mCherry fused with a targeting sequence of subunit IV of COX prior to shRNA transfection. PDI was used as an ER marker. Nuclei were stained with Hoechst (blue). Pearson’s correlation coefficient was calculated to measure colocalization of ER and mitochondria on mCherry positive cells. n > 60 cells/group. Scale bar = 10 μm. ( D ) Transmission electron microscopy of negative control (NC; left) and NgBR knockdown KD (right) VSMCs. Arrowheads indicate the MAMs. The minimum ER-mitochondrial distance was quantified. n > 80 ER-mitochondria contact points/group. Scale bar = 500 nm. * p < 0.05, ** p < 0.01.

Article Snippet: For analysis of the MAM formation [ ], cells were transfected with plasmids expressing mCherry fused with a targeting sequence of subunit IV of COX (from GeneChem).

Techniques: Transfection, Expressing, Incubation, Plasmid Preparation, Sequencing, shRNA, Marker, Staining, Transmission Assay, Electron Microscopy, Negative Control, Knockdown

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