Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 1. Endosomes and lysosomes form giant structures in the periphery of the oocyte plasma membrane. (A) Metaphase II (MII) oocytes were fixed and co-stained with anti-RAB5 and anti-LAMP1 antibodies. The schematic diagram indicates the location of oocyte chromosomes and metaphase plate in the MII oocytes. Arrowheads indicate the positions of oocyte chromosomes. Magnified regions (right) are indicated by yellow boxes. (B) Embryos at

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 2. The giant structures are enlarged in the periphery of the metaphase II (MII) oocyte plasma membrane (PM) during oocyte maturation. Germinal vesicle (GV) oocyte, MII oocyte, early 2-cell (E2C: 24 hr post-fertilization [hpf]), and late 2-cell (L2C: 40 hpf) embryos were fixed and stained with anti-LAMP1 antibody. Reconstituted three-dimensional objects for the LAMP1-positive organelles in 9–10 oocytes for each stage from more than three independent experiments were further analyzed for number, size, and distribution. (A) Averaged total number of LAMP1-positive objects per

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 3. Endosomal-lysosomal organelles form assembly structures, ELYSA. (A) The internal structure of the giant structure in metaphase II (MII) oocytes was analyzed by correlative light and electron microscopy (CLEM) analysis using anti-RAB7 and anti-LAMP1 antibodies. A merged image of immunostaining and electron microscopy (EM) is indicated (left), and a toluidine blue staining image of the adjacent thin section (right) is indicated. Black arrowheads indicate the giant structures positive for RAB7/LAMP1/toluidine blue staining. Note that RAB7/LAMP1 staining is indicated in a

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Electron Microscopy, Immunostaining, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 4. An autophagy regulator LC3 is detected in endosomal-lysosomal organellar assembly (ELYSA). Germinal vesicle (GV) (A) and metaphase II (MII) (B) oocytes were fixed and co-stained with anti-LC3 and anti-LAMP1 antibodies. Magnified regions (right) are indicated by yellow boxes. DNA was stained with Hoechst 33342. Arrowheads indicate the positions of oocyte chromosomes. Maximum intensity projection of confocal images at an axial scan range of 80 µm is shown as Z-projection images.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 5. Enlargement of endosomal-lysosomal organellar assembly (ELYSA) and its redistribution to the cell periphery occur in an actin cytoskeleton- dependent manner. Germinal vesicle (GV) oocytes were incubated for in vitro maturation (IVM) for 18 hr with or without actin polymerization inhibitors (10 μM latrunculin A or cytochalasin B) or a tubulin inhibitor (20 μM nocodazole), then fixed and co-stained with anti-LC3 and anti-LAMP1 antibodies. The reconstituted objects for LAMP1-positive organelles in 10 oocytes for each treatment from more than three independent experiments were

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Incubation, In Vitro, Staining

Journal: eLife

Article Title: Endosomal-lysosomal organellar assembly (ELYSA) structures coordinate lysosomal degradation systems through mammalian oocyte-to-embryo transition

doi: 10.7554/elife.99358

Figure Lengend Snippet: Figure 6. Assembly of endosomal-lysosomal organellar assembly (ELYSA) during migration and limited interaction with the acidification machinery. (A) Snapshots of a time-lapse observation for EGFP fluorescence of germinal vesicle (GV) oocytes injected with Lamp1-EGFP and V1A-mCherry mRNA are indicated. Maximum intensity projection of confocal images with a height of 40 μm (bottom half of the oocyte) are shown. Magnified perinuclear regions (right) are indicated by yellow boxes. Arrowheads indicate assemblies that adhere to each other in the subsequent frame. (B) Maximum intensity projection of deconvolved confocal images at an axial scan range of 80 µm is shown as Z-projection images. Magnified regions are indicated by yellow boxes (a, b) and the LAMP1-EGFP organelle regions are indicated by yellow dotted line.

Article Snippet: The following primary antibodies were used: rabbit monoclonal anti- RAB5 (C8B1; 3547; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti- RAB7 (D95F2; 9367; Cell Signaling Technology), rat monoclonal anti- mouse LAMP1 (1D4B; sc- 19992; Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti- rat LC3 (Clones 4E12; M152- 3; Medical and Biological Laboratories, Tokyo, Japan).

Techniques: Migration, Fluorescence, Injection

Journal: bioRxiv

Article Title: Depletion of endomembrane reservoirs drives phagocytic appetite exhaustion in macrophages

doi: 10.1101/2024.07.31.605905

Figure Lengend Snippet: A. Reconstructed micrographs of LAMP1 staining (grayscale) in resting and macrophages that engulfed filamentous Legionella (red). Phagosome-associated LAMP1 was defined as LAMP1 that colocalized onto and to the periphery of Legionella . Dashed lines contours the cell. Free lysosomes were interpreted as LAMP1 puncta not proximal to phagosomes. Scale bar = 10 µm. B. Quantification of LAMP1-puncta per cell as described in A. C, H. Macrophages were given no particles (Mock phagocytosis, lanes 1-4) or allowed to engulf BSA-anti-BSA-opsonized magnetic beads (Magnetic beads, lanes 5-8) for 2 h, followed by 1 h incubation to elicit maturation. Phagosomes were then magnetically isolated, washed with PBS, and analyzed with Western Blot. LAMP1 (C) and VAMP3 (H) abundance were probed as a proxy for lysosome and endosome content, respectively. GAPDH was used as a loading control in whole cell lysates (WCL). Post-magnetic lysate (PML) is content remaining in lysate after magnetic separation; Wash fractions (W); Magnetic phagosome fraction is represented by M. D, I : Quantification of percent LAMP1 (D) and VAMP3 (I) remaining in lysate after magnetic separation. Percent remaining protein is defined as the percent ratio of GAPDH-normalized protein content in the PML fraction (lanes 2 and 6 for mock phagocytosis and magnetic beads, respectively) to GAPDH-normalized protein content in the WCL fraction (lanes 1 and 5 for mock phagocytosis and magnetic beads, respectively). E, J : Analysis in C and H, respectively, but normalized to mock phagocytosis to account for experimental variation in absolute values. Data represented as mean ± STD of N=4 independent experiments. Sample means were compared using two-tailed, paired Student’s t-test. *: p<0.05; **: p<0.01 (D, I) or one sample t and Wilcoxon test (E, J). F. Macrophages were transfected to express VAMP3-GFP (magenta) and then were given no particles (resting) or allowed to engulf IgG-opsonized beads for 2 h. Cytosol was demarcated with CFSE staining (green). Phagosome-associated VAMP3 was defined as VAMP3 that colocalized to the phagosome periphery as defined by black void in CFSE resulting from the bead contour. Free endosomes were interpreted as small, VAMP3 puncta not proximal to phagosomes. Scale bar = 10 µm. G. Quantification of VAMP3-puncta per cell as described in E. Data represented as mean ± SEM of N=3 independent experiments using 24-40 individual cells per condition per replicate. Data was analysed two-tailed, paired Student’s t-test.

Article Snippet: Afterwards, cells were incubated with a 1:100 dilution of rat anti-mouse LAMP1 antibodies (clone 1D4B, Developmental Studies Hybridoma Bank) for 1 h at 37 °C, washed three times with PBS, followed by incubation for 1 h at room temperature with 1:1000 fluorescently tagged secondary antibodies, and subjected to another round of washes with PBS.

Techniques: Staining, Magnetic Beads, Incubation, Isolation, Western Blot, Control, Two Tailed Test, Transfection