Journal: The Journal of Cell Biology

Article Title: Intracellular TRPA1 mediates Ca 2+ release from lysosomes in dorsal root ganglion neurons

doi: 10.1083/jcb.201603081

Figure Lengend Snippet: Localization of intracellular TRPA1 channels to lysosome-like organelles. (A and B) Representative images captured by an N-SIM superresolution microscope showing immunofluorescent staining for the subcellular localization of TRPA1 in WT DRG neurons. TRPA1-positive staining was prominently colocalized with labeling for the lysosome marker LAMP1 (A) but not that for the vesicle marker VAMP2 (B). For LAMP1, the curved boxes along the plasma membrane were straightened, enlarged, and are shown in the middle panels in A with arrows pointing to colocalized puncta. The rectangular boxes were enlarged and are shown in the bottom panels in A for intracellular puncta. (C) Representative immuno-electron micrographs of subcellular organelles in DRG neurons labeled with antibodies for LAMP1 (10 nm; large arrowheads) and TRPA1 (6 nm; small arrowheads). (D) Representative immuno-electron micrographs of plasma membrane localization of TRPA1 (left two panels). The TRPA1 label was absent from clear vesicles (arrow) and clathrin-coated pits (asterisk; right two panels). Images are representative of three independent cultures. Bars: (A [top] and B) 5 µm; (A, middle and bottom) 2 µm; (C and D) 200 nm.

Article Snippet: Images were captured using an N-SIM superresolution microscope system with 78-nm resolution in the x and y axes and 300 nm in the z axis (Nikon).

Techniques: Microscopy, Staining, Labeling, Marker, Clinical Proteomics, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity

doi: 10.1073/pnas.1901176116

Figure Lengend Snippet: Myosin Va teams transport intracellular cargo through networks of actin. (A) Lipid-bound cargo is produced and packaged at the interior of the cell within the Golgi. These cargos are first transported along microtubule tracks, followed by handoff to MyoVa for distribution and final delivery to sites of secretion at the cell membrane. (B) Superresolution, 3D STORM reconstruction of an in vitro actin network. Actin filaments are strung between silica beads of varying diameters, which support the network and maintain a 3D organization. Color represents z position. (Scale bar: 2 µm.) (C) Overlay of 350-nm vesicle trajectory (magenta) by teams of MyoVa within a 3D actin filament network (colored by z position). (Scale bar: 1 µm.)

Article Snippet: 3D STORM images were acquired using a Nikon N-STORM superresolution microscope system with excitation of Alexa-647 phalloidin-labeled actin by 647- and 405-nm lasers.

Techniques: Produced, In Vitro

Journal: The Journal of Cell Biology

Article Title: Visualization of long-lived proteins reveals age mosaicism within nuclei of postmitotic cells

doi: 10.1083/jcb.201809123

Figure Lengend Snippet: Mechanism of NPC turnover in nondividing quiescent cells. (A) Single pore imaging of Nup93 turnover in quiescent cells. C2C12 cells stably expressing RITE-tagged (MF) Nup93 were induced into quiescence and tag switch initiated. Cells were fixed and stained before (0 Days) and 7 and 14 d after tag switch. Cells were then imaged using structured illumination superresolution microscopy. Representative images display Myc (red) and Flag (green) signal overlaid. Scale bar represents 1 µm. (B) Quantification of Nup turnover in quiescent cells. Identical experiments were performed using Nup96 and Nup133 as described in A. Myc (Old) and Flag (New) pore numbers were then quantified at each time point and plotted. Error bars represent 95% confidence intervals. (C) MIMS imaging of long-lived NPCs. 15 N-labeled mice chased with 14 N food for 6 mo were perfused and fixed, tissues dissected and prepared for EM, and 80-nm-thick brain sections mounted on silicon wafers were imaged by scanning EM (SEM; left) and MIMS (right). Images were aligned, NPCs identified in SEM images (arrows), and corresponding MIMS signals highlighted (arrows). Yellow arrows represent NPCs with high 15 N signal, and the white arrow is an NPC with low 15 N signal. Data shown is from one mouse and is representative of three scanned neurons. (D) Nup93 turnover in Pom121 knockdowns. C2C12 cell lines were constructed that expressed both RITE-tagged (MF) Nup93 and an inducible shRNA targeting Pom121. Cells were placed into quiescence and tag switch induced with (orange bars) and without (gray bars) Pom121 knockdown induction. Cells were fixed, stained, and imaged before (0 Days) and 14 d after tag switch. Intensity of the flag signal (new) was normalized to background intensity and plotted. Error bars represent 95% confidence intervals. (E) Nup93 turnover with ESCRT-III knockdowns. C2C12 cell lines were constructed as in D, but expressing a nontargeting (luciferase) or ESCRT-III targeting inducible shRNAs (Chmp3 and Chmp2a). Turnover was quantified as described in . **, P < 0.01; ***, P < 0.001 calculated using two-tailed unpaired t tests.

Article Snippet: For superresolution imaging, experiments were performed as described above, but with cells seeded on high-performance cover glass, fixed, stained, mounted as described in RITE in U2OS above, and imaged using superresolution SIM (Elyra PS.1; Zeiss).

Techniques: Imaging, Stable Transfection, Expressing, Staining, Microscopy, Labeling, Construct, shRNA, Knockdown, Luciferase, Two Tailed Test