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: Rab18 promotes lipid droplet (LD) growth by tethering the ER to LDs through SNARE and NRZ interactions

doi: 10.1083/jcb.201704184

Figure Lengend Snippet: Rab3GAP1/2 controls the activity and LD localization of Rab18. (A) Endogenous Rab18 (green) was associated with LDs (red) in 3T3-L1 preadipocytes. Bars: 10 µm; (insets) 2 µm. (B) Rab18 was enriched in LD fraction isolated from 3T3-L1 preadipocytes. ADRP, LD marker; GRP94, a microsomal marker; β-tubulin, a cytosol marker. (C) Rab18 (green) was associated with LD (blue) at early stage of LD biogenesis. Red, endogenous ACSL3. Bars: 10 µm; (insets) 2 µm. (D) Representative SIM superresolution images showing the localization of HA-Rab3GAP2 (red) and GFP-Rab18 (green) on LDs (blue). Bars: 10 µm; (insets) 1 µm. (E) Rab18 (green) was not localized to LDs (blue) in Rab3GAP1 - or Rab3GAP2 -deficient cells. Red, ACSL3. Bars: 10 µm; (insets) 2 µm. (F) Rab18 was not enriched in the LD fraction in Rab3GAP1 -deficient cells. (G and H) Reduced mature LDs and the presence of supersized LDs in Rab3GAP1/2 -deficient cells. Bars: 10 µm; (insets) 2 µm. Histogram in H showing the mean number of LDs in each diameter in G. Mean ± SD; n = 13 for control cells, n = 20 for Rab3GAP1 KO cells, n = 16 for Rab3GAP2 KO cells; ***, P < 0.001 by one-way ANOVA with Tukey post hoc tests. (I) Increased LD (green) sizes in Rab3GAP1 -deficient adipocytes. Bars: 10 µm; (insets) 5 µm. All experiments were performed at least twice.

Article Snippet: Structured illumination microscopy (SIM) images were obtained using a Nikon N-SIM superresolution microscope with SR Plan Apo 100× oil immersion objective (NA 1.49) and Andor iXon 897 EMCCD at room temperature.

Techniques: Activity Assay, Isolation, Marker