Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca 2+ -activated K + channels in bronchial smooth muscle cells

doi: 10.1152/ajplung.00331.2019

Figure Lengend Snippet: Large-conductance Ca2+-activated K+ channel α-subunit (BKα) and BKγ1 form molecular complex on the cell surface in mouse bronchial smooth muscle cells (mBSMCs). A: coimmunoprecipitation assay was performed using rat BSMs (rBSMs). Lysates were precipitated with anti-BKα antibody and blotted using either anti-BKα or anti-BKγ1 antibody. Similar results were obtained from 3 independent experiments. Two rats were used for each experiment. Lane 1, lysates from rBSM; lane 2, samples precipitated with control resin that cannot bind to antibodies; lane 3, samples precipitated with resin that binds to anti-BKα antibody. B: total internal reflection fluorescence (TIRF) imaging of mBSMCs, in which BKα and BKγ1 were labeled with each antibody. Fluorescent signals from particles corresponding to BKα, BKγ1, and colocalization are shown in green, red, and yellow, respectively. Merged image was overlapped with a cell image. C: ratio of BKα particles localized alone or colocalized with BKγ1 to total BKα particles in mBSMCs (BKα alone, 134 particles and colocalized, 214 particles from 15 cells).

Article Snippet: Fluorescently labeled cells were observed by using confocal (AIR, Nikon, Tokyo, Japan) or total internal reflection fluorescence (TIRF) microscopes (Nikon).

Techniques: Co-Immunoprecipitation Assay, Fluorescence, Imaging, Labeling

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Activity-dependent filopodial extension drives the increase in footprint surface area in neurosecretory cells. A, B, Bovine chromaffin (A) and PC12 (B) cells expressing GFP-GPI were examined by TIRF microscopy, imaged at 2 Hz and stimulated with Ba2+ (2 mm) or vehicle treated as indicated. Top, Time-lapse of the GFP-GPI fluorescence in the footprint. Bottom, GPI-GFP footprint area (green) with an overlay of the pretreatment area (white with red outline). Scale bars, 5 μm. Arrowheads indicate outgrowing filopodia. C, Changes in surface area over time were plotted as indicated. D, Average change in footprint area (normalized to initial area before treatment) for stimulated chromaffin and PC12 cells and vehicle-treated controls 400 s after stimulation (n = 6/10/8/5). E, Bovine chromaffin cells were treated with vehicle or 2 mm BaCl2 for 8 min and the region of the cell containing the cell footprint was analyzed by electron microscopy. Note the numerous filopodia emerging from the footprint in stimulated cells. The inset shows a region of membrane extension containing SGs (arrowheads). Proximity to the plasma membrane is confirmed by the profiles of clathrin-coated pits containing ruthenium red stain, indicative of continuity with the plasma membrane (arrows). Bottom, Outlines of the cells in the top. Left: Ratio of outline length to visible surface before and stimulation. Right: Counts of objects outside the cell surface in unstimulated and stimulated conditions (n = 6).

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Activity Assay, Expressing, Microscopy, Fluorescence, Electron Microscopy, Staining

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Vesicular fusion does not account for the entire footprint enlargement. SG granule fusion was measured in bovine chromaffin cells expressing NPY-mCherry. Cells were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation (black arrow) with Ba2+ (2 mm). A, Individual fusion events were defined as the disappearance of NPY-positive SGs, followed by a cloud-like dispersal of NPY-mCherry to the extracellular space, corresponding to a spike in overall NPY-mCherry intensity. B, SG fusion was measured in bovine chromaffin cells expressing NPY-mCherry and VAMP2-pHluorin. Cells were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). C, Validation of the NPY-mCherry assay described in A by comparison with VAMP2-pHluorin overall fluorescence intensity change. To count fusion events with VAMP2-pHluorin, we determined the average fluorescence intensity change due to alkalinization of the pHluorin moiety during a fusion event by calculating the difference in fluorescence intensity between two adjacent frames before and during a fusion event and averaging over a number of events. We then assigned this value to each individual fusion event detected using the NPY-mCherry assay (orange). Note that these results do not significantly differ from the direct VAMP2-pHluorin fluorescence measurement (green). D, Chromaffin and PC12 cells were cotransfected with NPY-mCherry and VAMP2-pHluorin and analyzed by TIRF microscopy. Arrowheads in the inset indicate vesicles where both markers colocalize. The graph depicts the percentage of VAMP2-pHluorin-positive vesicles that also contain NPY-mCherry and vice versa (n = 7). E, Chromaffin and PC12 cells cotransfected with GFP-GPI and NPY-mCherry and stimulated with Ba2+ (2 mm). The extent of footprint enlargement based on the actual GFP-GPI measurements (green) was compared with the estimated membrane increase based on the measured number of SG fusion events (NPY-mCherry assay) multiplied by the predicted membrane surface area of a 300 nm SG (orange; n = 4–6). Note that the actual footprint increase is significantly larger than that of the estimate based on fusion alone.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Expressing, Microscopy, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Footprint expansion does not depend on regulated exocytosis. NPY-mCherry was expressed in PC12 cells, DKD-PC12 cells, and DKD-PC12 cotransfected with Munc18–1-GFP. A, Cells were imaged at 2 Hz by TIRF microscopy before and for 7 min after 2 mm Ba2+ stimulation and fusion numbers were counted and normalized to footprint area. Note the rescue of exocytosis elicited by Munc18–1-GFP reexpression in DKD-PC12 cells. B, GFP-GPI was expressed in DKD-PC12 cells and DKD-PC12 cotransfected with (untagged) Munc18–1. Cells were examined by TIRF microscopy and imaged at 2 Hz before and during Ba2+ (2 mm) stimulation. Top, Time-lapse of the GFP-GPI fluorescence in the footprint. Bottom, GPI-GFP footprint area (green) with an overlay of the prestimulation area (white with red outline). Scale bars, 5 μm. C, Footprint area change for GFP-GPI-transfected DKD PC12 cells expressing empty vector or Munc18–1 and wild-type (wt)-PC12 cells after Ba2+ (2 mm) stimulation (400 s). Note that all three populations exhibit similar increases in footprint area. The numbers in the bars indicate the number of cultures analyzed.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence, Transfection, Expressing, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Interfering with actin polymerization and myosin II function reduces footprint enlargement. Bovine chromaffin (A, B) and PC12 (C, D) cells coexpressing GFP-GPI and NPY-mCherry were treated with cytochalasin D (CytoD) or blebbistatin (Blebbi) for 20 min before examination by time-lapse TIRF microscopy. Cells were then stimulated with Ba2+ and imaged at 2 Hz. A, B, Time-lapse profile of the GFP-GPI fluorescence in the footprint of chromaffin (A) or PC12 (B) cells treated as indicated. Bottom, GPI-GFP footprint area (green) with an overlay of the prestimulation area (white with red outline). Scale bars, 5 μm. B, D, Footprint area change under indicated conditions 400 s after Ba2+ stimulation or vehicle treatment for chromaffin (B) and PC12 cells (D) based on either the actual GFP-GPI measurements (black bars) or the corresponding estimated (est.) footprint increase based on fusion events (white bars). Note that for both blebbistatin and cytochalasin D treatments in chromaffin cells, the measured and estimated footprint area changes do not differ significantly. The numbers inside the bars indicate the numbers of cultures analyzed.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Newly added footprint area contains high levels of F-actin. A, PC12 cells cotransfected with Lifeact-RFP (LA-RFP; red) and GFP-GPI (green) were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). Bottom, Ratio of Lifeact-RFP:GFP-GPI fluorescence in pseudocolor to indicate changes in F-actin concentration. B, 3D surface intensity plot of Lifeact-RFP:GFP-GPI ratio before and 400 s after stimulation, highlighting the increased F-actin concentration (red) at the edge of the footprint and the decrease in F-actin in the center of the footprint. C, Quantification of Lifeact-RFP and GFP-GPI ratios using regions of interest from the center or the expanding edge of PC12 cell footprints (n = 3). After stimulation, the Lifeact-RFP:GFP-GPI ratio in the initial footprint area decreases while simultaneously increasing in the added footprint area.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: SGs move into the newly added footprint area. A, Time-lapse of PC12 cells cotransfected with NPY-mCherry(NPY-Ch) and GFP-GPI, imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). Top, NPY-mCherry-positive SGs appear outside the initial area (green dashed line) and are evenly distributed across the entire footprint (green outline). Bottom and side, x-y-t reconstruction of NPY-mCherry-positive SGs, with new SGs appearing in both the initial and the newly added area after stimulation. Note that the SG density (number of granules per unit area) remains constant over time (small inset) in all areas. B, Time-lapse TIRF microscopy of a bovine chromaffin cell cotransfected with Lifeact-GFP (LA-GFP) and NPY-mCherry showing the distribution of F-actin and SGs before and 400 s after stimulation. Right, Trajectories of SGs in the added footprint membrane area show that they are entering the newly forming area that also contains Lifeact-GFP positive structures (bottom). Scale bar, 5 μm. C,D, Maps of NPY-mCherry positive SGs trajectories in a Ba2+-stimulated chromaffin cell color coded for time after stimulation (C) and square displacement (D), as indicated. The white outline indicates the footprint area before stimulation. E, Mean square displacement (MSD) analysis of SGs in the original footprint and added areas. Note that there is no significant difference in mobility between either SG populations. F, SGs undergo regulated exocytosis in the newly added area. Time-lapse profile of a bovine chromaffin cell transfected with Lifeact-GFP (LA-GFP) and NPY-mCherry (red outline of the initial footprint area) stimulated with Ba2+ at t = 0 and imaged at 2 Hz. Arrowheads depict a SG entering an added area and undergoing fusion (pseudocolored in the inset). In the merged insets, the GFP channel is replaced with an outline of the limit of the footprint. Scale bar, 5 μm. G, Quantification of fusion events per area in both the original footprint and the added area. During footprint expansion, starting 120 s after stimulation, the number of fusions/area in the new area does not differ significantly from those in the initial footprint. Scale bar, 5 μm.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Transfection