Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: cAMP increases Na+/K+-ATPase activity, protein abundance at the plasma membrane and the distance traveled by the Na+/K+-ATPase-containing vesicles in A549-GFPα1 cells. (A) A549-GFPα1 cells were incubated in the absence (CT) or presence of 50 μM forskolin (FSK) for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three experiments. (B) A549-GFPα1 cells were incubated as in A, and the Na+/K+-ATPase abundance at the basolateral plasma membrane was determined by western blot of the BLM fraction using a specific antibody against GFP. E-cadherin was used as a loading control. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (C) The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Vesicles were randomly selected from those that showed plus-end-directed displacement. Left panel shows a representative image of A549-GFPα1 cells. Arrowhead indicates the vesicle whose trajectory is shown in the right panel before (CT) and after FSK treatment (FSK). (D) Average contour length traveled by the vesicles as a function of time. The black line represents control vesicles; at 60 seconds, upon addition of FSK (red line), the vesicles move at a faster rate. The average contour length is determined by averaging over many trajectories as described in the Materials and Methods. **P<0.01; ***P<0.001. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Activity Assay, Incubation, Western Blot, Labeling, Single-particle Tracking, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The three isoforms of myosin-V are expressed in A549 cells. (A) RT-PCR using mRNA obtained from A549 and HeLa cells. Primers used for the amplification are described in supplementary material Table S6. (B) Cell lysates from A549 and HeLa cells were obtained and analyzed by western blot with specific antibodies against the three myosin-V isoforms. A representative western blot is shown. (C) The particulate fraction (100,000 g pellet) of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. Rab5 and Rab7 are used as markers of early and late endosomes, respectively. (D) Gradients obtained in C were scanned and the marker content was digitally quantified as indicated. Results are expressed as percentage of the total amount of protein.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Marker

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and myosin-Vc colocalize with Na+/K+-ATPase. (A) A549-GFPα1 cells were incubated in the absence or presence of 50 μM FSK for 10 minutes, basolateral membranes (BLM) and intracellular compartments (IC) were isolated and the Na+/K+-ATPase abundance was determined by western blot using a specific antibody against GFP. E-cadherin and actin were used as loading controls for the BLM and IC fractions, respectively. Graph represents mean ± s.e.m. of three experiments. A representative western blot is shown. (B) The IC fraction of A549-GFPα1 cells was loaded onto a flotation sucrose gradient and eight fractions were recovered. The distribution of the proteins of interest was analyzed by western blotting with specific antibodies. A representative western blot is shown. C+, positive control.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Incubation, Isolation, Western Blot, Positive Control

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a myosin-Va stalk-tail. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Va that has a m-cherry-tag (red) (m-cherry-DN-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (B) Average contour length traveled by the vesicles in A as a function of time. The black line represents the control vesicles; FSK was added at time 60 seconds and is represented as a red line. (C) Live imaging of A549-GFPα1 cells (green) transiently transfected with a dominant-negative myosin-Vc that has a m-cherry-tag (red) (m-cherry-DN-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of one vesicle before (CT) and after forskolin treatment (FSK). (D) Average contour length traveled by the vesicles in C as a function of time. The black line represents control vesicles; FSK was added at 60 seconds and is represented as the red line. Scale bars: 10 μm and 2 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, Imaging, Transfection, Dominant Negative Mutation, Labeling

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: The average speed of Na+/K+-ATPase-containing vesicles moving towards the cell periphery is increased in cells expressing a shRNA against myosin-Va. (A) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Va that has a m-cherry-tag (red) (m-cherry-sh-Va). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions. (B) Live imaging of A549-GFPα1 cells (green) transiently transfected with a shRNA against myosin-Vc that has a m-cherry-tag (red) (m-cherry-shRNA-Vc). The movement of the GFP-labeled particles was recorded. Upper panels show a representative image of the transfected A549-GFPα1 cells. Lower panels show the tracking of the movement of two vesicles (arrowheads) under control (CT) conditions (C). Graph represents the average contour length traveled by the vesicles as a function of time, calculated as described in methods. The black line represents the m-cherry-sh-Va vesicles and the red line, the m-cherry-sh-Vc vesicles. (D) A549-GFPα1 cells were transfected with a shRNA against myosin-Va or myosin-Vc, cell lysates were isolated and the myosin-Va (left panel) or myosin-Vc (right panel) abundance was determined by western blot using specific antibodies. E-cadherin and tubulin were used as loading controls. Scale bars: 10 μm and 4 μm (magnified images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Expressing, shRNA, Imaging, Transfection, Labeling, Isolation, Western Blot

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Dominant-negative myosin-Va mimics cAMP-mediated Na+/K+-ATPase increased activity and recruitment to the plasma membrane in A549-GFPα1 cells. (A) Stable clones expressing myosin-Va tail (DN-Va) and myosin-Vc tail (DN-Vc) were generated as described. Expression of the constructs in the permanent clones was analyzed by western blotting using and antibody against the V5 tag. A representative western blot is shown. (B) A549-GFPα1 cells (CT) and A549-GFPα1 cells permanently transfected with DN-Va and DN-Vc were incubated in the absence or presence of 50 μM FSK for 10 minutes and the Na+/K+-ATPase activity was measured as 86Rb+ uptake. Graph represents mean ± s.e.m. of three different experiments. (C) Control (CT), DN-Va and DN-Vc cells were incubated in the absence or presence of 50 μM FSK for 10 minutes and western blots of the basolateral membrane fraction were performed using a specific antibody against GFP. E-cadherin was used as loading control. A representative western blot is shown. *P<0.05; **P<0.01; n.s., not significant; u.s., unstimulated.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Dominant Negative Mutation, Activity Assay, Clone Assay, Expressing, Generated, Construct, Western Blot, Transfection, Incubation

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Myosin-Va and the Na+/K+-ATPase-containing vesicles colocalize. A549-GFPα1 cells were fixed, permeabilized and blocked. Myosin-Va was visualized by using an anti-myosin-Va antibody and a secondary antibody labeled with Alexa Fluor 568. GFP was directly visualized. Cellular distribution of Na+/K+-ATPase-GFPα1 and myosin-Va was analyzed using a Zeiss LSM 510 laser-scanning confocal microscope and colocalization (blue) was determined using the LSM 510 Meta software.

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Labeling, Microscopy, Software

Journal: Journal of Cell Science

Article Title: Myosin-Va restrains the trafficking of Na + /K + -ATPase-containing vesicles in alveolar epithelial cells

doi: 10.1242/jcs.046953

Figure Lengend Snippet: Microtubules and actin filaments are involved in Na+/K+-ATPase traffic. (A) Live imaging of A549 cells incubated with 10 μM nocodazole for 3 hours. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the microtubule cytoskeleton in control (left) and nocodazole (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and nocodazole (right) conditions. (B) Live imaging of A549 cells incubated with 5 μM cytochalasin D (Cyto D) for 1 hour. The movement of the GFP-labeled particles was recorded as Metamorph stacks and vesicle trajectories were obtained by single-particle tracking using Metamorph software. Upper panels show a representative immunofluorescence of the actin cytoskeleton under control (left) and cytochalasin D (right) conditions. Lower panel shows the tracking of the movement of one vesicle in control (left) and cytochalasin D (right) conditions. (C) Average contour length traveled by the vesicles as a function of time. The blue line represents the control vesicles; the black line, cells treated with cytochalasin D and the red line, cells treated with nocodazole. Scale bars: 10 μm and 2 μm (inset images).

Article Snippet: Cell culture Human A549 (ATCC CCL 185) and HeLa cells (ATCC CCL 2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mM HEPES.

Techniques: Imaging, Incubation, Labeling, Single-particle Tracking, Software, Immunofluorescence

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet:

Article Snippet: A20 and A20 D1.3 B cells (3 × 10 6 cells) were transiently transfected using the AMAXA nucleofector kit V (Lonza, #VCA-1003) or the Ingenio electroporation kit (Mirus, #MIR 50117) with 1.5 μg of either control siRNA (ON-TARGETplus Non-Targeting Pool, Dharmacon, #D-001810-01-05) or Actr3 siRNA (SMARTpool ON-TARGETplus, Dharmacon, #L-046642-01-0005), or with 2 μg of plasmid DNA encoding either F-tractin-GFP ( ) or myosin IIA-GFP (Addgene, #38297) ( ).

Techniques: Sequencing, Immunofluorescence, Western Blot, Single-particle Tracking, Flow Cytometry, Labeling, Cell Isolation, Electroporation, Recombinant, Software

Journal: eLife

Article Title: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation

doi: 10.7554/eLife.44574

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit anti-pERK , Cell Signaling Technologies , #9101 , Western blot 1:1000.

Techniques: Sequencing, Immunofluorescence, Western Blot, Single-particle Tracking, Flow Cytometry, Labeling, Cell Isolation, Electroporation, Recombinant, Software

Journal: Bioengineering & Translational Medicine

Article Title: Fibrin‐specific poly(N‐isopropylacrylamide) nanogels for targeted delivery of tissue‐type plasminogen activator to treat thrombotic complications are well tolerated in vivo

doi: 10.1002/btm2.10277

Figure Lengend Snippet: Overview of t issue‐type plasminogen activator‐fibrin‐specific nanogels (tPA‐FSN) particle design and characterization. (a) Core‐shell nanogel composition and schematic of tissue‐type plasminogen activator (tPA) loading into FSNs. (b) Representative atomic force microscopy (AFM) images of core and core‐shell nanogels. (c) Representative height traces of single particles (depicted with a red line through the center) from AFM images of core and core‐shell nanogels. (d) Particle size distribution of hydrodynamic diameter measurements from the core and core‐shell nanogels utilizing NanoSight particle tracking analysis software. At least 10 8 core and core‐shell nanogels were tracked. (e) Fibrin binding assay of FSNs, tPA‐FSNs, control sheep immunoglobulin G (CS‐IgG) particles, and tPA‐CS‐IgG particles at 0, 0.001, 0.01, 0.05, 0.1, and 0.2 mg/ml concentrations on fibrin‐coated wells or negative control 2% powdered milk in phosphate‐buffered saline (MPBS) wells ( n = 6–9/group). Mean ± SD is shown. Data were analyzed via a two‐way analysis of variance with a Tukey's post hoc test using a 95% confidence interval. a: p < 0.0001 b: p < 0.001 compared to IgG control particle types

Article Snippet: Nanogel size was characterized via NanoSight particle tracking analysis (Malvern Panalytical) and AFM (Asylum Research).

Techniques: Microscopy, Software, Binding Assay, Control, Negative Control, Saline

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet: ( A ) Schematic representation of IRE1 with a C-terminal HaloTag, the construct used for tagging IRE1 at the endogenous locus. IF1 L and IF2 L refer to the primary dimerization and oligomerization interfaces of the lumenal domain, respectively. ( B ) RT-PCR analysis of stress-dependent XBP1 mRNA splicing in WT U-2 OS cells, IRE1 knock-out (KO) U-2 OS cells, and U-2 OS cells in which IRE1 has been fully edited with a C-terminal HaloTag. Tm indicates treatment with 5 μg/ml tunicamycin. ( C ) Immunoblot of UPR activation in response to 5 μg /ml tunicamycin (left) and 100 nM thapsigargin (right) treatments in the three cell lines shown in panel B. ( D ) Maximum intensity projections of representative spinning-disk confocal images of live cells expressing endogenously tagged IRE1-HaloTag, labeled with the JF549 dye. Regions shown with yellow boxes are enlarged below. ( E ) Same as D, except the cells have been treated with 5 μg/ml tunicamycin for 5 hr. Figure 1—source data 1. Annotated uncropped gel used to generate . Figure 1—source data 2. Raw uncropped gel used to generate . Figure 1—source data 3. All annotated uncropped gels used to generate . Figure 1—source data 4. Raw uncropped gel of immunoblot against IRE1 and phospho-IRE1 in . Figure 1—source data 5. Raw uncropped gel of immunoblot against XBP1 in . Figure 1—source data 6. Raw uncropped gel of immunoblot against PERK and actin in . Figure 1—source data 7. Raw uncropped gel of immunoblot against ATF4 in . Figure 1—source data 8. Raw uncropped gel of immunoblot against ATF6 in . Figure 1—source data 9. Raw uncropped gel of immunoblot against CHOP in .

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Western Blot, Activation Assay, Expressing, Labeling

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet: ( A ) Immunoblot showing IRE1 expression levels and UPR activation in WT U-2 OS cells, IRE1 KO U-2 OS cells, partial KO cells used as the parental cell line for generating HaloTag knock-ins, and two clones of endogenously labeled HaloTag (with high and low IRE1 expression levels). Note the shift in protein size due to the addition of the HaloTag and the absence of a WT IRE1 band in the two clones on the right. ( B ) Flow cytometry analysis of the low-and high-expressing clones shown in panel A. Cells were labeled with 5 nM JF549-HaloTag dye for 1 hr prior to the start of the flow cytometry experiment. Note the unimodal intensity distributions of both clones, ruling out the possibility that the lower-expressing clone simply contains a bimodal mixture of low- and high-expressing cells. Error bars represent 95% confidence intervals. Figure 1—figure supplement 1—source data 1. Annotated uncropped gel used to generate . Figure 1—figure supplement 1—source data 2. Raw uncropped gel of immunoblot against IRE1 and phospho-IRE1 in . Figure 1—figure supplement 1—source data 3. Raw uncropped gel of immunoblot against XBP1 in . Figure 1—figure supplement 1—source data 4. Raw uncropped gel of immunoblot against PERK in . Figure 1—figure supplement 1—source data 5. Raw uncropped gel of immunoblot against ATF4 and CHOP in . Figure 1—figure supplement 1—source data 6. Raw uncropped gel of immunoblot against ATF6 in . Figure 1—figure supplement 1—source data 7. Raw uncropped gel of immunoblot against actin in .

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Western Blot, Expressing, Activation Assay, Clone Assay, Labeling, Flow Cytometry

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet: Single-particle tracking data showing stress-dependent oligomerization of the high- and low-expressing IRE1-HaloTag clones. IRE1 in the lower-expressing clone remains dimeric in unstressed cells, while the shift to higher-order oligomers upon stress is less prominent than in the higher-expressing clone. Each data point represents a single cell. Error bars represent 95% confidence intervals. Figure 4—figure supplement 1—source data 1. Raw uncropped gel of immunoblot against IRE1, XBP1s, and GAPDH of . Figure 4—figure supplement 1—source data 2. Raw uncropped gel of immunoblot against PERK, ATF4, and CHOP of . Figure 4—figure supplement 1—source data 3. Raw uncropped gel of immunoblot against ATF6 of .

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Single-particle Tracking, Expressing, Clone Assay, Western Blot

Journal: eLife

Article Title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

doi: 10.7554/eLife.74342

Figure Lengend Snippet:

Article Snippet: Antibody , Anti- ATF6 (Mouse monoclonal) , Proteintech , 66563–1 , WB (1:1000).

Techniques: Cloning, CRISPR, Knock-Out, Expressing, Recombinant, Plasmid Preparation, Transfection, Sequencing, Software, Diffusion-based Assay, Single Particle