Journal: Retrovirology

Article Title: Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus

doi: 10.1186/s12977-015-0215-z

Figure Lengend Snippet: Post-fusion decay of HIV-1 YFP-Vpr signal. a , d ASLVpp co-labeled with the core-associated YFP-Vpr ( green ) and a releasable content marker Gag-imCherry ( dark red ) were pre-bound in the cold to CV-1/TVA950 ( a – c , g ) or A549/TVA950 ( d – f ) cells expressing the ASLV receptor TVA950. Entry was initiated by introducing warm buffer, and cells were maintained at 37 °C for 45 min and imaged every 3–5 s. Fusing viruses were detected by the near-instantaneous disappearance of mCherry from double-labeled particles (marked by white circles in a and d ). White dashed lines show the boundaries of cell nuclei. b , c Fluorescence intensity profiles (total fluorescence of YFP-Vpr and Gag-imCherry) obtained by single ASLVpp tracking in CV-1-derived cells. e , f Fluorescence intensity profiles for YFP-Vpr and Gag-imCherry obtained by single ASLVpp tracking in an A549-derived cell. g An example of YFP-Vpr and Gag-imCherry signals from a non-fusing particle selected from an experiment carried out in the presence of the ASLV fusion inhibitor R99 (50 μg/ml). Black dashed lines outline different YFP decay profiles occurring without ( c , e ) and with a lag ( b , f ) after the release of mCherry. Here and in Fig. , the abrupt ending of fluorescence traces occurs due to the inability to track faint YFP/GFP-Vpr puncta using particle tracking software, as the signal approaches the background level

Article Snippet: Human alveolar adenocarcinoma A549, human embryonic kidney HEK293T/17, and African green monkey kidney CV-1 cell lines were obtained from the ATCC (Manassas, VA, USA).

Techniques: Labeling, Marker, Expressing, Fluorescence, Derivative Assay, Software

Journal: Retrovirology

Article Title: Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus

doi: 10.1186/s12977-015-0215-z

Figure Lengend Snippet: Nuclear accumulation of YFP-Vpr correlates with virus input. a , b Varying dilutions of YFP-Vpr/Gag-imCherry labeled ASLVpp were pre-bound to CV-1/TVA950 ( a ) or A549/TVA950 ( b ) cells in the cold followed by incubation at 37 °C for 2 h in the presence of 20 μΜ MG132. The nuclear YFP-Vpr was measured in fixed samples in the absence ( filled circles ) or in the presence ( open circles ) of 50 μg/ml R99 peptide. Data are means and SEM from four image fields. Linear regression lines are shown. c ASLVpp fusion-mediated nuclear accumulation of YFP-Vpr in CV-1/TVA950 cells in the absence ( filled circles ) or in the presence ( gray circles ) of 20 μΜ MG132. Data represent the ratio of nuclear YFP-Vpr to the signal prior to initiation of fusion. d The fusion efficiency of ASLVpp/YFP-Vpr/Gag-imCherry with CV-1/TVA950 or A549/TVA950 cells, as determined by single particle tracking. Bars are means and SEM from three experiments each, with total number of dual-labeled viral particles indicated. e Correlation between the fraction of viral YFP-Vpr accumulated in the nucleus after 2 h at 37 °C (see the legend to a ) and the slope of viral fusion (BlaM signal) vs the viral p24 input (see Additional file : Figure S6). Every point represents a distinct ASLVpp or VSVpp preparation. f ASLVpp ( lanes 1, 2 ) and VSVpp ( lanes 3, 4 ) were produced using the wild-type HIV-1 R9ΔEnv backbone and co-labeled with YFP-Vpr (and BlaM-Vpr to enable measurements of virus-cell fusion shown in Fig. 5a). The viruses either contained ( odd lanes ) or lacked ( even lanes ) Gag-imCherry. Equal amounts of p24 from virus preparations were subjected to SDS-PAGE and blotted for p24 (HIV IG antibody) or YFP-Vpr (GFP antibody). The loading order: 1 ASLVpp/YFP-Vpr, 2 ASLVpp/YFP-Vpr/Gag-imCherry, VSVpp/YFP-Vpr and 4 VSVpp/YFP-Vpr/Gag-imCherry. The numbers are the respective fractions (%) of viral YFP-Vpr that entered the nucleus in live cell experiments within 2 h

Article Snippet: Human alveolar adenocarcinoma A549, human embryonic kidney HEK293T/17, and African green monkey kidney CV-1 cell lines were obtained from the ATCC (Manassas, VA, USA).

Techniques: Virus, Labeling, Incubation, Single-particle Tracking, Produced, SDS Page

Journal: Retrovirology

Article Title: Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus

doi: 10.1186/s12977-015-0215-z

Figure Lengend Snippet: YFP-Vpr shedding is rate-limiting for nuclear entry and is not modulated by capsid stability. a Nuclear GFP-Vpr and Hoechst fluorescence before and after GFP photobleaching. b Fluorescence recovery after photobleaching. Circles are normalized means and SEM of 10 nuclei. Inset The GFP-Vpr signal recovery after photobleaching ( circles ), the line is a double-exponential fit to the data. c Kinetics of Vpr nuclear accumulation in CV-1/TVA950 and A549/TVA950 cells in the presence or absence of 50 μg/ml of R99 peptide. d Kinetics of single ASLVpp fusion and YFP-Vpr shedding in CV-1- or A549-derived cells measured as the time-point of mCherry disappearance from dual-labeled ASLVpp and complete loss of YFP-Vpr, respectively. Circles represent normalized cumulative plots for signal disappearance from ASLVpp. Lifetimes of post-fusion cores were measured as the difference in disappearance times of mCherry and YFP signals for the same particle. e Synchronized fusion of ASLV from endosomes. ASLVpp was allowed to enter CV-1-derived cells for 45 min at 37 °C in the presence of 70 mM NH 4 Cl. Viral fusion was initiated by replacing NH 4 Cl with imaging buffer, and the kinetics of fusion (release of mCherry) and loss of YFP-Vpr was measured ( left axis ). The corresponding appearance of YFP-Vpr in the nucleus in the same imaging field was determined as a fold-increase over that prior to initiation of synchronous fusion from endosomes ( open circles , right axis ). f CV-1/TVA950 cells inoculated with YFP-Vpr-labeled VSVpp containing either the wild-type (WT) HIV-1 capsid (R9 backbone) or one of the two capsid mutants, K203A (destabilizing) and 5Mut (stabilizing). The amount of cell-bound viruses was equalized based on cell-associated YFP-Vpr fluorescence and viruses were allowed to fuse for 2 h at 37 °C in the presence of 20 μΜ MG132. The nuclear YFP-Vpr signal was measured at indicated time intervals and normalized to the value at 2 h. Data-points are means and SEM from four image fields each

Article Snippet: Human alveolar adenocarcinoma A549, human embryonic kidney HEK293T/17, and African green monkey kidney CV-1 cell lines were obtained from the ATCC (Manassas, VA, USA).

Techniques: Fluorescence, Derivative Assay, Labeling, Imaging

Journal: Retrovirology

Article Title: Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus

doi: 10.1186/s12977-015-0215-z

Figure Lengend Snippet: Cellular GFP-Vpr mobility analysis by fluorescence correlation spectroscopy. a Representative autocorrelation curves for monomeric and tetrameric GFP expressed in A549 cells, as well as for nuclear GFP-Vpr delivered through fusion of ASLVpp co-labeled with GFP-Vpr and Gag-imCherry. b Diffusion coefficients obtained by curve fitting the autocorrelation plots in a , as described in “ ” and in Additional file : Figure S10. Monomeric and tetrameric GFP curves were fit with a 3D single-component diffusion model, whereas the GFP-Vpr curves could only be fit with a 2-component diffusion equation. The faster diffusion coefficient D 1 was assumed to correspond to an GFP-Vpr monomer and was fixed for curve fitting purposes in order to obtain D 2 coefficient. Similar analysis was performed for GFP-Vpr expressed in A549 cells by transient transfection. Data are means and SEM from 6 to 12 experiments. Possible reasons for the unexpectedly large difference in D for a monomer and a tetramer compared to the predicted D − 1/(MW) 1/3 relationship are discussed in

Article Snippet: Human alveolar adenocarcinoma A549, human embryonic kidney HEK293T/17, and African green monkey kidney CV-1 cell lines were obtained from the ATCC (Manassas, VA, USA).

Techniques: Fluorescence, Spectroscopy, Labeling, Diffusion-based Assay, Transfection

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: ( A ) Ex vivo primary murine splenic B cells were treated with 100 μM CK-689 or CK-666 for 1 hr. SPT was then carried out as in , using Cy3-labeled Fab fragments of antibodies to CD19. Single-particle trajectories from a representative cell are plotted using a color-coded temporal scale (left panels). Scale bars: 5 µm. Diffusion coefficients (center panels) and the diameter of maximum displacement (confinement diameter, right panels) over the 10 s period of observation were calculated for each track and cumulative frequency curves are shown. The dots on the curves indicate the median values. ****p<0.0001; Kolmogorov-Smirnov test. ( B–E ) Primary murine B cells were pre-treated with 100 μM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the single-chain anti-Ig κ surrogate Ag. Cells were fixed at the indicated time points and stained with an antibody that recognizes the surrogate Ag and with an antibody that recognizes phosphorylated CD19. Representative cells are shown ( B ). Scale bars: 2 µm. For each B cell, the total fluorescence intensity of clustered pCD19 was calculated. Beeswarm plots in which each dot represents one cell are plotted with the median (red line) and interquartile ranges (red box) for >125 cells per time point from a representative experiment ( C ). For each cell in ( C ), the fraction of total pCD19 fluorescence that overlaps with BCR-Ag microclusters was quantified by calculating the Manders’ coefficient ( D ). For each cell in ( C ), the total fluorescence intensity of pCD19 that was within BCR-Ag microclusters in cells was quantified ( E ). ****p<0.0001; ***p<0.001; ns, not significant; Mann-Whitney U test.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Ex Vivo, Labeling, Single Particle, Diffusion-based Assay, Expressing, Staining, Fluorescence, MANN-WHITNEY

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: ( A ) Primary murine B cells were treated with CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the anti-Ig κ surrogate Ag. The cells were fixed at indicated times and stained for surrogate Ag (anti-Ig κ ) and pCD19. For each B cell, the total fluorescence intensity of clustered pCD19 was calculated. For each condition, the median pCD19 fluorescence intensity was determined for >15 cells per experiment. For each experiment, the median pCD19 fluorescence intensity for CK-666-treated cells was expressed as a percent of the median value for CK-689-treated cells (=100%). This ratio is plotted for four independent experiments. ( B ) Primary murine B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then stimulated with 10 µg/ml soluble anti-Ig κ for the indicated times. pCD19 and total CD79a (loading control) immunoblots are shown (left panels) and the pCD19/total CD79a ratios are graphed (right panels). Dotted red line corresponds to the pCD19/total CD79a ratio value for unstimulated CK-689-treated B cells. Representative data from one of three experiments. See for full blots. ( C ) The co-localization of pSyk with BCR-Ag clusters is not dependent on Arp2/3 complex activity. The co-localization of pSyk and Ag in the cells that were analyzed in was quantified using Manders’ coefficient.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Expressing, Staining, Fluorescence, Control, Western Blot, Activity Assay

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: Images of blots that were cropped for presentation in , , and are shown. The portions of the blots that were shown in the indicated figures are outlined by a red dashed box. Molecular weight markers are shown in kDa. ( A ) Full blots for and . Primary murine B cells were pre-treated with CK-689 (lanes 1–5) or CK-666 (lanes 6–10) for 1 hr then stimulated with COS-7 APCs expressing anti-Ig κ (left) or with soluble anti-Ig κ (right) for 0, 3, 5, 15 or 30 min. The upper blots were probed with anti-pCD79 antibodies and the lower blots with anti-CD79a antibodies. ( B ) Full blots for , an additional independent experiment carried out as in ( A ). ( C ) Full blots for . Primary murine splenic B cells were treated with DMSO (lane 1), CK-689 (lane 2), CK-666 (lane 3) for 1 hr, or stimulated with anti-Ig κ antibodies for 5 min (lane 4). The blots were probed with anti-pAkt plus anti-pERK antibodies (upper blot) or with anti-ERK plus anti-Akt antibodies (lower blot). ( D ) Full blots for . Primary murine B cells were pre-treated with CK-689 (lanes 1–5) or CK-666 (lanes 6–10) for 1 hr then stimulated with soluble anti-IgΚ for 0, 3, 5, 15 or 30 min. The left blot was probed with anti-pCD19 antibodies and the right blot with anti-CD79a antibodies as a loading control.

Article Snippet: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

Techniques: Molecular Weight, Expressing, Control

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: Filters were incubated overnight at 4°C with antibodies against Arp3 (Santa Cruz, #sc-15390; 1:1000), Arp2 (abcam, #ab128934; 1:1000), p34 (Millipore, #07–227; 1:1000), actin (Santa Cruz, #sc-47778; 1:5000), or CD79a ( ; 1:5000), or with the following antibodies from Cell Signaling Technologies: pCD79a (#5173; 1:1000); pCD19 (#3571; 1:1000); CD19 (#3574; 1:1000); pERK (#9101; 1:1000), ERK (#9102; 1:1000), pAkt (#9271; 1:1000), or Akt (#9272; 1:1000).

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