Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: ( a,g,j ) Schematic representation of the SNAP-tagged constructs. ( b,h,k ) Representative images of single CHO cells, stably transfected with the corresponding labeled protein and visualized by TIRF-M. Scale bar, 10 μm. The first 100 frames of the cell in b are shown in . Inserts correspond to higher magnification images of the areas in the white boxes. ( c,i,l ) Representative intensity distributions of fluorescent spots identified over the first 10-frame time window of TIRF illumination of CHO cells, stably transfected with the corresponding construct and labeled with Alexa546-BG. Number of identified particles, n = 5770 ( c ), 6252 ( f ) and 6458 ( i ). Data were fitted with a mixed Gaussian model. A mixed Gaussian fit after partial photobleaching (dotted line) was used to estimate the intensity of a single fluorescent molecules in each image sequence. ( d ) Individual trajectories of moving SNAP-D 2L receptors were identified from the entire recording of the cell shown in ( b) . The insert shows a higher magnification that illustrates the random nature of the diffusive process. ( e ) Representative plot of the average mean square displacement (MSD) (mean ± s.d.) versus the time interval (δt) for the trajectories shown in ( a ). The plot is linear (r 2 = 0.99 – linear fit (blue)), over a 3-s timescale, which is consistent with receptor movement following a random walk, and it shows no evidence for anomalous diffusive behavior. ( f ) Distribution of the diffusion coefficients of the receptor particles tracked in ( d ).

Article Snippet: Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface ® Alexa Fluor ® 546; New England Biolabs).

Techniques: Construct, Stable Transfection, Transfection, Labeling, Sequencing, Diffusion-based Assay

Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: ( a ) 48 sequential frames of two Alexa546-labeled SNAP-D 2L receptors showing transient dimer formation (frame rate of 19.32 fps) (also shown in ). ( b ) Intensity profile (blue) of the marked fluorescent SNAP-D 2L receptor shown in ( a) , compared to background intensity (grey). ( c ) A histogram of the lifetimes of 120 SNAP-D 2L receptor dimers taken from trajectories similar to those in ( a) and collected in 0.5 s bins. The solid line represents a one-phase exponential fit for a mean lifetime of 0.50 s (95% confidence interval: 0.44–0.60). ( d ) Effect of the size of SNAP-D 2L receptors on their lateral diffusion. The diffusion coefficients (D lat ) of the analyzed receptor particles ( n ) are shown (monomers −0.104 ± 0.052 μm 2 s −1 , n = 412 from 3 cells and dimers −0.075 ± 0.027 μm 2 s −1 , n = 373 from 3 cells. Data represent mean ± s.d. The difference, determined by an unpaired t-test (**** p -value < 0.0001) is significant and shows that the receptor mobility is negatively correlated with the size of the receptor complexes.

Article Snippet: Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface ® Alexa Fluor ® 546; New England Biolabs).

Techniques: Labeling, Diffusion-based Assay

Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: ( a ) Representative images of a single CHO cell stably expressing the SNAP-D 2L receptor and seeded on HF-treated glass slides, labeled with Alexa546-BG and visualized by TIRF-M. The insert corresponds to higher magnification image of the area in the small white box. ( b ) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in a. The plot is linear (r 2 = 0.99 – linear fit (blue)), over a 3-s timescale, which is consistent with receptor movement following a random walk. ( c ) The distribution of the diffusion coefficients of the analyzed receptor particles ( n ) are shown ( n = 4409, 5 cells - HF-treated slide; n = 4409, 8 cells - non HF-treated slide) and revealed no evidence for anomalous diffusive behavior as a result of HF-treatment. ( d ) Representative intensity distribution of fluorescent spots identified over the first 10-frame time window of TIRF-illumination. Data were fitted with a mixed Gaussian model (sum of two Gaussian functions). ( e ) Representative TIRF-M images of CHO cells stably transfected with SNAP-D 2L receptor, incubated in iso-osmotic (300 mOsm) (left) and hypo-osmotic PBS (108 mOsm) (right) for 2 h and labeled with Alexa546-BG. ( f,g ) Imaging of a region of membrane protrusions of CHO cells stably transfected with the SNAP-D 2L receptor, incubated in hypo-osmotic (108 mOsm) PBS for 2 h and labeled with Alexa546-BG in epi-illumination ( f ) and TIRF-illumination ( g ). ( h ) Representative images of one membrane protrusion of a CHO cell stably expressing the labeled SNAP-D 2L receptor and visualized by TIRF-M. ( f ) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in ( h ). The plot is linear (r 2 = 0.99 – linear fit (blue)), over a 2,5-s time scale and the calculation of the average diffusion coefficient D lat of 0.077 ± 0.007 μm 2 s −1 (mean ± s.d., 16 regions of membrane protrusions of 8 cells) revealed no evidence for anomalous diffusive behavior in the membrane protrusions. Scale bars, 10 μm.

Article Snippet: Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface ® Alexa Fluor ® 546; New England Biolabs).

Techniques: Stable Transfection, Expressing, Labeling, Diffusion-based Assay, Transfection, Incubation, Imaging

Journal: Scientific Reports

Article Title: Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

doi: 10.1038/srep33233

Figure Lengend Snippet: ( a ) Chemical structures of monovalent ligands ( 1a–c ), bivalent ligands ( 2a–c ), and control ligands ( 3a,b ). ( b ) Monomer/dimer ratios calculated from fitted fluorescence intensity distributions of Alexa546-labeled SNAP-D 2L receptors incubated with monovalent ( 1a,b ), bivalent ( 2a,b ), control ( 3a,b ) ligands using a mixed Gaussian model ( , ). Data represent mean ± s.d. of n analysed cells ( n = 16 for 1a , 8 for 1b , 8 for 2a , 16 for 2b , 6 for 3a and 8 for 3b . ( c ) Average diffusion coefficients (D lat ) of the corresponding ligand-SNAP-D 2L receptor complexes of the same analyzed cells in ( b ). Data in ( b,c ) represent mean ± s.d., Statistical analysis was performed by an unpaired t -test (** p -value < 0.01, **** p -value < 0.0001) and showed that the receptor mobility is negatively correlated with the size of the receptor complexes. ( d–g ) Comparison of the apparent lifetimes of particle colocalization of the monomeric SNAP-CD86 and dimeric SNAP-CD28 control proteins proteins ( e and f respectively) and the SNAP-D 2L receptor in the absence and presence of the monovalent ligand 1a or bivalent ligand 2a ( g, h , and f respectively). ( d ) Representative intensity profile of one trajectory which showed intensity doubling from the beginning of the particle tracking followed by one step intensity change which was used to calculate the colocalization time of two particles. ( e–i ) The apparent lifetime of particle colocalizations (τ; 95 confidence interval) was calculated by fitting colocalization time data with a one-phase exponential decay function. 120 trajectories like those shown in a were analyzed from 8 different cells in ( e ), 4 in ( f ), 6 in ( g ), 6 in ( h ) and 4 in ( i ), respectively.

Article Snippet: Cells were washed two times with phenol red-free DMEM/F12 supplemented with 10% FBS and were labeled 30 min at 37 °C with 1 μM Alexa546-BG (SNAP-Surface ® Alexa Fluor ® 546; New England Biolabs).

Techniques: Fluorescence, Labeling, Incubation, Diffusion-based Assay

Journal: bioRxiv

Article Title: MRAP2 modifies the signaling and oligomerization state of the melanocortin-4 receptor

doi: 10.1101/2024.04.09.588099

Figure Lengend Snippet: (A) Representative images from TIRF microscopy of the basolateral membrane of HEK293AD cells expressing SNAP-tagged MC4R labeled using Alexa Fluor 647 (gray) and Nb35-eYFP (green). Images were taken pre-(top) and 4 min post-(bottom) stimulation with 1 µM NDP-α-MSH for cells expressing SNAP-MC4R alone or with MRAP2 (at a ratio of 1:4) and indicate recruitment of Nb35 (green intensity). (B) Kinetics of recruitment of Nb35 to the SNAP-MC4R–Gs complex, without (black; 5 transfections, 51 single cells) and with MRAP2 (1+4) (turquoise; 5 transfections, 55 cells). Shown are the normalized fluorescence intensities recorded at the membrane in the eYFP channel; the shaded area represents SEM. (C) Mean concentration-response curves of Rluc8-miniGs recruitment to the plasma membrane localization sensor rGFP-CAAX upon MC4R stimulation with α-MSH for 45 minutes, in the presence or absence of MRAP2 or RAMP3, at the indicated ratio with respect to MC4R, in transiently transfected HEK293-SL cells. Normalized data are expressed as mean ± SEM of three independent experiments. (D) EC50 values from the miniGs recruitment BRET experiments. Data are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons post-hoc test (*** = p < 0.001).

Article Snippet: Prior to microscopy imaging experiments, coverslips with cells expressing SNAP-MC4R and MRAP2 were labeled with 1 μM SNAP-Surface Alexa Fluor 647 dye in complete DMEM (Pan Biotech) for 25 min and kept in the incubator at 37 °C and 5 % CO 2 .

Techniques: Microscopy, Membrane, Expressing, Labeling, Transfection, Fluorescence, Concentration Assay