Additional file 2: Figure S2. of Automatic detection of diffusion modes within biological membranes using back-propagation neural network

- Comparison of the percentage of decision using the BPNN, Hidden Markov Modeling (HMM)-Bayes, Bayesian Information Criterion (BIC) or Support Vector Machines (SVM) algorithms. 200 simulated trajectories of 300 frames mimicking diffusion within plasma membranes, including one directed motion segment with velocity randomly ranging from 1 to 3 μm/s and one confinement segment with diameters ranging from 0.5 and 1.2 μm, were analyzed with BPPN, HMM-Bayes, BIC or SVM. Within a trajectory each 50 frames segment is always localized at the same position. The diffusion coefficient D is 0.25 μm2/s and the integration time 100 ms. A 30 nm localization noise Pn was added to the trajectory (see Material and Methods section). The percentage of decision based on BPNN corresponds to the number of positive decision for a specific motion mode detected for a given frame over 200 trajectories and normalized to 1 or-1 for confined (light grey) or directed (dark grey) trajectories, respectively. The HMM-Bayes and the BIC algorithms can only detect directed or confined segments within a trajectory, respectively. The tables at the bottom detail the performance of the 4 algorithms in terms of sensitivity and specificity for detecting confined and directed motion modes in the range of parameters tested in this study (D = 0.25 μm2/s, 1 μm/s < v < 3 μm/s, 0.5 μm < L < 1.2 μm). (PDF 400 kb

(A) Immunoprecipitation experiments in WT PC3 cells or in cells overexpressing CD9 (PC3/CD9) or a nonpalmitoylated form of CD9 (PC3/CD9)

Biotin-labeled cells were lysed in Brij97 and incubated with anti-CD9, anti-CD81, or anti-α5 antibodies (the latter is used as a negative control). Immunoprecipitated proteins were detected using peroxidase-coupled streptavidin. (B) Immunofluorescence images of PC3/CD9 living cell basal membrane by TIRF microscopy at 37°C. Cells were incubated with the anti-CD9 Cy3B-conjugated antibody SYB-1 (middle; green in the merge image) and with various antibodies labeled with Atto647N (left; red in the merge images) and raised against (top to bottom) CD81, CD9P-1, the α5 chain of integrin, CD55, or CD46. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p

(A) Time lapse showing a simultaneous single-molecule tracking of two differentially labeled CD9 molecules with a Fab fragment conjugated with Atto647N (red) or with Cy3B (green); see Video 2 (available at )

(B) Representative trajectory of CD9 dynamic colocalization. The parts of trajectories where the fluorescence signal of two particles overlap at least for one pixel (160 nm) are encircled in gray and magnified in the ellipse underneath (colored arrows indicate the trajectory direction). (C) Quantitative analysis of single-molecule colocalization. Two particles were considered spatially colocalized when at least one pixel of their fluorescence signals was overlapped during at least seven frames corresponding to 700 ms (the two molecules were colocalized during 24 frames in the time lapse shown in A). Different combinations of proteins were tested: CD9/CD9 on cells treated or not treated with MβCD, CD9/CD9, and irrelevant pairs such as CD9/CD55, CD55/CD55, and CD46/CD46.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p

(left) ADC distribution of CD9 in control cells (CD9), cells treated with MβCD (CD9 MβCD), cells treated with MβCD loaded with Chl (CD9 MβCD–Chl), or cells transfected with nonpalmitoylated CD9 (CD9)

50% of the membrane Chl was removed by MβCD treatment, and MβCD–Chl treatment increased the Chl content to 130% as compared with control cells. All of the palmitoylation sites have been mutated in CD9 cells. (right) Histograms (open boxes) representing the percentage of each diffusion mode of the molecules as compared with the total number of trajectories (B, Brownian; C, confined; M, mixed). The gray part corresponds to the proportion of trajectories associated with TEAs (identified with the ensemble membrane labeling) for each diffusion mode.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p

(A) ADC distribution and mean value (±SD) of CD55 molecules labeled with Atto647N-conjugated mAb 12A12

D is the mean value of the ADC calculated from a linear fit of the MSD-τ plot, and the dashed line delineates two different populations corresponding to pure confined trajectories (lower ADC) or mixed and Brownian trajectories. (B) Histograms (open boxes) representing the percentage of each CD55 diffusion mode as compared with the total number of trajectories. The gray part corresponds to the proportion of trajectories associated with TEAs (identified with the ensemble membrane labeling) for each diffusion mode (B, Brownian; C, confined; M, mixed). Compare with . (C) Trajectories of a single CD55 molecule. The inset is a magnification of the transient confinement area delineated by the boxed area.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p

(A) Distribution of the ADC of CD9, CD55, and CD46 treated or not treated with MβCD (∼50% of the membrane Chl was removed)

CD55 is a raft marker, and CD46 is excluded from rafts and TEAs. Mean values of ADC of all the molecules are available in . (B) Comparison of trajectories (thin white lines) in living PC3 cells before (left) or after (right) MβCD treatment. Bars, 7.5 μm.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p