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

The A and B helices of CD81 LEL confer CD9/CD81 chimeric molecules the ability to support infection by <i>P. yoelii</i> sporozoites.

<p>A: Amino acid sequence alignment of CD81, CD9, and chimeras. Only the sequence of the LEL is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices is indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined. CD81 LEL residues presumably in contact with the SEL are indicated with an asterisk. Open circles shows residues known to be involved in the interaction with HCV E2 glycoprotein. B and C: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras and infected two days later with <i>P. yoelii</i> sporozoites. After two days incubation, the number of EEF-infected cells was determined by immunofluorescence in triplicate wells. Results are expressed as mean±s.d. **, p<0.01 and *, p<0.05 as compared to CD9-transfected cells.</p

A CD81 mAb binds poorly to the non-functional mutant VVD (135–137)→AAA but does not block infection.

<p>A: Hepa 1–6 cells were transfected with the indicated construct in pEGFP-N3 and analyzed for the surface expression and recognition of the transgene by several CD81 mAb using flow-cytometry analysis. Data are expressed as mean fluorescence intensity. In this experiment, the antibodies were used at 20 µg/ml (JS64, M38, JS81) or at 1/100 ascitic fluid dilution (all other mAbs). B: HepG2-A16/CD81 cells were infected with <i>P. yoelii</i> sporozoites in the presence of the indicated mAbs at 25 µg/ml except when otherwise indicated. All mAbs are directed to CD81 except TS9 which is a CD9 mAb and does not inhibit <i>P. yoelii</i> infection.</p

3D structure of CD81 LEL.

<p>The drawing of CD81 LEL (PDB #1g8q) was generated in MolMol. Four helices (A, C, D, E) are drawn in red while the B helix, crucial for <i>P. yoelii</i> infection is displayed in blue. The black balls indicate the CCG ubiquitous motif. The crucial D137 as well as D138 and D139 are in purple while V135 and V136 are in royal blue. Residues V146, T149, F150, T153 and L154 putatively involved in contact with the SEL are indicated in dark blue. T163, F186 and D196 residues, in yellow, have been reported to play a role in the HCV E2 glycoprotein binding to CD81-LEL. Residues V135, V136, T163, F186 and D196 projected backward, behind the drawing plane. The two disulfides bridges are colored light coral. Hydrophilic residues K144, K148 and E152 located on the top of the B helix are in green. The SEL, in cyan, is in front of the drawing plane.</p

The VVD (135–137)→AAA and DDD (137–139)→AAA mutants unable to support infection by <i>P. yoelii</i> sporozoites interact with CD9P-1 and EWI-2.

<p>CHO cells were transiently transfected with WT or mutant CD81 plasmids (in pEGFP-N3), together with a CD9P-1 (top) or a EWI-2 (bottom) cDNA. After 48 h, the cells were lysed with digitonin and immunoprecipitations with antibodies against CD81, CD9P-1 and EWI-2 were performed. After electrophoresis and transfer, the membranes were incubated with biotin-labelled mAbs to CD81 (TS81), CD9P-1 (1F11) and EWI-2 (8A12). The mutants are designed as follows: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA</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

21 residues of CD81 in a CD9 backbone are sufficient to render hepatocytic cells susceptible to <i>P. yoelii</i> sporozoites infection.

<p>A: Amino acid sequence alignment of CD9, CD81 and chimeras. Only the sequence of the large extracellular loop of the different chimeras is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices are indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined B: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras and infected two days later with <i>P. yoelii</i> sporozoites. After two days incubation, the number of EEF-infected cells in triplicate wells was determined by immunofluorescence. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells. C: HepG2-A16 cells stably expressing CD81, CD9, CD81ccg9 or CD9[81B] were infected with <i>P. yoelii</i> sporozoites. After two days incubation, the number of EEF-infected cells was determined in triplicate wells by immunofluorescence. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells.</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) 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