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) 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

(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) 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

Recruitment, Assembly, and Molecular Architecture of the SpoIIIE DNA Pump Revealed by Superresolution Microscopy

<div><p></p><p>ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in <i>Bacillus subtilis</i>, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.</p></div

SpoIIIE localization at superresolution.

<p>(a) SpoIIIE is observed during all stages of the cell cycle. Individual cells were recognized and classified as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio-1001557-g001" target="_blank">Figure 1f</a>. Pixel size was 110 nm. From each 55 ms image, we automatically determined the localization of each single molecule in the image by using MTT <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio.1001557-Serge1" target="_blank">[57]</a>. Each of these localizations is called a single-molecule event. In our pointilist representation, each single-molecule event is represented by a single green dot, whereas membrane stain is shown in white (see SM10 in Methods S1 for more details). Cells without septum were classified as stage 1 (vegetative/pre-divisional, left panel). Cells having a symmetric division septum were classified as stage 2 (division, middle panel), whereas those showing an asymmetric septum (at 1/5^th or 4/5^th of the total cell length) were classified as stage 3 (sporulating, right panel). (b) SpoIIIE clusters were automatically detected and classified depending on their size and composition. FWHM, full width at half maximum. (c) Analysis of the cluster size distribution versus the number of single-molecule events shows two distinct cluster types: PALM-limited clusters (red dots) have a size equal or smaller (∼45 nm FWHM) than the resolution of PALM in our conditions and contain a large number of events (>1,000), whilst dynamic clusters (orange dots) are large (>100 nm FWHM) and contain fewer events (<1,000). (d) The size of PALM-limited clusters is independent of cell cycle stage and the most typical size is ∼45 nm FWHM. (e) PALM-limited cluster sizes as a function of imaging time (total time used to image each single cluster) show that these clusters are extremely stable.</p

SpoIIIE clusters assemble asymmetrically in sporulation septa.

<p>(a) PALM imaging of SpoIIIE (green dots) in sporulating cells overlaid with an epi-fluorescence image of the membrane (white, top panel). Intensity profiles across the direction perpendicular to the septum were used to determine the precise localization of the septal plane, which was used to calculate the distance of each single-molecule detection to the center of the septum and to automatically partition the cell into forespore (yellow) and mother cell (red). Using this partition, individual PALM-limited clusters and single-molecule events were classified as belonging to the mother cell or the forespore compartments. (b) Histogram of PALM-limited cluster localizations with respect to the center of the septum (red columns) in sporulating cells with flat septa and undergoing DNA translocation (<i>N</i> = 43). Black dotted line indicates the position of the septum. A Gaussian distribution was fitted to the data (blue solid line). SpoIIIE PALM-limited clusters preferentially localize on the mother cell side of the sporulation septum. (c) Histogram of single-molecule localizations in PALM-limited clusters (<i>N</i> = 43) in sporulating cells undergoing DNA translocation, and Gaussian fit (blue dotted line). In sporulating cells, the distribution of SpoIIIE with respect to the septum is asymmetric and biased towards the direction of the mother cell. (d) Histogram of localizations of single molecules in PALM-limited clusters of dividing cells (<i>N</i> = 71) and Gaussian fit (blue dashed line). During division, the distribution of SpoIIIE with respect to the septum is symmetric and unbiased. (e) Distance of SpoIIIE PALM-limited clusters to the center of asymmetric septa versus the amount of translocated DNA. Open blue circles represent individual distance values for individual clusters, and black squares the average distance for all clusters detected at each particular percentage of DNA translocated. The relative distance increases linearly with the amount of DNA in the forespore until ∼45% of DNA translocated, and thereon remains constant at ∼50 nm. Solid black line is a guide to the eye. (f) The size (FWHM) of individual PALM-limited clusters in the directions parallel or perpendicular to the asymmetric septa were calculated and plotted against each other to evaluate cluster symmetry (squares). The overwhelming majority of clusters are symmetric (green squares) with only a small minority being longer in the direction parallel to the septum (black squares). The dotted line is a guide to the eye.</p

SpoIIIE is recruited to future sites of septation and localizes to the leading edge of closing septa.

<p>(a–c) Statistics of SpoIIIE clusters in vegetative/pre-divisional (<i>N</i> = 705), dividing (<i>N</i> = 174), and sporulating (<i>N</i> = 2 13) cells. Clusters were automatically classified as dynamic, PALM-limited, or mixed (cells containing both cluster types). Cells with less than 50 detected events were classified as “empty.” Cells with more than one PALM-limited cluster were classified independently from those containing a single one. The proportion of single PALM-limited clusters increases from vegetative/pre-divisional to dividing cells, and is maximal in sporulating cells. (d–e) The normalized coordinates of each localized event (axial and longitudinal coordinates) were used to calculate the localization probability distribution (heat maps) of SpoIIIE for each cluster type in vegetative/pre-divisional cells. Normalized localization probability distributions were calculated for the first quartile of the cell and then reflected into the other three quartiles to impose mirror symmetry in the axis perpendicular and parallel to the cell axis. The relative average number of clusters detected in each pixel of the grid is color-coded according to the color bar (right). White lines represent cells outlines. (d) Dynamic clusters distribute homogeneously over the cell membrane. (e) In contrast, in cells in which sporulation was induced, PALM-limited clusters specifically localize to future sites of asymmetric septation. (f–h) 3D-SIM imaging of <i>B. subtilis</i> cells in the early phases of sporulation at different stages of septal constriction. Axial projections showing the distribution of FM4-64-stained membranes (red, i), fluorescently labeled SpoIIIE (ii), and a merged image (iii). A 3D reconstruction is obtained by calculating a constant fluorescence intensity profile (iv). (f) SpoIIIE localizes to future sites of asymmetric division before the onset of septal constriction as a single cluster with a size <100 nm (resolution limit in 3D-SIM). (g) In cells early in septal constriction, SpoIIIE often shows a ring-like distribution probably due to the intrinsic dynamics of the closing septal ring. (h) In cells advanced in septum constriction, SpoIIIE specifically localizes to the leading edge of the invaginating septum. A line scan (v) of the fluorescence signal across the closing septum (white dotted line in panel iii) shows that SpoIIIE fluorescence (green) is always internal to the fluorescence of the membrane (red). Scale bar, 400 nm.</p