Journal: PLoS Computational Biology

Article Title: Asymmetric PTEN Distribution Regulated by Spatial Heterogeneity in Membrane-Binding State Transitions

doi: 10.1371/journal.pcbi.1002862

Figure Lengend Snippet: (A) Fluorescent images of Dictyostelium discoideum cells expressing PTEN-Halo ( top ) and PTEN G129E -Halo ( bottom ) labeled with TMR-conjugated HaloTag ligand. Cells moved leftward. Scale bar, 5 µm. (B) Asymmetric distribution of PTEN G129E -Halo on the membrane upon stimulation with a cAMP gradient. The cell was treated with 5 µM Latrunculin A ( top ). The asterisk indicates the position of the pipette tip containing 1 µM cAMP ( bottom ). Scale bar, 5 µm. (C) Single molecules of PTEN G129E bound to the membrane of a migrating cell. The arrow indicates the direction of movement. Scale bar, 5 µm. (D) Typical trajectories of single PTEN G129E molecules observed at the pseudopod ( left ) and tail ( right ). (E) Dissociation curves of PTEN G129E molecules observed at the pseudopod and tail. Fitting curves are from Eq. S11 using the parameter values described in . (F) Distributions of displacement during 33 ms of observation at the pseudopod and tail. Fitting curves are from assuming a three-state model. Diffusion coefficients and their proportions are described in and . (G) The time series of displacement of a PTEN G129E molecule over a 33 ms window (excerpted from the whole trajectory observed at the tail). (H) Autocorrelation function calculated from the time series of displacements for 10 molecules observed at the tail. The fitting function is y = a *exp(− Kt )+ b with K = 1.64 s −1 . See also Movie S1.

Article Snippet: The plasmid was generated from the extrachromosomal expression of HaloTag-fusion PTEN proteins (PTEN-Halo) (Promega, Japan) and introduced into Ax2 cells by electroporation .

Techniques: Expressing, Labeling, Transferring, Diffusion-based Assay

Journal: PLoS Computational Biology

Article Title: Asymmetric PTEN Distribution Regulated by Spatial Heterogeneity in Membrane-Binding State Transitions

doi: 10.1371/journal.pcbi.1002862

Figure Lengend Snippet: (A) Fluorescent images of Dictyostelium discoideum cells expressing PTEN G129E –Halo (upper panel) or PTEN G129E;Δ15 –Halo (lower panel) labeled with a TMR-conjugated HaloTag ligand (left). Fluorescence intensity along a white line was measured (right). Red bars indicate the area corresponding to the membranes. Scale bar, 10 µm. (B) The dissociation curve of all PTEN G129E;Δ15 molecules ( open circles ) and decay profiles of three subpopulations ( crosses ) fitted to Eqs. S11 and S12, respectively ( solid lines ). (C) The distribution of displacement during a 33 ms window obtained from the trajectories ( open circle ) and fitting function , assuming 1 ( small dotted line ), 2 ( large dotted line ) or 3 states ( solid line ). ( inset ) AIC values show at least three states are required to explain the data. (D) Kinetic model describing the state transitions and membrane dissociation of PTEN G129E;Δ15 in non-polarized cells. The difference in membrane-to-cytoplasm ratio of fluorescence intensity is related to the difference in membrane association rate (s −1 ) between PTEN G129E and PTEN G129E;Δ15 which dissociated from the membrane at the same rate on average.

Article Snippet: The plasmid was generated from the extrachromosomal expression of HaloTag-fusion PTEN proteins (PTEN-Halo) (Promega, Japan) and introduced into Ax2 cells by electroporation .

Techniques: Expressing, Labeling, Fluorescence

Journal: eLife

Article Title: Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

doi: 10.7554/eLife.55667

Figure Lengend Snippet: ( A ) Growth of strains bearing SWR1 , H2A.Z ( HTZ1 ) and H2B ( HTB1 ) fusions to HaloTag. Saturated cultures at optical density 1.0 were spotted (1:5 serial dilutions) on CSM plates with or without 1% formamide or 150 mM HU (hydroxyurea) and incubated for 2–3 days at the indicated temperatures. WT and mutant strains htz1Δ and swr1Δ are shown for comparison. ( B ) Cells expressing H2A.Z-Halo and Swr1-Halo were stained with JF646 and the cell lysate was resolved on SDS-PAGE. Fluorescent scan of duplicate lanes show specific labeling of Halo-tagged H2A.Z and Swr1 proteins. ( C ) SDS-PAGE shows 10 nM JF646 saturates H2A.Z-Halo in yeast cells in a routine 2 hr staining period. ( D ) Normalized histogram and two-component Gaussian fit for HaloTag (fused to NLS). The slow fraction is 5%. ( E ) Top: Profile of H2A.Z-Halo diffusivity in biological replicate, same condition as experiment. ( F ) Profile of H2A.Z-Halo diffusivity in cells stained with JF552. All molecules tracked with JF552 dye except ( E ), which was tracked with JF552.

Article Snippet: The plasmid for HaloTag (Halo) fusions was generated by cloning HaloTag (Promega) in the pBluescript SK (-) vector followed by insertion of a KanMx cassette (Kanamycin) or NatMx cassette (Nourseothricin), following standard procedures ( ).

Techniques: Incubation, Mutagenesis, Expressing, Staining, SDS Page, Labeling

Journal: eLife

Article Title: Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

doi: 10.7554/eLife.55667

Figure Lengend Snippet: ( A, B ) Normalized histograms and two-component Gaussian fits for H2A.Z-Halo ( A ) and Halo-H2B ( B ) show the log diffusion coefficient distributions. The Gaussian fit for HaloTag is shown for reference (‘Halo only’ in A). ( C, D ) Normalized histograms and two-component Gaussian fits for Swr1-Halo in WT cells ( C ) and the swc2Δ mutant ( D ). Solid line: sum of two-component fit; dashed line: individual component. Percent value of the slow component along with Bootstrap resampling errors and the number of trajectories ( n ) are indicated. ( E ) Cumulative distribution functions (CDF) of 10 ms displacements. ( F ) Spot-On results with fitting errors showing fractions of chromatin-bound molecules derived from modeling CDFs over 10–50 ms intervals. All molecules tracked with JF646 dye except Halo only, which was imaged with JF552.

Article Snippet: The plasmid for HaloTag (Halo) fusions was generated by cloning HaloTag (Promega) in the pBluescript SK (-) vector followed by insertion of a KanMx cassette (Kanamycin) or NatMx cassette (Nourseothricin), following standard procedures ( ).

Techniques: Diffusion-based Assay, Mutagenesis, Derivative Assay