NCp7 cosediments with 80S ribosomes.

<p>(A) Sucrose gradient fractionation profile of purified 80S ribosomes (0.9 μM) incubated with NCp7 (13.3μM). Ribosome/NCp7 ratio was about 1/15. The peak fractions (8–9) were precipitated and further analyzed by western blot. (B) Western blot of fractions 8–9 from sucrose gradient fractionations performed with only NCp7 peptide (lanes 1); only 80S ribosomes (lanes 2); and 80S ribosomes and NCp7, together (lanes 3). NCp7 and ribosomal proteins were detected with polyclonal NCp7 and RpS7 antibodies, and monoclonal RpL26 antibodies. As a control, 90 nM of NCp7 peptide was loaded (4).</p

FRAP-based estimation of diffusion coefficient values (A) and mobile fraction (B) of eGFP and NCp7-eGFP in the cytoplasm and in the nucleus.

<p>FRAP-based estimation of diffusion coefficient values (A) and mobile fraction (B) of eGFP and NCp7-eGFP in the cytoplasm and in the nucleus.</p

FCS measurements in eGFP and NCp7-eGFP expressing HeLa cells.

<p>(A) Experimental autocorrelation function (blue) of NCp7-eGFP in HeLa cells fitted with a model for free (green) and anomalous (red) 3D diffusion. The residuals indicate that a better fit was obtained with the anomalous diffusion model. (B) Comparison of autocorrelation curves for eGFP and NCp7-eGFP diffusion in the cytoplasm of HeLa cells. Fits (solid lines) were performed with the anomalous diffusion model. (C) Histogram of the brightness analysis for eGFP and NCp7-eGFP (N = 16).</p

Confocal images (A, C) and RICS-based diffusion maps (B, D) of eGFP and NCp7-eGFP in HeLa cells.

<p>The color coded images (B, D) represent the values of the diffusion coefficients measured in the cell. The blue colors at the cell borders are artifacts, due to averaging with the exterior of the cells. Confocal images for the same cells (A, C) are given to identify the cell compartments and contour.</p

NCp7-eGFP dynamics in HeLa cells monitored by RICS.

<p>(A) A series of confocal images of eGFP and NCp7-eGFP expressing cells was acquired. A 128x128 pixel region was analyzed by calculating the two-dimensional spatial autocorrelation function represented as a spatial correlation surface (B) that was fitted by a 3D diffusion model (C), revealing the values of the diffusion coefficients and the number of diffusing molecules.</p

Intracellular distribution of NCp7-eGFP.

<p>(A) Amino acid sequence of NCp7. Confocal images of HeLa cells expressing transiently eGFP (B) or NCp7-eGFP (C, D). Comparison with the localization of DNA labeled by 1.6 μM Hoechst 33342, (C) and RNA labeled by 1 μM Pyronin Y. The cyan color of the merge panel in (C) indicates colocalization of NCp7 with DNA in the nucleus. The nearly uniform yellow color of the merge panel in (D) indicates a strong colocalization of NCp7 with RNA all over the cell.</p

Diffusion coefficients (D) and anomalous coefficients (α) inferred from FCS and RICS measurements of eGFP and NCp7-eGFP expressing cells.

<p>The D and α values are given as means +/- SD for 800 correlation curves in 16 cells (FCS) and 40 measurements in 10 cells (RICS).</p><p>Diffusion coefficients (D) and anomalous coefficients (α) inferred from FCS and RICS measurements of eGFP and NCp7-eGFP expressing cells.</p

Vacuolar morphologies quantification in yeast cells producing MTM1.

<p>The <i>ymr1Δ</i> cells expressing either wild-type MTM1 or the different MTM1 mutants from either pVV200 (2 µ, overexpression) or pVV204 (CEN, expression) plasmid were analyzed. For each strain, 300 to 600 cells were observed by microscopy (DIC and FM4-64) and sorted into one of the three categories: unilobar large or giant (in white), small one or two lobes (in grey) and more than two lobes or fragmented (in black) vacuoles. The main vacuolar phenotype of the non-transformed <i>ymr1Δ</i> mutant cells is fragmented vacuoles with more than two lobes. Histograms are the mean of three independent experiments and show the proportion of each category in the different transformed yeast cells.</p

FRAP experiments in eGFP and NCp7-eGFP expressing cells.

<p>(A) Time lapse sequence of typical FRAP measurements in the cytoplasm of an NCp7-eGFP expressing cell. The bleached region is highlighted by the red circle. Normalized fluorescence recovery curves of (B) eGFP and (C) NCp7-eGFP in the cytoplasm. For both eGFP and NCp7-eGFP, the distribution of residuals indicated a much better fit of the recovery curves with a double exponential as compared to a single exponential fit.</p

FCCS measurements on HeLa cells expressing RpL26-eGFP and NCp7-mCherry.

<p>The green, red and black curves denote the autocorrelation curve of RpL26-eGFP in the green channel, the autocorrelation curve of NCp7-mCherry in the red channel, and the cross-correlation curve between the two channels, respectively. As a negative control, the blue curve corresponds to the cross-correlation between eGFP and mCherry proteins co-expressed in HeLa cells. The close to zero value of the FCCS curve of the negative control not only shows that the two fluorescent proteins do not diffuse together, but also that there is marginal spectral bleed-through between the green and red channels. The solid lines correspond to the fit of the curves to the anomalous 3 D diffusion model. Diffusion constants of 5.6 (+/- 0.7) μm²/s, 6 (+/- 3) μm²/s and 4 (+/- 3) μm²/s were obtained for RpL26-eGFP (green), NCp7-mCherry (red) and the RPL26-eGFP/NCp7-mCherry complex (black), respectively.</p