Design and reporting principle of NANOMS.

<p>FRET-biosensor design of the three different NANOMS. (<b>A</b>) The myristoylated N-terminal membrane-targeting motifs of mouse Gα_i2 (residues 1–35), human Yes (1–17)- and human Src (1–16)-kinases were genetically fused to the N-terminus of fluorescent proteins mCFP or mCit. The sequence of the employed membrane-targeting motifs can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066425#pone.0066425.s006" target="_blank">Table S2</a></b>. (<b>B</b>) Intracellular processing involves cleavage of the N-terminal methionine (grey) by methionine amino-peptidase (Met-AP), NMT-mediated myristoylation on glycine 2 (yellow) and depending on the motif cysteine-palmitoylation (red). (<b>C</b>) Lipid modified reporters spontaneously organize into plasma membrane nanocluster. Tight packing of membrane targeted donor (mCFP)- and acceptor (mCit)-fluorophores (blue and yellow squares, respectively) in nanocluster leads to FRET. FRET can decrease due to loss of nanoclustering or cytoplasmic redistribution of the NANOMS after inhibitor treatment. As membrane anchorage is required for the functioning of myristoylated proteins, NANOMS report on functional membrane anchorage.</p

Cherry-picked chemical compound library screen with Yes-NANOMS.

<p>(<b>A</b>) Chemical structures of chemical compounds that were included in the cherry-picked chemical library. (<b>B</b>) BHK21 cells were transfected with Yes-NANOMS and screened with shown chemical compounds at a final concentration of 10 µM/mL. FRET-response of Yes-NANOMS to the chemical compounds is represented with E_max values. Block line indicates the average E_max and the error bars denote the s.e.m (n≥4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.</p

NANOMS reports on RNAi-mediated depletion of NMT.

<p>(<b>A</b>) HEK293 EBNA cells transiently expressing Yes-NANOMS and (<b>B</b>) HEK293 cells transiently expressing Gi2-NANOMS were treated with NMT1 or NMT2 specific siRNAs or control siRNA. Knock-down efficiencies are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066425#pone.0066425.s004" target="_blank">Figure S4</a></b>. The characteristic E_max-value was determined on flow cytometric FRET data. The error bars denote the s.e.m (n = 4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.</p

NANOMS report on chemical inhibition of NMT.

<p>(<b>A</b>) FRET-responses of Yes-, Src- and Gi2-NANOMS transfected BHK cells treated with 4 µM of the specific NMT inhibitor DDD85646. The error bars denote the s.e.m (n = 5). Samples were statistically compared with the untreated control. See Methods for more information on statistical analysis. (<b>B</b>) Confocal sensitized acceptor FRET-imaging of Yes-NANOMS expressed in BHK cells. Cells were treated as indicated. Top row shows acceptor channel images, and bottom row FRET images. The look-up table shows the FRET-index FR, color coded with high FRET levels in black and yellow (value 1) indicating no FRET. Scale bar is representative for all images and corresponds to 10 µm. (<b>C</b>) Dose-response curves of the effect of DDD85646 on the E_max values of Yes- and Src-NANOMS expressed in BHK cells (n = 6).</p

Rescue of underprenylation in REP1 knockdown cells by REP1 but not by REP2.

<p>(A) REP1 knockdown cells were transfected with rat REP1-myc, REP2-myc or the vector control. The cell lysate was subjected to the <i>in </i><i>vitro</i> prenylation with BGPP to quantitate the levels of unprenylated Rabs. (B) Quantification of unprenylated Rabs in REP1 knockdown cells transfected with REP1 or REP2. The Western Blot signal of the unprenylated Rabs was normalized to the β-actin signal to account for differences in loading. REP1 knockdown cells transfected with vector only were taken as reference representing maximal underprenylation of Rabs in this model and therefore shown as 100%.The graph represents the mean of three independent experiments (± SEM). P-levels are denoted above the bars and were determined with the two-tailed Student’s t-test.</p

Analysis of Rab prenylation rates <i>in</i><i>vivo</i> and <i>in</i><i>vitro</i>.

<p>(A-B) Analysis of Rab prenylation status after blocking and releasing prenylation <i>in </i><i>vivo</i>. HeLa cells were treated with compactin for 24h and then incubated with GGOH for different time periods. Cells were lysed and subjected to <i>in </i><i>vitro</i> prenylation with BGPP and recombinant RabGGTase and REP for 6h. (A) Degree of prenylation for each Rab was determined by mass spectrometry. The decrease in signal from the timepoint 0h to 5h was determined by label-free spectral counting and converted into the degree of prenylation for each Rab 5 hours after GGOH addition. The graph represents the mean of three independent experiments (±SEM). (B) Streptavidin-HRP Western blot detection of unprenylated Rabs in the cellular lysates at different timepoints after GGOH addition to compactin-treated cells. The cellular lysates were prenylated with BGPP and RabGGTase as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081758#pone-0081758-g003" target="_blank">Figure 3 (C)</a><i>In </i><i>vitro</i> prenylation of lysate from compactin-treated HeLa cells transfected with different GFP-Rabs. <i>In </i><i>vitro</i> prenylation reaction was stopped after an hour and subjected to Western blot analysis visualized by infrared Odyssey scanning for total GFP-Rab (GFP/red) and for prenylated biotin-labeled GFP-Rab (biotin/green). Representative blots are shown. (D) The graph represents the percentage of prenylated GFP-Rabs after an hour <i>in </i><i>vitro</i> prenylation reaction normalized to complete (overnight) prenylation. Means of three independent experiments are shown (±SEM).</p

Schematic representation of <i>in</i><i>vitro</i> prenylation assay.

<p>(A) Cell lysates are subjected to <i>in </i><i>vitro</i> prenylation using a recombinant Rab prenylation machinery and the biotin-labeled analogue of GGPP, BGPP. Only those Rabs unprenylated in the cell will be labeled in the assay and can be visualized or purified through the biotin-tag. (B) Western blot analysis of cell lysate from untreated and compactin-treated BHK cells after <i>in </i><i>vitro</i> prenylation with BGPP. Unprenylated Rabs are detected via the biotin-tag and actin serves as a loading control. BGPP: biotin-geranyl-pyrophosphate, GGPP: geranylgeranyl-pyrophosphate. Note that compactin treated sample is underloaded and the actual level of unprenylated RabGTPases are higher than they appear from comparison of the band’s intensities. </p

Nanocomposite films based on cellulose nanofibrils and water-soluble polysaccharides

All-polysaccharide composite films were prepared from native, unmodified cellulose nanofibrils (CNF) mixed with various natural water-soluble polysaccharides like carboxymethyl cellulose, galactoglucomannan, xyloglucan and guar gum. Composite films were manufactured by pressurized filtration and hot pressing. The mechanical properties of the films were systematically evaluated in the dry and the wet state. GG was furthermore selectively oxidized using galactose oxidase (EC 1.1.3.9), and the effect of the degree of oxidation on the final composite film properties was shown. It was found that all the tested polysaccharides increased the strength and toughness of the dry composite films at 2 weight percent (wt.%) addition to CNF. After soaking the samples for 24 h in water, striking differences between the samples were found: already at 2 wt.% CMC the wet strength of the composite films diminished, while the uncharged polysaccharides improved the wet strength. For example, the addition of 2 wt.% GGM increased Young's modulus by a factor of 1.3, the tensile strength by a factor of 2.8, and the toughness by a factor of 3.4. The results are discussed in relation to the amount of water absorbed in the films and possible reasons for the improved properties are suggested.Peer reviewe

Localization is an indicator of prenylation.

<p>MALDI mass spectrometry comparison of unmodified (gray) and <i>in </i><i>vitro</i> prenylated (black) Citrine-Rab7 (A) and Citrine-Rab27a (B). Average protein masses are indicated at the peaks. Prenylated Citrine-Rab7:REP1 (C) and prenylated Citrine-Rab27a:REP1 (D) were microinjected into compactin treated A431 cells. Membrane targeting of Rab proteins was detected by imaging the cells 20 min post-injection. Scale bars represent 10 µm.</p