Nanoparticles Associate with Intrinsically Disordered RNA-Binding Proteins

Nanoparticles are capable of penetrating cells, but little is known about the way they interact with intracellular proteome. Here we show that inorganic nanoparticles associate with low-complexity, intrinsically disordered proteins from HeLa cytosolic protein extracts in nondenaturing <i>in vitro</i> nanoparticle pull-down assays. Intrinsic protein disorder associates with structural mobility, suggesting that side-chain flexibility plays an important role in the driving of a protein to nanoparticle absorption. Disordered protein domains are often found in a diverse group of RNA-binding proteins. Consequently, the nanoparticle-associated proteomes were enriched in subunits of RNA-processing protein complexes. In turn, this indicates that within a cell, nanoparticles might interfere with protein synthesis triggering a range of cellular responses

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.

<p>(<b>A</b>) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (<b>B–C</b>) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (<b>B</b>) or SMC1 (<b>C</b>) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen-1003719-g004" target="_blank">Figure 4B–C</a>. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (<b>D</b>) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (<b>E</b>) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (<b>D</b>), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s005" target="_blank">Figure S5A</a>). (<b>F</b>) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (<b>G</b>) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (<b>D</b>). Similar results were obtained for SMC1 IPs from colhicine-treated cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s006" target="_blank">Figure S6</a>). (<b>H</b>) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (<b>I</b>) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion.</p

NAP1 and PP2A act antagonistically in cohesin cycle.

<p>(<b>A</b>) Analysis of mitotic chromosomes from colchicine-treated S2 cells after knockdown of NAP1, PP2A or both factors. We quantified the frequency of resolved (blue), unresolved (red) sister chromatids and loss of centromeric cohesion (Cen. Loss; green). Concomitant depletion of NAP1 and PP2A resulted in a statistically significant increase of the frequency of resolved chromatids compared to the NAP1 knockdown, as determined by χ²-test (n>30, from 3 biological replicates). For the corresponding Western blot analysis see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s007" target="_blank">Figure S7A</a>. (<b>B</b>) Representative example of mitotic chromosomes from colhicine-treated S2 cells depleted for NAP1, PP2A or for both proteins. DNA visualized by DAPI staining is shown in red. Centromers are indicated by arrowheads, whereas loss of centromeric cohesion is indicated by full arrows. (<b>B′</b>) The localization of SA (green) on mitotic chromosomes same as in (<b>B</b>) was determined by indirect immunofluorescence. (<b>C</b>) RAD21 (green) localization on mitotic chromosomes. (<b>D</b>) MeiS332 (green) localization on mitotic chromosomes. (<b>E</b>) Depletion of PP2A restores SA phosphorylation in cells lacking NAP1. Western blot analysis of SA IPed from either mock-treated S2 cells or after knockdown (KD) of NAP1, PP2A or both proteins under normal (top panel) or denaturing (middle panel, (d)IP) conditions from asynchronously dividing cells (− colhicine) or colhicine treated cells (bottom panel, + colhicine). Blots were probed with antibodies against SA, phosphorylated serine, PP2A or NAP1. After NAP1 knockdown, SA phosphorylation levels drop substantially. Whereas depletion of PP2A alone does not affect bulk SA phosphorylation, concomitant knockdown of PP2A and NAP1 neutralized the effect of NAP1 depletion, leading to restored levels of phosphorylated SA. Antibodies against phosphoSer recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (<b>F</b>) Analysis of mitotic chromosomes from colchicine-treated S2 cells after over-expression (OE) of GFP (Mock), NAP1, PP2A, both NAP1 and PP2A or the catalytic mutant PP2A^H59Q. Quantification of mitotic phenotypes was as described above (A). Overexpression of PP2A, but not PP2A^H59Q, resulted in significant increase of the frequency of unresolved chromatids. The PP2A over-expression phenotype was rescued by co-expression of NAP1, as determined by χ²-test (n>30, from 3 biological replicates). For the corresponding Western blot analysis see . Representative examples of mitotic chromosomes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s007" target="_blank">Figure S7C</a>–D.</p

NAP1 and cohesin co-localize on chromatin.

<p>(<b>A</b>) Distribution of NAP1 and SA proteins on <i>Drosophila</i> salivary gland polytene chromosomes visualized by indirect immunofluorescence with antibodies against NAP1 (green) and SA (red). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Split images and merge for red and green channels are shown. Regions harboring the <i>Enhancer of split</i> (<i>E(spl)</i>) gene cluster, <i>Broad Complex</i> (<i>BrC</i>), <i>Eip75B</i> and chromocenter (c) are indicated. (<b>B</b>) Genomic view of NAP1 (green), SA (red) and SMC1 (blue) ChIP-chip enrichment profiles at <i>Enhancer of Split E(spl)</i> NOTCH inducible gene cluster. Filtered binding sites (FDR<0.01) are indicated as bars below the respective profiles. ChIP-chip enrichment scores, genomic coordinates and genes are indicated. Regions examined by ChIP-qPCR are indicated by arrows. (<b>C</b>) Venn diagram depicting the overlap between SMC1, SA and NAP1 binding loci. The overlap between SMC1 and SA loci is 3.7 times greater (p<0.001) than expected by random chance. The overlap between NAP1 and cohesin loci derived from shared SMC1 and SA filtered peaks is 5.2 times greater (p<0.001) than expected by random chance. (<b>D</b>) ChIP-qPCR analysis of SA binding to genomic sites harboring <i>E(spl)</i>, <i>cut</i>, <i>Eip75B</i> and <i>BrC</i> genes selected from ChIP-chip profiles (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen-1003719-g003" target="_blank">Figure 3B</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s003" target="_blank">Figure S3A</a>–C). ChIP enrichments after SA (red bars) and Mock (black bars) RNAi knockdowns (KD) were expressed relative to signals from mock-treated cells. For mock treatment we used dsRNAs directed against GFP. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#s2" target="_blank">Results</a> are based on 3 biological replicates and error bars represent standard error of mean (S.E.M.). (<b>E</b>) ChIP-qPCR analysis of NAP1 binding to genomic loci. Analysis as described above.</p

Loss of NAP1 compromises cohesin removal from mitotic chromosome arms.

<p>(<b>A</b>) Indirect immunofluorescent analysis of SA (green) binding to mitotic chromosomes from mock-treated or NAP1 knockdown S2 cells. DNA visualized by DAPI is shown in red. The centromeric localization of SA in mock-treated cells is indicated by arrowheads. Upon NAP1 KD, there is a dramatic accumulation of SA on the mitotic chromosome arms in ∼80% of cells. (<b>B</b>) Indirect immunofluorescent analysis of RAD21 (green) binding to mitotic chromosomes from S2 cells. Analysis as described above. RAD21 accumulates on mitotic chromosome arms in ∼80% of cells depleted for NAP1. (<b>C</b>) Binding of SA to mitotic chromosomes from wild type or <i>nap1^KO1</i> larval brain cells. (<b>D</b>) Binding of RAD21 to mitotic chromosomes from larval brain cells. ∼85% of <i>nap1^KO1</i> larval brain cells show accumulation of SA and RAD21 on mitotic chromosome arms.</p