From Basic Principles of Protein–Polysaccharide Association to the Rational Design of Thermally Sensitive Materials

Biology resolves design requirements toward functional materials by creating nanostructured composites, where individual components are combined to maximize the macroscale material performance. A major challenge in utilizing such design principles is the trade-off between the preservation of individual component properties and emerging composite functionalities. Here, polysaccharide pectin and silk fibroin were investigated in their composite form with pectin as a thermal-responsive ion conductor and fibroin with exceptional mechanical strength. We show that segregative phase separation occurs upon mixing, and within a limited compositional range, domains ∼50 nm in size are formed and distributed homogeneously so that decent matrix collective properties are established. The composite is characterized by slight conformational changes in the silk domains, sequestering the hydrogen-bonded β-sheets as well as the emergence of randomized pectin orientations. However, most dominant in the composite’s properties is the introduction of dense domain interfaces, leading to increased hydration, surface hydrophilicity, and increased strain of the composite material. Using controlled surface charging in X-ray photoelectron spectroscopy, we further demonstrate Ca ions (Ca2+) diffusion in the pectin domains, with which the fingerprints of interactions at domain interfaces are revealed. Both the thermal response and the electrical conductance were found to be strongly dependent on the degree of composite hydration. Our results provide a fundamental understanding of the role of interfacial interactions and their potential applications in the design of material properties, polysaccharide–protein composites in particular

Genome-wide analysis of small nucleolar RNAs of <i>Leishmania major</i> reveals a rich repertoire of RNAs involved in modification and processing of rRNA

<div><p>Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of <i>Leishmania major</i> small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related <i>Trypanosoma brucei</i>, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications.</p></div

Exosome secretion affects social motility in <i>Trypanosoma brucei</i>

<div><p>Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite <i>Trypanosoma brucei</i>, exosome secretion is induced by stress that affects <i>trans</i>-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, <i>Vps36</i>, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites.</p></div

Exosome secretion affects social motility in <i>Trypanosoma brucei</i> - Fig 5

<p><b>(A) Immunogold TEM analysis demonstrating the location ZC3H41 in ILVs within MVBs.</b> Cells carrying the <i>SmD1</i> silencing construct, and the YFP-VPS36 construct were used. <b>(a, b)</b> un-induced (-Tet) or <b>(c-g)</b> silenced for 40 hrs. (+Tet) were used to prepare Cryo-EM sections, and were subjected to immunogold analysis. Imaging was performed using antibody to ZC3H41. <b>(B)</b> as in <b>(A)</b>, but using anti-GFP antibody to detect the VPS36. The scale bars are indicated.</p

The SL RNA exosomes are distinct from stress granules and nanotubes.

<p>Exosomes were prepared from <i>SmD1</i>-silenced cells grown in medium containing FCS free of bovine exosomes, after 2 days of silencing. The exosomes were fractionated on a 15–50% sucrose gradient at 39,900 rpm in a SW41 rotor for 20 hr. Fractions (500 μl) were subjected to SEM and western analyses as well as to primer extension to monitor SL RNA. <b>(A) Fractionation of vesicles on the sucrose gradient.</b> The density of the fractions are given and SEM images derived from fractions 13–14 and fractions 17–18 are presented. <b>(B) Western analysis.</b> Fractions 10–21 were subjected to western analysis using the indicated antibodies. <b>(C) The location of SL RNA</b>. RNA was extracted from the different fractions and subjected to primer extension.</p

Identification of SL RNA associated proteins.

<p><b>(A) Identification of proteins purified by affinity-selection</b>. Cells containing the <i>SmD1</i> silencing construct were induced for 48h, as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006245#ppat.1006245.ref009" target="_blank">9</a>]. An extract was prepared from 2×10⁹ cells. The extract was separated on a Superdex 200 column, and SL RNA containing fractions were subjected to affinity selection as described in Materials and Methods. Proteins obtained from the control experiment lacking the selecting oligonucleotide (-Oligo) and proteins from the affinity selected particles (+Oligo) were extracted from the streptavidin beads, separated on a 12% acrylamide SDS gel, and stained with silver. <b>(B) <i>ZC3H41</i>silencing.</b> Cell lines expressing the <i>ZC3H41</i> stem-loop silencing construct were induced for 48 hrs. Cells (~10⁶ cells/ lane) were subjected to western analysis using ZC3H41 and PTB1 antibodies. <b>(C) <i>ZC3H41</i> is an essential gene for trypanosome survival.</b> Cells were either induced (+Tet) or un-induced (-Tet), and growth was monitored. The arrow indicates the time of tetracycline addition. The number of un-induced cells is designated by triangles, and of induced cells by squares. <b>(D) ZC3H41 binds loosely to SL RNA.</b> Cells expressing TAP-Myc-His- ZC3H41 fusion protein and the <i>SmD1</i>-silencing construct were silenced for 48 hrs. Cells (1.5×10⁹) were UV irradiated, as described in Materials and Methods. Extracts prepared from control (-UV) cells, and cells following UV irradiation were affinity selected on IgG beads. The RNA was extracted from the beads and analyzed by primer extension with SL and U3 RNAs specific primers. T, Total extract (5%); S, supernatant after removing the IgG beads (5%); P, the entire RNA sample bound to beads. The position of the cap-4 modification is indicated. <b>(E) Depletion of ZC3H41 in <i>SmE/ ZC3H41</i> silenced cells.</b> Western analysis was performed, as described in panel B. <b>(F) Levels of SL RNA under <i>SmE</i> and <i>SmE/ ZC3H41</i> silencing.</b> 10 μg of total RNA was subjected to primer extension with primers specific to SL RNA, U4, and U3 snoRNAs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006245#ppat.1006245.s011" target="_blank">S1 Table</a>). The extension products were separated on a 6% denaturing gel. The identity of the cell line and the position of the modified cap nts are indicated. The statistical analysis represents the mean ± s.e.m of quantification from three independent experiments. **<i>P</i> <0.01, and ***<i>P</i> <0.005 compared to–Tet, using Student's <i>t</i>-test. <b>(G) Changes in localization of ZC3H41 and SL RNA during <i>SmD1</i> silencing.</b> Cells carrying the <i>SmD1</i> silencing construct were induced for the time indicated and subjected to <i>in situ</i> hybridization with SL RNA (red) and IFA with ZC3H41 antibodies (green). The nucleus was stained with DAPI. The merge was performed on DAPI staining and SL RNA hybridization and the time points of silencing are indicated.</p

Exosome secretion repels the migration of wild-type cells.

<p><b>Cells (~10</b>^<b>7</b><b>) were plated on semi solid agar containing tetracycline at a distance of 2.5 cm from each other.</b> The <i>SmD1 and SmD1/Vps36</i> silenced cells were plated on plates containing tetracycline. Pattern formation was analyzed 2 days after plating. The parasites from the plates were blotted, and the blot was stained with Ponceau and then reacted with anti-GPEET and anti-EP. The distance between the wild-type colony and the adjacent colony is presented. The statistical analysis represents the mean ± s.e.m **P< 0.01 comparing the distance between wild-type to wild-type, wild-type to <i>SmD1and</i> wild-type to <i>SmD1/Vps36</i> silenced cells based on three independent experiments.</p

Entry of exosomes to target cells.

<p><b>(A) Fluorescence of exosomes stained with DiL and containing the ZC3H41-GFP.</b> Exosomes (20μl, representing 1/10 of the exosome preparation from 10⁹ cells) were incubated at room-temperature with 1:1000 dilution of the DiL and were analyzed by ImageStream. <b>(a)</b> The number of particles versus their area is presented. The peak corresponding to exosomes is indicated. <b>(b) The overlap between the two fluorescent dyes. (c)</b> Single exosome fluorescence. The fluorescence of GFP and DiL was recorded in addition to the overlay. DIC, and the different fluorophores are indicated. <b>(B) Exosomes enter wild-type trypanosome cells. (a)</b> The exosomes described in <b>(A)</b> (40 μl) were incubated with 100μl containing 10⁶ cells. ImageStream analysis was performed after 10 minutes, and single-cell fluorescence of ZC3H41–GFP and DiL was monitored. <b>(b)</b> The level of internalization of the two fluorophores. <b>(C) Live imaging of cells incubated with ZC3H41-GFP exosomes and lysotracker.</b> Exosomes were prepared from 10⁹ cells, and 1/10 of the preparation (20 μl) was incubated with 100μl containing 10⁶ cells for 1 hr and then lysotracker was added, and cells were visualized.</p

Exosomes secreted from <i>SmD1</i> silenced cells.

<p><b>(A) TEM analysis of the exosome-enriched fraction from <i>SmD1</i> silenced cells.</b> Exosomes were purified, from 10⁹ cells after 40 hrs of silencing, and were subjected to TEM. <b>(a)</b> The entire field of exosomes; <b>(b)</b> and <b>(c)</b> show enlargement of a section indicated by a square on panel <b>(a). Exosome size distribution.</b> For the analysis, the size of 150 exosomes was determined from ten different images using ‘NIS-elements’ imaging Software (Nikon, USA). The data were analyzed by the one-sample Kolmogorov-Smirnov test (SPSS IBM, USA). The average size of each exosome was 106.1 ± 26.6 nm (p value for normal distribution, 0.69). <b>(B) RNA is secreted via exosomes.</b> Cells containing the <i>SmD1</i> silencing construct were silenced for the indicated times (hours). RNA was prepared from the cells and from exosomes as described in Materials and Methods. The RNA was subjected to primer extension. The products were separated on a 6% acrylamide denaturing gel. The cap-4 nts are indicated. <b>(C) Proteins are secreted with the exosomes.</b> Proteins were extracted from exosomes prepared from 10⁹ cells and were subjected to western analysis with the indicated antibodies. Total cell extract (20 μg) was used to monitor the specificity of the antibodies. <b>(D) Immunogold-SEM detecting ZC3H41 protein on the surface of the parasite.</b> Un-induced cells containing the <i>SmD1</i> silencing construct (-Tet) and <i>SmD1</i> silenced cells at the indicated time points were subjected to immunogold staining with specific anti-ZC3H41 antibodies. Backscatter images are presented; scale bars are indicated.</p

TEM analysis of <i>SmD1</i> silenced cells.

<p>Cells before and after silencing of <i>SmD1</i> (40 hrs) were fixed, and ultra-thin sections were prepared and examined by TEM. The different ultra-structures are indicated. MVB, multivesicular bodies; ILV, intraluminal vesicles; M, mitochondrion; ER, Enodoplasmic reticulum; scale bar, 500 nm. (<b>A and B</b>) Un-induced cells (-Tet). (<b>C-G</b>) induced cells. Section showing MVB containing ILVs. <b>(C-D)</b> MVB located near the plasma membrane, and secreted exosome present on the cell surface. <b>(E-F)</b> EVs secreted from the cell membrane and flagella pocket. <b>(G)</b> Larger vesicles emerging from the flagellar membrane. The white arrow-heads indicate the ILVs and black arrow-heads the EVs.</p