An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-crosslinking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein:His6– Xpr–GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activitywas observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization

Impact of polystyrene nanoparticles on marine diatom Skeletonema marinoi chain assemblages and consequences on their ecological role in marine ecosystems.

Marine diatoms have been identified among the most abundant taxa of microorganisms associated with plastic waste collected at sea. However, the impact of nano-sized plastic fragments (nanoplastics) at single cell and population level is almost unknown. We exposed the marine diatom Skeletonema marinoi to model polystyrene nanoparticles with carboxylic acid groups (PS-COOH NPs, 90 nm) for 15 days (1, 10, 50 mu g/mL). Growth, reactive oxygen species (ROS) production, and nano-bio-interactions were investigated. No effect on diatom growth was observed, however Dynamic light scattering (DLS) demonstrated the formation of large PS aggregates which were localized at the diatoms' fultoportula process (FPP), as shown by TEM images. Increase production of ROS and reduction in chain length were also observed upon PS NPs exposure (p < 0.005). The observed PS-diatom interaction could have serious consequences on diatoms ecological role on the biogeochemical cycle of carbon, by impairing the formation of fast-sinking aggregates responsible for atmospheric carbon fixation and sequestration in the ocean sea floor. S. marinoi exposure to PS NPs caused an increase of intracellular and extracellular oxidative stress, the reduction of diatom's chain length and the adhesion of PS NPs onto the algal surface

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX

Introduction: In the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed “pollensomes”, are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification. Methods: To do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics. Results: These analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes. Discussion: The presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles

Neisserial adhesin A (NadA) binds human Siglec-5 and Siglec-14 with high affinity and promotes bacterial adhesion/invasion

ABSTRACT Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the K D value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection. IMPORTANCE Bacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death. </jats:sec

Small extracellular vesicles released from germinated kiwi pollen (pollensomes) present characteristics similar to mammalian exosomes and carry a plant homolog of ALIX

IntroductionIn the last decade, it has been discovered that allergen-bearing extracellular nanovesicles, termed “pollensomes”, are released by pollen during germination. These extracellular vesicles (EVs) may play an important role in pollen-pistil interaction during fertilization, stabilizing the secreted bioactive molecules and allowing long-distance signaling. However, the molecular composition and the biological role of these EVs are still unclear. The present study had two main aims: (I) to clarify whether pollen germination is needed to release pollensomes, or if they can be secreted also in high humidity conditions; and (II) to investigate the molecular features of pollensomes following the most recent guidelines for EVs isolation and identification.MethodsTo do so, pollensomes were isolated from hydrated and germinated kiwi (Actinidia chinensis Planch.) pollen, and characterized using imaging techniques, immunoblotting, and proteomics.ResultsThese analyses revealed that only germinated kiwi pollen released detectable concentrations of nanoparticles compatible with small EVs for shape and protein content. Moreover, a plant homolog of ALIX, which is a well-recognized and accepted marker of small EVs and exosomes in mammals, was found in pollensomes.DiscussionThe presence of this protein, along with other proteins involved in endocytosis, is consistent with the hypothesis that pollensomes could comprehend a prominent subpopulation of plant exosome-like vesicles

Detection and localization of pectin methylesterase isoforms in pollen tubes of Nicotiana tabacum L.

Pectin methylesterases (PMEs) were detected in tobacco (Nicotiana tabacum) pollen tubes grown in vitro. Seven PME isoforms exhibiting a wide isoelectric-point (pI) range (5.3-9.1) were found in crude extracts of pollen tubes. These isoforms were mainly retrieved in supernatants after low- and high-speed separation of the crude extract. Two isoforms, with pIs 5.5 and 7.3 and molecular weight about 158 kDa, were detected by immunoblotting with anti-flax PME antiserum. Localization of pectins and PME isoforms in pollen tubes was investigated by immunogold labelling with JIM5 monoclonal antibodies and anti-flax PME antiserum, respectively. In germinated pollen grains, two PME isoforms were mainly detected in the exine, Golgi apparatus and secretory vesicles. In pollen tubes the same two PME isoforms were distributed along the outer face of the plasma membrane in the vicinity of the inner layer of the cell wall, in the Golgi and around secretory vesicles. In pollen grains, PME isoforms were, in some cases, mixed with acidic pectins m proximity to the outer surface of the plasma membrane. In pollen tubes the presence of PMEs inside secretory vesicles carrying esterified pectins supports the hypothesis that, during pollen tube growth, PMEs could be transferred by secretory vesicles in a precursor form and be activated at the tip where exocytosis takes place

Comparative ecotoxicity of polystyrene nanoparticles in natural seawater and reconstituted seawater using the rotifer Brachionus plicatilis

The impact of nanoplastics using model polystyrene nanoparticles (PS NPs), anionic (PS-COOH) and cationic (PS-NH2), has been investigated on the marine rotifer Brachionus plicatilis, a major component of marine zooplanktonic species. The role of different surface charges in affecting PS NP behaviour and toxicity has been considered in high ionic strength media. To this aim, the selected media were standardized reconstituted seawater (RSW) and natural sea water (NSW), the latter resembling more natural exposure scenarios. Hatched rotifer larvae were exposed for 24\uc2&nbsp;h and 48\uc2&nbsp;h to both PS NPs in the range of 0.5\ue2\u80\u9350\uc2&nbsp;\uce\ubcg/ml using PS NP suspensions made in RSW and NSW. No effects on lethality upon exposure to anionic NPs were observed despite a clear gut retention was evident in all exposed rotifers. On the contrary, cationic NPs caused lethality to rotifer larvae but LC50values resulted lower in rotifers exposed in RSW (LC50=2.75\uc2\ub10.67\uc2&nbsp;\uc2\ub5g/ml) compared to those exposed in NSW (LC50=6.62\uc2\ub10.87\uc2&nbsp;\uc2\ub5g/ml). PS NPs showed similar pattern of aggregation in both high ionic strength media (RSW and NSW) but while anionic NPs resulted in large microscale aggregates (Z-average 1109 \uc2\ub1 128\uc2&nbsp;nm and 998\uc2\ub167\uc2&nbsp;nm respectively), cationic NP aggregates were still in nano-size forms (93.99 \uc2\ub1 11.22\uc2&nbsp;nm and 108.3 \uc2\ub1 12.79\uc2&nbsp;nm). Both PDI and Z-potential of PS NPs slightly differed in the two media suggesting a role of their different surface charges in affecting their behaviour and stability. Our findings confirm the role of surface charges in nanoplastic behaviour in salt water media and provide a first evidence of a different toxicity in rotifers using artificial media (RSW) compared to natural one (NSW). Such evidence poses the question on how to select the best medium in standardized ecotoxicity assays in order to properly assess their hazard to marine life in natural environmental scenarios