Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM)*

Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca²⁺ efflux pump, plasma membrane Ca²⁺ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4–64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.     

Copepod grazing during an iron-induced diatom bloom in the Antarctic Circumpolar Current (EisenEx): I. Feeding patterns and grazing impact on prey populations

Feeding activity, selective grazing and the potential grazing impact of two dominant grazers of the Polar Frontal Zone, Calanus simillimus and Rhincalanus gigas, and of copepods < 2 mm were investigated with incubation experiments in the course of an iron fertilized diatom bloom in November 2000. All grazers were already actively feeding in the low chlorophyll waters prior to the onset of the bloom. C. simillimus maintained constant clearance rates and fed predominantly on diatoms. R. gigas and the small copepods strongly increased clearance and ingestion of diatoms in response to their enhanced availability. All grazers preyed on microzooplankton, most steadily on ciliates, confirming the view that pure herbivory appears to be the exception rather than the rule in copepod feeding. The grazers exhibited differences in feeding behavior based on selectivity indices. C. simillimus and R. gigas showed prey switching from dinoflagellates to diatoms in response to the phytoplankton bloom. All grazers most efficiently grazed on large diatoms leading to differences in daily losses for large and small species, e.g. Corethron sp. or Thalassionema nitzschioides. Species-specific diatom mortality rates due to grazing suggest that the high feeding activity of C. simillimus prior to and during the bloom played a role in shaping diatom population dynamics.     

Identification and comprehensive analysis of the CLV3/ESR‐related (CLE) gene family in Brassica napus L.

• The CLE (CLAVATA3/ESR) gene family, encoding a group of small secretory peptides, plays important roles in cell‐to‐cell communication, thereby controlling a broad spectrum of development processes. The CLE family has been systematically characterized in some plants, but not in Brassica napus.
• In the present study, 116 BnCLE genes were identified in the B. napus genome, including seven unannotated, six incorrectly predicted and five multi‐CLE domain‐encoding genes. These BnCLE members were separated into seven distinct groups based on phylogenetic analysis, which might facilitate the functional characterization of the peptides.
• Further characterization of CLE pre‐propeptides revealed 31 unique CLE peptides from 45 BnCLE genes, which may give rise to distinct roles of BnCLE and expansion of the gene family. The biological activity of these unique CLE dodecamer peptides was tested further through in vitro peptide assays. Variations in several important residues were identified as key contributors to the functional differentiation of BnCLE and expansion of the gene family in B. napus. Expression profile analysis helped to characterize possible functional redundancy and sub‐functionalization among the BnCLE members.
• This study presents a comprehensive overview of the CLE gene family in B. napus and provides a foundation for future evolutionary and functional studies.

     

Downscaling indicators of forest habitat structure from national assessments

Downscaling is an important problem because consistent large-area assessments of forest habitat structure, while feasible, are only feasible when using relatively coarse data and indicators. Techniques are needed to enable more detailed and local interpretations of the national statistics. Using the results of national assessments from land-cover maps, this paper demonstrates downscaling in the spatial domain, and in the domain of the habitat model. A moving window device was used to measure structure (habitat amount and connectivity), and those indicators were then analyzed and combined with other information in various ways to illustrate downscaling.     

Alleviation of sodicity stress on rice genotypes by Phosphorus fertilization

Rice seedlings transplanted into sodic soil are exposed to an excess of potentially toxic ions as well as nutritional imbalance, both of which adversely affect their growth and yield. The present study was aimed to investigate the beneficial effects of fertilization with phosphorus and potassium on the plants at varying sodicity levels and also the response of genotypes with known variability in their tolerance to sodicity. In pot-house experiments during two seasons, the alleviating effects of P and K fertilization on three rice genotypes were examined at four sodicity levels. Seedlings of CSR13 and Jaya (both moderately tolerant to sodicity), died by 25–35 days after transplanting in sodic soils of pH 9.7–9.9 where Olsen's P was 12.5 and 14.8 kg/ha, respectively. However, there was no problem of survival or growth in these soils when Olsen's P was 17.6 and 20.8 kg/ha. Depletion in P from 12.0 kg to 10 kg resulted in some mortality of the seedlings even at pH 9.1. Sodicity tolerant genotype CSR10, did show some survival and growth even at pH 9.9 with Olsen's P at 14.8 kg/ha (without P fertilization) which suggests that differences in tolerance to sodicity which exist at genotypic level are not masked by low P. None of the three genotypes showed any survival problem at pH 8.0 and 8.1 with Olsen's P at 8.5 and 8.7 kg/ha, respectively. Seedlings in P fertilized sodic soils not only produced significantly more new roots but also higher root biomass than those in unfertilized sodic soils and these roots seem to have some control on Na uptake as reflected by low Na concentration in the shoots. Thus, P fertilization not only improved P and K status of plants but also reduced the concentration of potentially toxic Na ions in shoots, resulting in better survival, growth and yield. Although fertilization with K alone did improve shoot K content, it had no significant effect on reducing Na. So the mortality of the seedlings or grain yield in K fertilized sodic soils was as good as in control and this could be explained on the basis of lack of any significant difference in Na concentrations in shoots between these two treatments.     

Early ontogeny of Adinia xenica (Pisces,Cyprinodontiformes): 2. Implications of embryonic resting interval for larval development

Synopsis Larval development commences with first exogenous feeding, and ends with final remodelling of caenogenetic structures into the definitive organs of juvenile and adult. For the intertidal cyprinodontid Adinia xenica this generally corresponds to the interval between hatching and completion of scalation. The final step of the embryo period is a resting interval of variable duration. Embryos were induced to hatch after 2 and 10 days of this near arrest. Although the general pattern of larval development was the same for both groups, differences were observed in the rates and order of calcification of skeletal elements, fin differentiation and growth, and scalation. For example, embryos hatching 8 days later in the resting interval were already pattially calcified, but completed calcification at a slower rate than the group hatching after 2 days. These differences may be due to effects of the duration of the resting interval itself; or they may reflect genetic variation of which age at hatching is only one manifestation.     

A segmentation clock patterns cellular differentiation in a bacterial biofilm

1. Download : Download high-res image (171KB)
2. Download : Download full-size image