Nutrients,light and primary production by phytoplankton and microphytobenthos in the eutrophic,turbid Westerschelde estuary (The Netherlands)

Abiotic factors and primary production by phytoplankton and microphytobenthos was studied in the turbid Westeschelde estuary. Because of the high turbidity and high nutrient concentrations primary production by phytoplankton is light-limited. In the inner and central parts of the estuary maximum rates of primary production were therefore measured during the summer, whereas in the more marine part spring and autumn bloom were observed. Organic loading is high, causing near anaerobic conditions upstream in the river Schelde. Because of this there were no important phytoplankton grazers in this part of the estuary and hence the grazing pressure on phytoplankton was minimal. As this reduced losses, biomass is maximal in the river Schelde, despite the very low growth rates.On a number of occasions, primary production by benthic micro-algae on intertidal flats was studied. Comparison of their rates of primary production to phytoplankton production in the same period led to the conclusion that the contribution to total primary production by benthic algae was small. The main reason for this is that the photosynthetic activity declines rapidly after the flats emerged from the water. It is argued that CO₂-limitation could only be partially responsible for the noticed decrease in activity.     

TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

Background

RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.

Methods and Results

TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105^−/− mice resulting in a higher proliferation rate. In RP105^−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm² vs.1947±278 µm²,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105^−/− mice this effect was more pronounced (10316±1243 µm² vs.4208±555 µm²,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm²,p<0.05), whereas expression of the single factors RP105 or MD1 had no effect.

Conclusion

RP105 is a potent inhibitor of post-interventional neointima formation.     

Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By coimmunoprecipitation we show that ICAP-1 and ROCK form complexes in cells and that ICAP-1 contains two binding sites for ROCK. In cells transfected with both ICAP-1 and ROCK, the proteins colocalized at the cell membrane predominantly in lamellipodia and membrane ruffles, but also in retraction fibers. ROCK was not found at these sites when ICAP-1 was not co-transfected, indicating that ICAP-1 translocated ROCK. In lamellipodia ICAP-1 and ROCK colocalized with endogenous beta1 integrins and this colocalization was also observed with the isolated ICAP-1 PTB domain. The plasma membrane localization of ROCK did not depend on beta1 integrin ligation or ROCK kinase activity, and in truncated ROCK proteins it required the presence of the ICAP-1-binding domain. To show that the interaction was direct, we measured fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) fused to ICAP-1 and yellow fluorescent protein (YFP) fused to ROCK. FRET was observed in lamellipodia in cells that were induced to spread. These results indicate that ICAP-1-mediated binding of ROCK to beta1 integrin serves to localize the ROCK-I kinase to both the leading edge and the trailing edge where ROCK affects cell migration.     

The Redox Potential of the Plastoquinone Pool of the Cyanobacterium Synechocystis Species Strain PCC 6803 Is under Strict Homeostatic Control

A method is presented for rapid extraction of the total plastoquinone (PQ) pool from Synechocystis sp. strain PCC 6803 cells that preserves the in vivo plastoquinol (PQH₂) to -PQ ratio. Cells were rapidly transferred into ice-cold organic solvent for instantaneous extraction of the cellular PQ plus PQH₂ content. After high-performance liquid chromatography fractionation of the organic phase extract, the PQH₂ content was quantitatively determined via its fluorescence emission at 330 nm. The in-cell PQH₂-PQ ratio then followed from comparison of the PQH₂ signal in samples as collected and in an identical sample after complete reduction with sodium borohydride. Prior to PQH₂ extraction, cells from steady-state chemostat cultures were exposed to a wide range of physiological conditions, including high/low availability of inorganic carbon, and various actinic illumination conditions. Well-characterized electron-transfer inhibitors were used to generate a reduced or an oxidized PQ pool for reference. The in vivo redox state of the PQ pool was correlated with the results of pulse-amplitude modulation-based chlorophyll a fluorescence emission measurements, oxygen exchange rates, and 77 K fluorescence emission spectra. Our results show that the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 is subject to strict homeostatic control (i.e. regulated between narrow limits), in contrast to the more dynamic chlorophyll a fluorescence signal.The photosynthetic apparatus of oxygenic phototrophs consists of two types of photosynthetic reaction centers: PSII and PSI. Both photosystems are connected in series, with electrons flowing from PSII toward PSI through an intermediate electron transfer chain, which comprises the so-called plastoquinone (PQ) pool, plastocyanin and/or cytochrome c₅₅₃, and the cytochrome b₆f complex. The redox potential of the PQ pool is clamped by the relative rates of electron release into and uptake from this pool. Within the PSII complex, electrons are extracted from water at the lumenal side of the thylakoid membrane and transferred to the primary accepting quinone (Q_A) at the stromal side. The electron is subsequently transferred to a PQ molecule in the secondary accepting quinone (Q_B) of PSII. The intermediate Q_B semiquinone, which is formed accordingly, is stable in the Q_B site for several seconds (Diner et al., 1991; Mitchell, 1993) and subsequently can be reduced to plastoquinol (PQH₂). The midpoint potential of Q_A reduction is approximately −100 mV (Krieger-Liszkay and Rutherford, 1998; Allakhverdiev et al., 2011), whereas the corresponding midpoint potential of the Q_B semiquinone is close to zero (Nicholls and Ferguson, 2013). PQH₂ equilibrates with the PQ pool in the thylakoid membranes, which has a size that is approximately 1 order of magnitude larger than the number of PSII reaction centers (Melis and Brown, 1980; Aoki and Katoh, 1983).PQ is a lipophilic, membrane-bound electron carrier, with a midpoint potential of +80 mV (Okayama, 1976), that can accept two electrons and two protons to form PQH₂ (Rich and Bendall, 1980). PQH₂ can donate both electrons to the cytochrome b₆f complex, one to low-potential cytochrome b₆, by which reduced high-potential cytochrome b₆ is formed, and one to the cytochrome f moiety on the lumenal side of the thylakoid membrane, where the two protons are released. High-potential cytochrome b₆ then donates an electron back to PQ on the stromal side of the membrane, rendering a semiquinone in the PQ-binding pocket on the cytoplasmic face of the b₆f complex ready as an acceptor of another electron from PSII, and reduced cytochrome f feeds an electron to a water-soluble electron carrier (i.e. either plastocyanin or cytochrome c₅₅₃) for subsequent transfer to the reaction center of PSI or to cytochrome c oxidase, respectively (Rich et al., 1991; Geerts et al., 1994; Schubert et al., 1995; Paumann et al., 2004; Mulkidjanian, 2010).Electron transfer through the cytochrome b₆f complex proceeds according to the Q-cycle mechanism (Rich et al., 1991). As a result, maximally two protons from the stroma are released into the lumen per electron transferred. This electrochemical proton gradient can be used for the synthesis of ATP by the ATP synthase complex (Walker, 1998). In PSI, another transthylakoid membrane charge separation process is energized by light. Electron transfer within the PSI complex involves iron-sulfur clusters and quinones and leads to the reduction of ferredoxin, the reduced form of which serves as the electron donor for NADPH by the ferredoxin:NADP+ oxidoreductase enzyme (van Thor et al., 1999). The ATP and NADPH generated this way are used for CO₂ fixation in a mutual stoichiometry that is close to the stoichiometry at which these two energy-rich compounds are formed at the thylakoid membrane. Normally, this ratio is ATP:NADPH = 3:2 (Behrenfeld et al., 2008).Photosynthetic and respiratory electron transport in cyanobacteria share a single PQ pool (Aoki and Katoh, 1983; Aoki et al., 1983; Matthijs et al., 1984; Scherer, 1990). Respiratory electron transfer provides cells the ability to form ATP in the dark, but this ability is not limited to those conditions. Transfer of electrons into the PQ pool is the result of the joint activity of PSII, respiratory dehydrogenases [in particular those specific for NAD(P)H and succinate], and cyclic electron transport around PSI (Mi et al., 1995; Cooley et al., 2000; Howitt et al., 2001;Yeremenko et al., 2005), whereas oxidation of PQH₂ is catalyzed by the PQH₂ oxidase, the cytochrome b₆f complex, the respiratory cytochrome c oxidase (Nicholls et al., 1992; Pils and Schmetterer, 2001; Berry et al., 2002), and possibly plasma terminal oxidase (Peltier et al., 2010). Multiples of these partial reactions can proceed simultaneously, including respiratory electron transfer during illumination (Schubert et al., 1995), which includes oxygen uptake through a Mehler-like reaction (Helman et al., 2005; Allahverdiyeva et al., 2013).Because of its central location between the two photosystems, the redox state of the PQ pool has been identified as an important parameter that can signal photosynthetic imbalances (Mullineaux and Allen, 1990; Allen, 1995; Ma et al., 2010; Allen et al., 2011). Yet, an accurate estimation of the in vivo redox state of this pool has not been reported in cyanobacteria so far. Instead, the redox state of the PQ pool is widely assumed to be reflected in, or related to, the intensity of the chlorophyll a fluorescence emissions (Prasil et al., 1996; Yang et al., 2001; Gotoh et al., 2010; Houyoux et al., 2011). Imbalance in electron transport through the two photosystems may lead to a loss of excitation energy and, hence, to a loss of chlorophyll a fluorescence emission (Schreiber et al., 1986). Therefore, patterns of chlorophyll a fluorescence (pulse-amplitude modulated [PAM] fluorimetry; Baker, 2008) have widely been adopted for the analysis of (un)balanced photosynthetic electron transfer and, by inference, for indirect recording of the redox state of the PQ pool. However, the multitude of electron transfer pathways in the thylakoid membranes of cyanobacteria (see above) makes it much more complex to explain PAM signals in these organisms than in chloroplasts (Campbell et al., 1998). Additional regulatory mechanisms of nonphotochemical quenching, via the xanthophyll cycle in chloroplasts (Demmig-Adams et al., 2012) and the orange carotenoid protein (Kirilovsky and Kerfeld, 2012) in cyanobacteria, and energy redistribution via state transitions (Allen, 1995; Van Thor et al., 1998) complicate such comparisons even further.Several years ago, an HPLC-based technique was developed for the detection of the redox state of PQH₂ in isolated thylakoids (Kruk and Karpinski, 2006), but these results have neither been related to physiological conditions nor to the results of chlorophyll a fluorescence measurements. In this report, we describe an adaptation of this method with elements of a method for estimation of the redox state of the ubiquinone pool in Escherichia coli (Bekker et al., 2007). This modified method allows for reliable measurements of the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 under physiologically relevant conditions. The method uses rapid cell lysis in an organic solvent to arrest all physiological processes, followed by extraction and identification of PQH₂ by HPLC separation with fluorescence detection. Next, we manipulated the redox state of the PQ pool with various redox-active agents, with inhibitors of photosynthetic electron flow, and by illumination with light specific for either PSII or PSI. The measured redox state of the PQ pool was then related to the chlorophyll a fluorescence signal and 77 K fluorescence emission spectra of cell samples taken in parallel and to oxygen-exchange rates measured separately. These experiments reveal that, despite highly fluctuating conditions of photosynthetic and respiratory electron flow, a remarkably stable redox state of the PQ pool is maintained. This homeostatically regulated redox state correlates poorly in many of the conditions tested with the more dynamic signal of chlorophyll a fluorescence emission, as measured with PAM fluorimetry. The latter signal only reflects the redox state of Q_A and not that of the PQ pool.     

Buoyancy regulation in Microcystis aeruginosa grown at different temperatures

Abstract The buoyancy regulation in light-limited cultures of the gas vacuolate cyanobacterium Microcystis aeruginosa AK1 was studied at three temperatures, 15, 20 and 28°C. At the two highest temperatures the organism remained buoyant during the entire light period, whereas at the lowest temperature the buoyancy was reduced at the start of the light period. With this temperature the buoyancy was lost during the light period. This reduced buoyancy was caused by an increase in ballast and a decrease in the gas vesicle volume. Buoyancy changes during a transient state with slow changes in temperatures, i.e., 1°C per day, were caused by changes in polysaccharide ballast. The gas vesicle volume showed no significant change during the transient state.
The maximal photosynthetic rate was dependent upon the growth and incubation temperature, whereas the light harvesting efficiency was independent of the temperature. The results are discussed in an ecological context.     

Cancer cells copy migratory behavior and exchange signaling networks via extracellular vesicles

Recent data showed that cancer cells from different tumor subtypes with distinct metastatic potential influence each other's metastatic behavior by exchanging biomolecules through extracellular vesicles (EVs). However, it is debated how small amounts of cargo can mediate this effect, especially in tumors where all cells are from one subtype, and only subtle molecular differences drive metastatic heterogeneity. To study this, we have characterized the content of EVs shed in vivo by two clones of melanoma (B16) tumors with distinct metastatic potential. Using the Cre‐LoxP system and intravital microscopy, we show that cells from these distinct clones phenocopy their migratory behavior through EV exchange. By tandem mass spectrometry and RNA sequencing, we show that EVs shed by these clones into the tumor microenvironment contain thousands of different proteins and RNAs, and many of these biomolecules are from interconnected signaling networks involved in cellular processes such as migration. Thus, EVs contain numerous proteins and RNAs and act on recipient cells by invoking a multi‐faceted biological response including cell migration.     

How light and biomass density influence the reproduction of delayed Saccharina latissima gametophytes (Phaeophyceae)

Kelp life-cycle transitions are complex and susceptible to various (a)biotic controls. Understanding the microscopic part of the kelp's lifecycle is of key importance, as gametophytes form a critical phase influencing, among others, the distributional limits of the species. Many environmental controls have been identified that affect kelp gametogenesis, whose interactive effects can be subtle and counterintuitive. Here we performed a fully factorial experiment on the (interactive) influences of light intensity, light quality, and the Initial Gametophyte Density (IGD) on Saccharina latissima reproduction and vegetative growth of delayed gametophytes. A total of 144 cultures were followed over a period of 21 d. The IGD was a key determinant for reproductive success, with increased IGDs (≥0.04 mg DW · mL⁻¹) practically halting reproduction. Interestingly, the effects of IGDs were not affected by nutrient availability, suggesting a resource-independent effect of density on reproduction. The Photosynthetically Usable Radiation (PUR), overarching the quantitative contribution of both light intensity and light quality, correlated with both reproduction and vegetative growth. The PUR furthermore specifies that the contribution of light quality, as a lifecycle control, is a matter of absorbed photon flux instead of color signaling. We hypothesize that (i) the number of photons absorbed, independent of their specific wavelength, and (ii) IGD interactions, independent of nutrient availability, are major determinants of reproduction in S. latissima gametophytes. These insights help understand kelp gametophyte development and dispersal under natural conditions, while also aiding the control of in vitro gametophyte cultures.     

Temperature-induced plasticity in membrane and storage lipid composition: thermal reaction norms across five different temperatures

Temperature is a key environmental factor inducing phenotypic plasticity in a wide range of behavioral, morphological, and life history traits in ectotherms. The strength of temperature-induced responses in fitness-related traits may be determined by plasticity of the underlying physiological or biochemical traits. Lipid composition may be an important trait underlying fitness response to temperature, because it affects membrane fluidity as well as availability of stored energy reserves. Here, we investigate the effect of temperature on lipid composition of the springtail Orchesella cincta by measuring thermal reaction norms across five different temperatures after four weeks of cold or warm acclimation. Fatty acid composition in storage and membrane lipids showed a highly plastic response to temperature, but the responses of single fatty acids revealed deviations from the expectations based on HVA theory. We found an accumulation of C_18:2n6 and C_18:3n3 at higher temperatures and the preservation of C_20:4n6 across temperatures, which is contrary to the expectation of decreased unsaturation at higher temperatures. The thermal response of these fatty acids in O. cincta differed from the findings in other species, and therefore shows there is interspecific variation in how single fatty acids contribute to HVA. Future research should determine the consequences of such variation in terms of costs and benefits for the thermal performance of species.     

A role for PtdIns(4,5)P2 and PIP5Kalpha in regulating stress-induced apoptosis

The phosphoinositide phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) is essential for many cellular processes and is linked to the etiology of numerous human diseases . PtdIns(4,5)P(2) has been indirectly implicated as a negative regulator of apoptosis ; however, it is unclear if apoptotic stimuli negatively regulate PtdIns(4,5)P(2) levels in vivo. Here, we show that two apoptotic-stress stimuli, hydrogen peroxide (H(2)O(2)) and UV irradiation, cause PtdIns(4,5)P(2) depletion during programmed cell death independently of and prior to caspase activation. Depletion of PtdIns(4,5)P(2) is essential for apoptosis because maintenance of PtdIns(4,5)P(2) levels by overexpression of PIP5Kalpha rescues cells from H(2)O(2)-induced apoptosis. PIP5Kalpha expression promotes both basal and sustained ERK1/2 activation after H(2)O(2) treatment, and importantly, pharmacological inhibition of ERK1/2 signaling blocks PIP5Kalpha-mediated cell survival. H(2)O(2) induces tyrosine phosphorylation and translocation of PIP5Kalpha away from its substrate at the plasma membrane, and both are dependent upon the activity of c-src family kinases. Furthermore, constitutively active c-src enhances tyrosine phosphorylation of PIP5Kalpha in vivo and is sufficient for the translocation of PIP5Kalpha away from the plasma membrane. These observations demonstrate that certain apoptotic stimuli initiate an essential signaling pathway during cell death, and this pathway leads to caspase-independent downregulation of PIP5Kalpha and its product PtdIns(4,5)P(2).