The Ca2+ response of a smart forisome protein is dependent on polymerization

Forisomes are giant self‐assembling mechanoproteins that undergo reversible structural changes in response to Ca²⁺ and various other stimuli. Artificial forisomes assembled from the monomer MtSEO‐F1 can be used as smart biomaterials, but the molecular basis of their functionality is not understood. To determine the role of protein polymerization in forisome activity, we tested the Ca²⁺ association of MtSEO‐F1 dimers (the basic polymerization unit) by circular dichroism spectroscopy and microscale thermophoresis. We found that soluble MtSEO‐F1 dimers neither associate with Ca²⁺ nor undergo structural changes. However, polarization modulation infrared reflection absorption spectroscopy revealed that aggregated MtSEO‐F1 dimers and fully‐assembled forisomes associate with Ca²⁺, allowing the hydration of poorly‐hydrated protein areas. A change in the signal profile of complete forisomes indicated that Ca²⁺ interacts with negatively‐charged regions in the protein complexes that only become available during aggregation. We conclude that aggregation is required to establish the Ca²⁺ response of forisome polymers.     

Rate of synthesis of βL-lipovitellin in the liver of immature chicks treated with 17β estradiol

Using a combination of radioimmunoprecipitation and SDS-polyacrylamide gel electrophoresis of the immunoprecipitate we studied the rate of synthesis of the heavy chain of β-lipovitellin in the liver of immature chicks. In male and female chicks the base-line synthesis of β_L-lipovitellin¹ was about 30 molecules per minute and per cell. Four days after a single injection of 40 mg estradiol/kg, as many as 48,000 molecules of β_L-lipovitellin were synthesized per minute and per diploid liver cell. The increase in the rate of β_L-lipovitellin synthesis could be correlated with an increase in membrane bound mRNA coding for β_L-lipovitellin.     

Interactions among tobacco sieve element occlusion (SEO) proteins

Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.     

Fusoselect: cell–cell fusion activity engineered by directed evolution of a retroviral glycoprotein

Membrane fusion plays a key role in many biological processes including vesicle trafficking, synaptic transmission, fertilization or cell entry of enveloped viruses. As a common feature the fusion process is mediated by distinct membrane proteins. We describe here ‘Fusoselect’, a universal procedure allowing the identification and engineering of molecular determinants for cell–cell fusion-activity by directed evolution. The system couples cell–cell fusion with the release of retroviral particles, but can principally be applied to membrane proteins of non-viral origin as well. As a model system, we chose a γ-retroviral envelope protein, which naturally becomes fusion-active through proteolytic processing by the viral protease. The selection process evolved variants that, in contrast to the parental protein, mediated cell–cell fusion in absence of the viral protease. Detailed analysis of the variants revealed molecular determinants for fusion competence in the cytoplasmic tail (CT) of retroviral Env proteins and demonstrated the power of Fusoselect.     

Proteins from the FLOWERING LOCUS T‐like subclade of the PEBP family act antagonistically to regulate floral initiation in tobacco

Flowering is an important agronomic trait that often depends on the integration of photoperiod, vernalization, gibberellin and/or autonomous signaling pathways by regulatory proteins such as FLOWERING LOCUS T (FT), a member of the phosphatidylethanolamine‐binding protein (PEBP) family. Six PEBP family proteins control flowering in the model plant Arabidopsis thaliana, and their regulatory functions are well established, but variation in the number and structural diversity of PEBPs in different species means their precise functions must be determined on a case‐by‐case basis. We isolated four novel FT‐like genes from Nicotiana tabacum (tobacco), and determined their expression profiles in wild‐type plants and their overexpression phenotypes in transgenic plants. We found that all four genes were expressed in leaves under short‐day conditions, and at least NtFT3 expression was restricted to phloem companion cells. We also found that the NtFT1, NtFT2 and NtFT3 proteins are floral inhibitors (atypical for FT‐like proteins), whereas only NtFT4 is a floral inducer. We were unable to detect the expression of these genes under long‐day conditions, suggesting that all four tobacco FT‐like proteins may control flowering in response to short days. Phylogenetic analysis of PEBP family proteins and their functions in different solanaceous species confirmed that gene duplication and divergence within the FT‐like clade has led to the evolution of antagonistic regulators that may help to fine‐tune floral initiation in response to environmental cues.     

Kinetic modeling of phototrophic biofilms: the PHOBIA model

A kinetic model for mixed phototrophic biofilms is introduced, which focuses on the interactions between photoautotrophic, heterotrophic, and chemoautotrophic (nitrifying) functional microbial groups. Biofilm-specific phenomena are taken into account, such as extracellular polymeric substances (EPS) production by phototrophs as well as gradients of substrates and light in the biofilm. Acid-base equilibria, in particular carbon speciation, are explicitly accounted for, allowing for the determination of pH profiles across the biofilm. Further to previous models reported in literature, the PHOBIA model combines a number of kinetic mechanisms specific to phototrophic microbial communities, such as internal polyglucose storage under dynamic light conditions, phototrophic growth in the darkness using internally stored reserves, photoadaptation and photoinhibition, preference for ammonia over nitrate as N-source and the ability to utilize bicarbonate as a carbon source in the absence of CO(2). The sensitivity of the PHOBIA model to a number of key parameters is analyzed. An example on the potential use of phototrophic biofilms in wastewater polishing is discussed, where their performance is compared with conventional algal ponds. The PHOBIA model is presented in a manner that is compatible with other reference models in the area of water treatment. Its current version forms a theoretical base which is readily extendable once further experimental observations become available.