A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic-inorganic hybrid materials in sponge primmorphs.

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nanoparticles were detected within intracellular vesicles resembling silicasomes (silica-rich cellular compartments) and as cytosolic clusters where they lent primmorphs fluorescent/magnetic properties. During spiculogenesis, the nanoparticles initially formed an incomplete layer around juvenile, intracellular spicules. In the mature, extracellular spicules the nanoparticles were densely arranged as a surface layer that rendered the resulting composite fluorescent and magnetic. By branching off the conventional route of solid-state materials synthesis under harsh conditions, a new pathway has been opened to a versatile platform that allows adding functionalities to growing spicules as templates in living cells, using nonbiogenic nanoscale building blocks with multiple functionalities. The magnet-assisted alignment renders this composite with its fluorescent/magnetic properties potentially suitable for application in biooptoelectronics and microelectronics (e.g., microscale on-chip waveguides for applications of optical detection and sensing).

An evolutionary perspective on the role of mesencephalic astrocyte-derived neurotrophic factor (MANF): At the crossroads of poriferan innate immune and apoptotic pathways.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a recently discovered family of neurotrophic factors. MANF can be secreted but is generally resident within the endoplasmic reticulum (ER) in neuronal and non-neuronal cells, where it is involved in the ER stress response with pro-survival effects. Here we report the discovery of the MANF homolog SDMANF in the sponge Suberites domuncula. The basal positioning of sponges (phylum Porifera) in the animal tree of life offers a unique vantage point on the early evolution of the metazoan-specific genetic toolkit and molecular pathways. Since sponges lack a conventional nervous system, SDMANF presents an enticing opportunity to investigate the evolutionary ancient role of these neurotrophic factors. SDMANF shares considerable sequence similarity with its metazoan homologs. It also comprises a putative protein binding domain with sequence similarities to the Bcl-2 family of apoptotic regulators. In Suberites, SDMANF is expressed in the vicinity of bacteriocytes, where it co-localizes with the toll-like receptor SDTLR. In transfected human cells, SDMANF was detected in both the organelle protein fraction and the cell culture medium. The intracellular SDMANF protein level was up-regulated in response to both a Golgi/ER transport inhibitor and bacterial lipopolysaccharides (LPS). Upon LPS challenge, transfected cells revealed a decreased caspase-3 activity and increased cell viability with no inducible Bax expression compared to the wild type. These results suggest a deep evolutionary original cytoprotective role of MANF, at the crossroads of innate immune and apoptotic pathways, of which a neurotrophic function might have arisen later in metazoan evolution.

Self-assembly and photocatalytic activity of branched silicatein/silintaphin filaments decorated with silicatein-synthesized TiO2 nanoparticles.

The fundamental mechanisms of biomineralization and their translation into innovative synthetic approaches have yielded promising perspectives for the fabrication of biomimetic and bioinspired organic-inorganic hybrid materials. In siliceous sponges, the enzyme silicatein catalyzes the polycondensation of molecular precursors to nano-structured SiO2 that is deposited on self-assembled filaments consisting of the two silicatein isoforms (silicatein-α and -ß) and the scaffold protein silintaphin-1. Due to its broad substrate specificity silicatein is also able to convert in vitro various other precursors to non-biogenic materials (e.g., hydrolysis of titanium bis(ammonium lactato)-dihydroxide [TiBALDH] and subsequent polycondensation to titania [TiO2]). In the present approach, silicatein was bioengineered to carry a protein tag (Arg-tag) that confers binding affinity to TiO2. Then, by combining Arg-tagged silicatein-α with silicatein-ß and silintaphin-1, self-assembled branched hybrid protein microfilaments were fabricated. Upon subsequent incubation with TiBALDH the filaments were decorated with TiO2 and assayed for photocatalytic activity through photodegradation of the dye methylene blue. This is the first approach that considers concomitant application of two silicatein isoforms for the synthesis of bioinspired organic-inorganic hybrid materials. It is also the first time that the biocatalytic activity of the enzymes has been combined with both the structure-providing properties of silintaphin-1 and a TiO2 affinity protein tag to fabricate self-assembled branched protein filaments as template for a silicatein-synthesized TiO2 photocatalyst. The TiO2-decorated filaments might be explored as a practical alternative to approaches where biotemplates have to be laboriously isolated from their original biological source prior to TiO2 immobilization.

Amorphous Ca²âº polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells.

Polyphosphate (polyP) is a physiologically occurring polyanion that is synthesized especially in bone-forming osteoblast cells and blood platelets. We used amorphous polyP nanoparticles, complexed with Ca(2+), that have a globular size of ∼100 nm. Because polyP comprises inorganic orthophosphate units that are linked together through high-energy phosphoanhydride bonds, we questioned whether the observed morphogenetic effect, elicited by polyP, is correlated with the energy-generating machinery within the cells. We show that exposure of SaOS-2 osteoblast-like cells to polyP results in a strong accumulation of mitochondria and a parallel translocation of the polyP-degrading enzyme alkaline phosphatase to the cell surface. If SaOS-2 cells are activated by the mineralization activation cocktail (comprising ß-glycerophosphate, ascorbic acid and dexamethasone) and additionally incubated with polyP, a tenfold intracellular increase of the ATP level occurs. Even more, in those cells, an intensified release of ATP into the extracellular space is also seen. We propose and conclude that polyP acts as metabolic fuel after the hydrolytic cleavage of the phosphoanhydride linkages, which contributes to hydroxyapatite formation on the plasma membranes of osteoblasts.

Cryptochrome in sponges: a key molecule linking photoreception with phototransduction.

Sponges (phylum: Porifera) react to external light or mechanical signals with contractile or metabolic reactions and are devoid of any nervous or muscular system. Furthermore, elements of a photoreception/phototransduction system exist in those animals. Recently, a cryptochrome-based photoreceptor system has been discovered in the demosponge. The assumption that in sponges the siliceous skeleton acts as a substitution for the lack of a nervous system and allows light signals to be transmitted through its glass fiber network is supported by the findings that the first spicules are efficient light waveguides and the second sponges have the enzymatic machinery for the generation of light. Now, we have identified/cloned in Suberites domuncula two additional potential molecules of the sponge cryptochrome photoreception system, the guanine nucleotide-binding protein ß subunit, related to ß-transducin, and the nitric oxide synthase (NOS)-interacting protein. Cryptochrome and NOSIP are light-inducible genes. The studies show that the NOS inhibitor L-NMMA impairs both morphogenesis and motility of the cells. Finally, we report that the function of primmorphs to produce reactive nitrogen species can be abolished by a NOS inhibitor. We propose that the sponge cryptochrome-based photoreception system, through which photon signals are converted into radicals, is coupled to the NOS apparatus.

Metazoan circadian rhythm: toward an understanding of a light-based zeitgeber in sponges.

In all eukaryotes, the 24-h periodicity in the environment contributed to the evolution of the molecular circadian clock. We studied some elements of a postulated circadian clock circuit in the lowest metazoans, the siliceous sponges. First, we identified in the demosponge Suberites domuncula the enzyme luciferase that generates photons. Then (most likely), the photons generated by luciferase are transmitted via the biosilica glass skeleton of the sponges and are finally harvested by cryptochrome in the same individual; hence, cryptochrome is acting as a photosensor. This information-transduction system, generation of light (luciferase), photon transmission (through the siliceous spicules), and photon reception (cryptochrome), all occur in the same individual. Therefore, we propose that this photoreception/phototransduction process might function as a nerve-cell-like signal transmitting system. This was corroborated by the fact that S. domuncula reacts to different wavelengths of light, originating from the sponge environment, with a differential gene expression of the transcription factor SOX. Recently, we succeeded in demonstrating that in sponges a light/dark controlled gene is expressed, which encodes for nocturnin, a protein showing poly(A)-specific 3'-exoribonuclease activity. Quantitative real-time polymerase chain reaction analyses revealed that primmorphs, 3D cell aggregates of sponge cells, after transfer from light to dark, show a 10-fold increased expression of the nocturnin gene. In contrast, the expression level of the gene encoding glycogenin decreases in the dark by three- to four-fold. It is concluded that sponges are provided with the molecular circadian clock protein nocturnin which is highly expressed in the dark. This finding together with the proposed light-transduction and spicule-based signaling system strongly supports the view that already the lowest metazoans, the sponges, have elements of a circadian rhythm, characteristic of higher metazoans.

Meta-analysis of the effects of human disturbance on seed dispersal by animals.

Animal-mediated seed dispersal is important for sustaining biological diversity in forest ecosystems, particularly in the tropics. Forest fragmentation, hunting, and selective logging modify forests in myriad ways and their effects on animal-mediated seed dispersal have been examined in many case studies. However, the overall effects of different types of human disturbance on animal-mediated seed dispersal are still unknown. We identified 35 articles that provided 83 comparisons of animal-mediated seed dispersal between disturbed and undisturbed forests; all comparisons except one were conducted in tropical or subtropical ecosystems. We assessed the effects of forest fragmentation, hunting, and selective logging on seed dispersal of fleshy-fruited tree species. We carried out a meta-analysis to test whether forest fragmentation, hunting, and selective logging affected 3 components of animal-mediated seed dispersal: frugivore visitation rate, number of seeds removed, and distance of seed dispersal. Forest fragmentation, hunting, and selective logging did not affect visitation rate and were marginally associated with a reduction in seed-dispersal distance. Hunting and selective logging, but not fragmentation, were associated with a large reduction in the number of seeds removed. Fewer seeds of large-seeded than of small-seeded tree species were removed in hunted or selectively logged forests. A plausible explanation for the consistently negative effects of hunting and selective logging on large-seeded plant species is that large frugivores, as the predominant seed dispersers for large-seeded plant species, are the first animals to be extirpated from hunted or logged forests. The reduction in forest area after fragmentation appeared to have weaker effects on frugivore communities and animal-mediated seed dispersal than hunting and selective logging. The differential effects of hunting and selective logging on large- and small-seeded tree species underpinned case studies that showed disrupted plant-frugivore interactions could trigger a homogenization of seed traits in tree communities in hunted or logged tropical forests.

A cryptochrome-based photosensory system in the siliceous sponge Suberites domuncula (Demospongiae).

Based on the light-reactive behavior of siliceous sponges, their intriguing quartz glass-based spicular system and the existence of a light-generating luciferase [Müller WEG et al. (2009) Cell Mol Life Sci 66, 537-552], a protein potentially involved in light reception has been identified, cloned and recombinantly expressed from the demosponge Suberites domuncula. Its sequence displays two domains characteristic of cryptochrome, the N-terminal photolyase-related region and the C-terminal FAD-binding domain. The expression level of S. domuncula cryptochrome depends on animal's exposure to light and is highest in tissue regions rich in siliceous spicules; in the dark, no cryptochrome transcripts/translational products are seen. From the experimental data, it is proposed that sponges might employ a luciferase-like protein, the spicular system and a cryptochrome as the light source, optical waveguide and photosensor, respectively.