Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials (APs) in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by several-fold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2–3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

The bone morphogenetic proteins (BMPs) constitute a subfamily of the transforming growth factor type beta (TGF-) supergene family. BMP-2 plays an important role not only in osteoblast differentiation but also in pattern formation during development. To determine the function of BMP-2 in Pinctada fucata development and hard tissue formation, we isolated a BMP-2 genomic DNA clone and the BMP-2 cDNA. The deduced BMP-2 sequence consisted of 447 amino acids. The BMP-2 gene was composed of three exons. The C-terminal portion (149 amino acids) had 86% and 66% identity to the Crassostrea gigas and the human BMP-2 sequence respectively. The 5 0 flanking promoter region contained putative glioma transcription factor (Gli) and retinoic acid receptor (RAR) responding elements. The BMP-2 gene was expressed strongly in the inner part of the mantle tissue, corresponding to the nacreous aragonite shell layer. This finding suggests that BMP-2 has a key role in nacreous layer formation.

The hard tissue of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the outer prismatic layer, bearing calcite, and the inner nacreous layer, bearing aragonite. An EDTA-insoluble fraction of the prismatic layer of P. fucata was extracted with urea. In-vitro crystallization experiments showed that this urea-soluble fraction contained the factor(s) that promoted the growth of calcite crystals. We purified a protein from this fraction and deduced the internal amino acid sequences EYDFDRPDPYDP and EYDFERPD. We performed 3' RACE using primer DPPF1, encoding EYDFDRPDPYDP, and an oligo-dT adapter primer and amplified a fragment of approximately 300 bp. We screened cDNA libraries using the 300 bp fragment and obtained two clones that we named prismin 1 and 2. Both cDNAs encode proteins of 51 amino acids. Homology searches revealed 91% amino acid identity between prismin 1 and 2. The synthetic peptide DFDRPDPYDPYDRFD, corresponding to the carboxy terminal region of prismin 1, has calcite growing activity and calcium binding capability, showing that the carboxy-terminal region is a functional domain. Prismin 1 is expressed strongly in the outer edge and in the inner part of the mantle tissue. However, immunoblot analysis revealed that prismin protein exists only in the prismatic layer, not in the nacreous layer, despite the presence of the mRNA. Therefore, we conclude that prismin is a novel prismatic layer-specific calcite growth factor.

Bone morphogenetic protein (BMP)-2 plays an important role in morphogenesis in both vertebrates and invertebrates. BMP-2 is one of the most powerful bioactive substances known to induce the osteogenic differentiation of mesenchymal cells. We examined the structural and functional conservation of Pinctada fucata BMP-2 in inducing osteogenesis in the murine mesenchymal stem cells, C3H10T1/2. Exposure of C3H10T1/2 cells to the recombinant mature fragment of Pinctada fucata BMP-2 resulted in osteoblastic differentiation. The sequence, SVPKPCCVPTELSSL, within the C-terminal portion of Pinctada fucata BMP-2, is homologous to the knuckle epitope of human BMP-2. This synthetic polypeptide was able to induce differentiation of C3H10T1/2 along the osteoblastic lineage, as confirmed by an increase in alkaline phosphatase activity, and the accumulation of calcium, as determined by von Kossa staining. Furthermore, using immunohistochemical staining, we observed an increased expression of collagen type I, osteopontin, and osteocalcin, which are known markers of osteogenesis. These results show that BMP-2 is conserved, not only in terms of its homology at the amino acid sequence, but also in terms of driving the formation of hard tissues in vertebrates and invertebrates.