Construction and composition of the squid pen from Doryteuthis pealeii

Author Posting. © University of Chicago Press, 2019. This article is posted here by permission of University of Chicago Press for personal use, not for redistribution. The definitive version was published in Messerli, M. A., Raihan, M. J., Kobylkevich, B. M., Benson, A. C., Bruening, K. S., Shribak, M., Rosenthal, J. J. C., & Sohn, J. J. Construction and composition of the squid pen from Doryteuthis pealeii. Biological Bulletin. 237(1), (2019): 1-15, doi:10.1086/704209.The pen, or gladius, of the squid is an internalized shell. It serves as a site of attachment for important muscle groups and as a protective barrier for the visceral organs. The pen’s durability and flexibility are derived from its unique composition of chitin and protein. We report the characterization of the structure, development, and composition of pens from Doryteuthis pealeii. The nanofibrils of the polysaccharide β-chitin are arranged in an aligned configuration in only specific regions of the pen. Chitin is secreted early in development, enabling us to characterize the changes in pen morphology prior to hatching. The chitin and proteins are assembled in the shell sac surrounded by fluid that has a significantly different ionic composition from squid plasma. Two groups of proteins are associated with the pen: those on its surface and those embedded within the pen. Only 20 proteins are identified as embedded within the pen. Embedded proteins are classified into six groups, including chitin associated, protease, protease inhibitors, intracellular, extracellular matrix, and those that are unknown. The pen proteins share many conserved domains with proteins from other chitinous structures. We conclude that the pen is one of the least complex, load-bearing, chitin-rich structures currently known and is amenable to further studies to elucidate natural construction mechanisms using chitin and protein.We thank John Dowling for financial support. We thank Kasia Hammar and Louie Kerr of the Marine Biological Laboratory Central Microscopy Facility for help obtaining scanning electron micrographs. We thank Bogdan Budnik and Renee Robinson from the Mass Spectrometry and Proteomics Resource Laboratory for their help and advice with protein identification. We thank Shin-Yi Marzano and Chenchen Feng of South Dakota State University for help with rapid amplification of cDNA ends. Funding for this work was provided by the Eugene and Millicent Bell Fellowship Fund in Tissue Engineering (MAM), an Agriculture and Biological Sciences Undergraduate Research Award (KSB), National Institutes of Health grant R01 GM101701 (MS), National Science Foundation grant IOS1557748 (JJCR), and Israel-United States Binational Science Foundation 2013094 (JJCR). Literature Cited2020-07-0

Exploring the Properties of Chitin for Promoting Tissue Engineering and Repair

Chitin is the second most abundant biopolymer found in nature and is present predominantly in the exoskeleton, endoskeleton, and cell wall of many organisms. It has great economic value for its properties and is extensively used for industrial and biomedical purposes. Chitin-protein composites in different organisms are usually localized in their external shells and are heavily mineralized. Self-assembly of shells from mollusks, arthropods and cephalopods is of fundamental interest to understand evolutionary diversity and learn construction mechanisms from nature to promote assembly of new materials with commercial applications. It is difficult to study natural assembly of complex chitin-protein composites, and very little is known so far. To learn how chitin and proteins are assembled into 3- dimensional weight-bearing structures it would be ideal to study a simple chitinous structure comprised of hydrated chitin without metals that could be studied at the cellular and molecular level from its embryonic origin and also easily extracted in bulk quantity from the adult. The pen or gladius of D. pealeii is one such example. While most chitinbearing structures are external to the organism’s anatomy, the pen of the squid is constructed internally. The pen is a translucent, tough, yet flexible skeletal element, involved with maintaining the structure of the mantle during jet propulsion. It is a multilamellar assembly of chitin and protein without mineralization or sclerotization. We have identified that the pen is constructed with chitin and very few proteins. We have studied the 20 most abundant embedded proteins in the chitin-protein composite in the pen. While we have identified some of these proteins, many of them remain to be characterized. We have used microscopy, CP-NMR, RACE PCR, and different bioinformatics tools to characterize and obtain full-length sequences for the proteins. We have used the transcriptome, proteome and genome of D. pealeii to assemble the building blocks of the gladius. We compare these protein sequences with the predicted sequences in the genomes of the elusive giant squid, Architeuthis dux, and the bobtail squid, Euprymna scolopes. The giant squid, like D. pealeii assembles a gladius, while the bobtail squid does not. This unique comparison has enabled us to identify protein sequences that appear unique to the construction of squid gladii. These efforts will be used to help identify the chemistry behind the cellular attachment to chitin as well as chemical modifications to promote chitin as a printable scaffold for tissue engineering