This Postdoctoral Research Fellowship award to Dr. Abigail Reft is supported by both the Directorate for Biological Sciences and the Office of International Science and Engineering at the National Science Foundation. During the 24-month fellowship, Dr. Reft will work on a project titled, "Apical structures and the molecular mechanics of nematocyst discharge" under the sponsorship of Dr. Suat Özbek at Heidelberg University in Heidelberg, Germany.
All members of the group Cnidaria (which includes sea anemones, corals, and jellyfish) produce microscopic stinging capsules called nematocysts. Nematocysts consist of a tubule enfolded within a capsule. Upon stimulation, the tubule discharges from the apex of the capsule and exposes spines borne on the tubule. At the apex of the capsule is one of three different structures, each one characteristic of different lineages within Cnidaria. The process by which the apical structure opens and discharge proceeds is known only for one form and little is known about the protein components or construction of any of the forms. This project seeks to understand these biologically important cellular products by documenting the proteins involved in making the apical structure and documenting discharge in nematocysts bearing each apical form.
This project will provide insight into the construction and evolution of one of the most complex structures produced by a single cell and will provide information on mechanics of these biological machines. After identifying the components in one of the apical forms, the presence of these components in the other two forms will be evaluated. Furthermore, the impact of different apical structures in the process of discharge will be determined. Through these endeavors, the common development, discharge processes, and "toolkit" of proteins specific to apical structures will be identified, allowing some inference of the ancestral form and discharge in nematocysts.
Morphological information will be shared on the Morphobank (www.morphobank.org/) to enable collaboration with other scientists studying the evolution of morphology. Furthermore, previous collaborations with institutions such as the Field Museum and Shedd Aquarium will be continued to educate the public and disseminate research results.The PI also plans to engage members of the host lab, including mentoring of students.
This award provides a unique opportunity for a US scientist to collaborate with foreign scientists, and utilize the unique facilities, expertise and experimental conditions available abroad.
All members of the group Cnidaria (which includes sea anemones, corals, and jellyfish) produce microscopic stinging capsules called nematocysts. Nematocysts are among the most complex structures produced by an individual cell and consist of a tubule enfolded within a capsule. Upon stimulation, the tubule discharges from the apex of the capsule and exposes spines borne on the tubule and releasing toxin. At the apex of the capsule is one of three different structures, each one characteristic of different lineages within Cnidaria. The model organism Hydra magnipapillata has nematocysts that have a lid structure at the apex of its nematocysts (called an operculum). The opening of this apex and the discharge of the tubule is one of the fastest reactions in nature, as the whole process can be as short as 700 ns, yet the exact structure and mode of function of the operculum was unknown. As a first step to understanding how the operculum is built and how it functions, in the project titled, "Apical structures and the molecular mechanics of nematocyst discharge" Dr. Abigail Reft worked under the sponsorship of Dr. Suat Özbek at Heidelberg University in Heidelberg, Germany to use molecular tools to isolate and identify components of the structure. First, the operculum was extracted (along with the spines that have known protein components) using chemicals that break down the cystine-created disulfide cross bridges between proteins in the capsule and tubule. Once the structure was isolated, the samples were then run on a SDS-PAGE gel which uses charge and size of the proteins to separate them. Finally, parts of the proteins (peptides) were then identified using mass spectrometry. The identified peptides where then determined to be parts of large proteins using a database of proteins in the species identified by earlier protein work. The result is a list a proteins that are localized to the operculum that can now be explored. In exploring these proteins, a few interesting patterns were determined. First, the list provided an opportunity to look at other cnidarian species to determine if any of these proteins were shared. In fact, some proteins had similar forms in the sea anemone Nematostella vectensis, even though this species has a different apical structure indicating that these might be shared among forms of apical structure and possibly are ancestral. However, many proteins had no analog in N. vectensis which likely indicates that these are particular to the operculum. Furthermore, the type of proteins found provide some clues as to how the operculum is structured. In particular, proteins that have domains that typically function in binding bicarbonate and chitin indicate that the operculum may be impregnated with these chemicals to give it structural strength. Additionally, an odd protein that has domains very similar to some found in fungi and is involved in breaking down plant cell walls was identified as one of the most abundant proteins in the operculum. This protein had been found in previous studies in Hydra, but no work had indicated what structure or process in which it is involved. This project localizes this protein to the operculum where it could play a role in the discharge process though further work is needed to identify the exact role it plays. However, as cnidarians without operculum do not appear to have this protein, or in fact any other metazoans (the only similar proteins are found in fungi) this is likely to be an interesting protein in function and in origin. The primary results of this project are a better understanding of the nature of the operculum which will be further explored, the identification of structurally interesting proteins that may be useful in other contexts, and the training of the postdoctoral fellow in molecular techniques and confocal microscopy. Through these endeavors, the common development and "toolkit" of proteins specific to apical structures was identified, allowing some inference of the ancestral form and discharge in nematocysts providing an opportunity to better understand this complex structure, and the process of discharge.
