Unlocking the role of the symbiotic community in the calcification process of Astrangia poculata

Unlocking the role of the symbiotic community in the calcification process of Astrangia poculata Zoe Dellaert1,2, Loretta Roberson2 1University of Chicago, Chicago, IL, 2Marine Biological Laboratory, Woods Hole, MA. Calcification is the process by which reef-building corals create their skeletons. This mechanism is still not wholly understood. Symbiotic and aposymbiotic colonies of Astrangia poculata were reared in 15oC, 27oC, or ambient conditions. Scanning electron microscopy (SEM) was used to describe how these physiological and environmental conditions impact skeletal structure. Buoyant weight data over time revealed that symbiont state and temperature both significantly affect growth rates. SEM of A. poculata skeletons revealed that aposymbiotic colonies appear to have a lower density of calcium carbonate at growing septal spines. Quantitative analysis of roughness of septal spines revealed that aposymbiotic colonies have a rougher surface texture than symbiotic colonies. This roughness trend is strongest in the colonies reared at 27oC, which were also the fastest growing colonies. Subsequently, scanning electron microscopy was used to examine the calicoblastic ectoderm of A. poculata. Initial results reveal that calicoblastic cells appear to form a fine mesh across the skeleton. SEM of both the skeleton and tissue revealed a pervasive presence of bioeroders in the skeleton. Light sheet microscopy using the L-SPI Single Plane Illumination system was used to confirm and characterize this community of bioeroders. Finally, we studied skeletons of the tropical corals Porites astreoides and Acropora cervicornis to understand how A. poculata skeletal structure compares to its tropical counterparts. Few studies have examined the skeleton of A. poculata or corals in general using SEM. These results unlock new insights into the skeletons of temperate corals and the associated community. Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago Site-Directed RNA Editing Using TadAs Rosalind Pan1,2, Juan Felipe Diaz Quiroz2, Joshua J.C. Rosenthal2 1The University of Chicago, 2Marine Biological Laboratory Site-Directed RNA Editing (SDRE) is a strategy to modify genetic information at the mRNA level. The SDRE system developed by the Rosenthal Lab catalyzes adenosine (A) to inosine (I) conversion using the Deaminase Domain (DD) of ADAR2 linked to λN peptides. The λN peptides interact with boxB hairpins located in guide RNAs (gRNA) that direct the DD to the target As. The system can precisely drive A-I conversion in mRNAs, but it has many limitations including sequence context dependency and off-target editing. In this study, we replaced ADAR with TadA, a different adenosine deaminase that catalyzes A-I conversion in E. coli tRNAs. This change will allow us to use random mutagenesis in bacteria to select for TadA variants with improved editing efficiency and novel substrate recognition. To investigate whether TadA fused to λN could edit RNA, we performed in cellula and in vitro editing assays using recombinant enzymes that contained TadA homo or heterodimers. For both assays, we designed an RNA substrate that contained several target As within sequence contexts preferred by TadAs, including a target site mimicking the tRNA anticodon loop (ACL) naturally edited by TadAs. In cellula results suggested that TadA-λN could edit at the ACL target site in the absence of gRNAs. The in vitro assay recapitulated ACL editing at high efficiency and revealed an additional editing site within a motif that partially overlaps the ACL sequence. This suggested that TadA-λN activity is dependent on specific neighbouring contexts but does not require the full tRNA ACL sequence. In vitro and in cellula results also showed that TadA-λN editing may be independent of binding to gRNAs. These preliminary data suggest that using TadA as the enzymatic component for SDRE is promising but it may be necessary to develop new strategies to direct TadA to specific target sites. Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago Differences in Axonal Growth of Dopaminergic Neurons Exhibited in Familial Parkinson’s Disease Mouse Model Olivia Morales1,2, Jack Tang3, Shobana Subramanian3, Rongmin Chen3, Elizabeth Jonas2,3 1The University of Chicago, 2Marine Biological Laboratory, 3Yale University Familial Parkinson’s Disease (PD) is a neurological disorder which results in tremors, bradykinesia, and stiffness of movement. PD is caused by the degeneration of dopaminergic neurons, which are nerve cells responsible for the production of dopamine. The dysfunction and death of dopaminergic neurons results in a lack of dopamine and disrupts normal motor function. How this degeneration of dopaminergic neurons occurs, however, is still not fully understood; PD can be due to either environmental or genetic conditions. One cause of familial PD is mutation of the gene for the protein DJ-1. Wild type DJ-1 functions to decrease the uncoupling of the mitochondrial inner membrane by binding to the β subunit of the ATP synthase and thus increasing the efficiency of ATP production. It is hypothesized that this increase in efficiency results in an increase in neuronal process outgrowth. Therefore, it is expected that tyrosine hydroxylase (TH +) dopaminergic neurons of model mice lacking DJ-1 will have impaired dopaminergic neuronal outgrowth. Comparing the intensity of TH staining in substantia nigra neuron axonal arbors within brain slices will determine whether this difference exists. McCarter Metcalf Fellowship Protein palmitoylation via a specific palmitoyl transferase facilitates Golgi dispersal observed with nicotine exposure William Ramos1,3; Okunola Jeyifous2,3; Anitha Govind2; William N. Green2,3 1The College of the University of Chicago; 2The Department of Neurobiology at the University of Chicago; 3Marine Biological Laboratory Our lab has observed dispersal occurring in nicotine treated neurons and human embryonic kidney cells (HEK) expressing α4β2-type nicotinic acetylcholine receptors (α4β2Rs). α4β2Rs bind nicotine with high affinity and initiate the additive process with nicotine binding. Typical GA morphology is observed as a set of membrane stacks in the soma of neurons. During nicotine-induced dispersal we find that the stacks disperse into mobile membranes throughout dendrites and axons. Preliminary data from our lab has implicated the palmitoyl transferase, DHHC2, in the downstream events after nicotine binding to α4β2Rs causing GA dispersal. The identification of DHHC2 as being involved in Golgi dispersal is also consistent with additional evidence from our lab that the posttranslational modification, palmitoylation, is part of the signaling that leads to GA dispersal. To further test whether DHHC is involved, we overexpressed DHHC2 in HEK cells and imaged for changes in GA morphology. Fluorescently tagged sialyltransferase 3 (eGFP-ST3) and DHHC2 (myc-DHHC2) were assayed on the Zeiss spinning disk confocal system with or without the expression of tagged α4β2Rs (HA-α4β2Rs). Preliminary results suggest that DHHC2 overexpression does facilitate GA dispersal and that the GA dispersal induced by DHHC expression occurs independent of α4β2R expression. Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago Examining symbiont selection and polyp connectivity in Astrangia poculata Juliette M. Thibodeau1,2, Mayra A. Sánchez-García2, Loretta M. Roberson2 1University of Chicago, Chicago, IL, 2Marine Biological Laboratory, Woods Hole, MA Given the recent acceleration in global coral reef decline, there is a growing interest in understanding resilient corals. Increasing ocean temperatures cause a breakdown of the symbiotic relationship between corals and the dinoflagellate Symbiodinium, leading to expulsion and eventually colony mortality due to lack of nutrition normally provided by symbionts. Astrangia poculata is distinctive in that it can survive in both symbiotic and aposymbiotic states, unlike most tropical corals that have an obligate relationship with Symbiodinium. It is therefore an exceptional model system to understand this symbiosis. Our study focuses on the reintroduction of Symbiodinium following a bleaching event and the connectivity between polyps in a colony. In this study, we reintroduced symbionts in a single polyp of naturally and chemically bleached colonies of A. poculata using two different clades of cultured Symbiodinium, as well as tissue from a symbiotic A. poculata colony. Image analysis was used to evaluate symbiont density following reintroduction. We found that successful reintroduction of symbionts was independent of how the coral was bleached and the clade of symbiont used, showing that tissue reinfection is the most effective method. This study demonstrates that reintroduction of symbionts is possible in a laboratory setting for A. poculata. We also found that there was a lack of polyp connectivity in grown A. poculata colonies as well as connectivity with surrounding polyps. This suggests that A. poculata polyps lose connectivity after development, unlike those in tropical corals like Acropora and Porites, species that maintain connectivity as developed polyps. These results offer insight into the symbiosis and colonial interaction of A. poculata and how they relate to wholly tropical corals, providing a deeper understanding of resilient corals in the face of climate change. Jeff Metcalf Summer Undergraduate Research Fellowship – The University of Chicago Identifying Markers of Tergal and Precoxal Tissues in Parhyale hawaiensis Eric Chen1,2, Heather Bruce2, Nipam Patel2 1The University of Chicago, 2Marine Biological Laboratory The origin of the insect wing as an evolutionary novelty is a longstanding problem in arthropod biology. One school of thought suggests that the wing was modified from a dorsal lobe (e.g.gill or plate) on the leg of ancestral crusta