Long-time lab member Zack Haley recently graduated from Ashland University and will be entering the Case Western Reserve University School of Dental Medicine this Fall. Zack started working in our lab during his sophomore year and took the lead on our current project characterizing how mammals and zebrafish regulate the expression of alpha crystallins, proteins involved in protecting cells from damage during physiological stress.
During his time in our lab Zack presented his research at a meeting of the Ohio Academy of Sciences and at two undergraduate research symposia at Ashland. He was a co-author on a poster I presented at last month’s Seattle meeting of the Association for Research in Vision and Ophthalmology. Check back soon for some news about the trip to Seattle. In the meantime, the zebrafish embryo below is data from our poster showing green fluorescent protein expressed under the control of a promoter for mouse alpha B-crystallin. This result shows that zebrafish cells are able to “read” this mouse DNA sequence and direct the expression of the gene under its control.
GFP protein expression driven in the zebrafish notochord by a mouse alpha B-crystallin promoter.
One of my undergraduate research students, Josh Allman, was recently accepted into a summer research program at Michigan State University in plant genomics funded by the National Science Foundation. Josh is doing his independent research in my lab on the regulation of alpha crystallin expression in zebrafish. Last summer he worked with me to genotype populations of the grass Phragmites to distinguish between native and invasive population. This work was part of a recent publication in the Journal of Chemical Ecology.
Josh will be returning to AU for his senior year with plans to enter graduate school in molecular biology or bioengineering.
The storms last week along the Outer Banks of the North Carolina Coast brought flooding in Kitty Hawk and again washed out route 12 through Pea Island. But the rough surf also brought some interesting marine animals onto the beach. Sunny, warm weather on Friday provided a good chance to check them out. In the photo below of the white baby ears you can see them still attached to their large muscular feet. Click on each image to see a larger version.
Assorted purse crabs and sea stars washed up from recent March storms
Purse crabs, Persephona punctata. Arranged with males on the left and females on the right.
White baby ears, Sinum perspectivum, arranged to show their muscular foot.
Phragmites stand in Duck, NC
Our lab had the opportunity to contribute to a new paper in the Journal of Chemical Ecology that shows the release of toxic gallic acid by the common reed Phragmites australis is not the key to its successful invasion of freshwater and brackish habitats in North America. A series of previous publications suggested that release of gallic acid might explain the reed’s ability to produce large stands and exclude native plants. However, work by our collaborators in the Ashland University Department of Chemistry with four Phragmites populations from Ohio and North Carolina found only trace amounts of gallic acid in the plant and none in surrounding soils.
Josh Allman, a junior conducting research in my lab, performed an analysis of a genomic region that differs between North American native Phragmites populations and the invasive strain. Josh’s restriction length polymorphism analysis showed that all four populations in our study were invasive and, according to past work, should contain gallic acid. The lack of gallic acid in these populations suggests that at a minimum gallic acid release is not a general explanation for the invasive success of Phragmites.
Students in Ashland University’s Marine Biology course collecting Phragmites samples in Nags Head, NC
Students in my spring 2012 marine biology course took part in the collection of samples from North Carolina during a 4-day field trip to the Outer Banks. Another student in my lab, Kelly Sullivan, is following up on this work by developing methods to study whether chemicals produced by Phragmites repel the snail herbivores in their habitat. Hopefully I can report back on that work soon.
A recent issue of the Journal of Experimental Biology included a summary of our PLoS One paper on thermal adaptation in the small heat shock protein alpha A crystallin. Our study identified single amino acids that affect alpha A crystallin’s ability to protect other proteins from stress by comparing the structure and function of this protein from six fish species living at different temperatures. We used this information to genetically engineer a zebrafish alpha A crystallin with the enhanced protective abilities found in the Antarctic toothfish.
Thanks to JEB and the author of the summary, Hans Merzendorfer of the University of Osnabruek, for a great explanation of our work.
Congratulations to former lab member Carrie LaCava who recently graduated from The Ohio State College of Veterinary Medicine and will begin practicing in southern Illinois. Carrie will be working in a practice with five other doctors conducting appointments and surgeries with both large and small animals, including farm visits.
While an undergraduate at Ashland University Carrie worked in my lab to produce the first proteomic map of the adult zebrafish lens using two-dimensional gel electrophoresis. Another student and I are now writing up a follow up study examining how the protein content of zebrafish lenses changes during the development of the lens. These studies will allow us to better understand the protein damage that causes lens cataracts, as well as provide foundational information on the roles that lens proteins called crystallins play in the development of the lens.
I’m excited to announce that our latest paper has been published in PLoS One. OK, this news is a little old since the paper came out March 29th, but with the end of the academic year, a trip to the ARVO eye meetings in Florida and the start of summer research with three undergraduate students in lab it has taken me some time to write this post.
Antarctic toothfish alpha A crystallin has evolved more flexibility to function at low temperatures
My lab studies the function of a family of small heat shock proteins called alpha crystallins, which plays a role in keeping the lens of the eye transparent and focusing light on the retina. These proteins are also found in other tissues like brain and muscle where they protect cells during physiological stress. Alpha crystallins keep other proteins from sticking to each other during this stress, which could otherwise cause a wide range of diseases such as Alzheimer’s and Parkinson’s. Recent research also shows that more alpha crystallin is made during many types of cancer, perhaps as a protective response by cells. We would like to better understand how alpha crystallins, and small heat shock proteins in general, protect other proteins and prevent disease. Understanding this function might allow us to design altered alpha crystallins with greater protective abilities. Our approach to studying alpha crystallin function is a bit unique as we examine these proteins in fishes. Why fishes? We use them as a model to dissect how natural selection has altered these proteins to function in different environmental settings. In particular, we looked at alpha crystallin function in fish species with different body temperatures to see how evolution has molded this protein to protect other proteins in bodies as different as the Antarctic toothfish (-2 degrees C) and the zebrafish (27 degrees C). This type of study is not possible in mammalian models like mice and rabbits whose body temperatures are all similar.
Structure of alpha A crystallin showing position of three amino acids that affect protective function
We hypothesized that the protective abilities of alpha A-crystallin, one of three alpha crystallins found in fishes, had evolved to function at the specific body temperature of the six fishes in our study. We found just that. When all six fish alpha A-crystallins were compared side by side at the same temperature, those from the cooler bodied fishes (like the Antarctic toothfish) were more flexible to compensate for the stabilizing effects of cold temperature. This greater flexibility allowed them to protect other proteins more readily than the comparatively stiffer alpha crystallins from the warmer species. By comparing the structure of all six proteins we identified three amino acid building blocks that differed between the cold and warm fishes that could cause this increase in protective function.
The most exciting part of our study was that when we took a zebrafish alpha A-crystallin and genetically engineered it to look like the Antarctic toothfish at the three amino acids, two of the changes increased its protective function. By comparing alpha crystallins from these six fish species we were able to identify specific parts of the protein that may be evolving to fine-tune protective ability to different body temperatures, and then showed experimentally that those changes are functionally significant. Because alpha A-crystallin is so well conserved between fishes and mammals we now want to see if similar changes will increase protection in the human version of this protein. While it may seem unusual to study a protein that causes human disease in a bunch of fish, our new study shows that this comparative approach can be quite effective. Stay tuned for updates.
Kayaking out of Manteo harbor with the Queen Elizabeth II in the background
This Thursday morning my marine biology class at Ashland University will be piling into two vans and trekking for 13 hours to the Oregon Inlet Campground on the outer banks of North Carolina for three days in the field. This is the fourth time I have taken this class to the Outer Banks for an end of semester trip, after previous expeditions to the much colder Assateague Island, Maryland. On past trips I have posted photos to this blog and shot video in the hopes of editing it together into a summary of our trips. But that editing never happened after the last trip as I succumbed to summer research and administrative duties.
Plans for this year are a bit different. Instead of relying primarily on this blog, I will be tweeting from the trip and am encouraging my students to do the same. They are required to keep field notes from the sites we visit, but I have offered them the option to keep those notes via Twitter. With 12 students and three faculty on the trip I hope to have a solid crowdsourced record of the trip. You can follow these tweets at #aubio412.
Once we get back I plan to turn the tweets, YouTube video and any other material into a story on Storify. Wish us luck.
Zach Haley, a junior biology major working in my lab on the regulation of alpha crystallin expression, gave his first research poster presentation on Tuesday at Ashland University’s Undergraduate Research and Creative Activity Symposium. This event gives students from all disciplines in the College of Arts and Sciences the opportunity to present their independent work in oral and poster sessions.
Zach is developing methods for using the zebrafish as a model to analyze the function of mammalian alpha crystallin promoters, the regions of the gene that determines when and where its protein is produced. Understanding promoter function can provide clues to how alpha crystallins are used in healthy cells, and why their levels are often increased or reduced during disease.
Why test mammalian genes in a zebrafish? The ability to produce large numbers of transparent zebrafish embryos in a relatively small facility makes this model species less expensive and faster to use than mice. Zach’s work will help to determine whether this approach is feasible.
- Zach Haley presents his work on alpha crystallin gene regulation
I thought it would be appropriate to break my long hiatus from this blog with an introduction of our latest lab members. Last time I posted we were seeing off three lab alumni to a job, graduate and professional schools. But the lab is full again.
From left to right in the photo (with a short description of their current projects) are Mary Brown (roles of alpha crystallins in the zebrafish lens), Lynette Vana (pesticide toxicity), Josh Allman (cloning of zebrafish genes involved in neural development) and Zach Haley (regulation of alpha crystallin expression). That is me in the back. The photo is taken in the scenic Rybolt greenhouse at Ashland University.
Check back for some exciting lab news later this week and future updates on our latest research.