NSF

SHARING MCB SCIENCE: REGULATING NITROGENASE FORMATION IN CYANOBACTERIA

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Cyanobacteria are blue-green colored microbes with a simple cellular structure (like bacteria) and the ability to convert sunlight into chemical energy through photosynthesis (like plants). They also perform nitrogen fixation, a process by which nitrogen is extracted from the air and converted into ammonia, using an enzyme (a specialized protein) called nitrogenase. Since ammonia is a potent plant fertilizer, cyanobacteria can live symbiotically with plants in a variety of soil, water, and marsh habitats – enabling some farmers to use cyanobacteria in place of traditional fertilizers to improve the yields of rice and other staple food crops. Because of its function in nitrogen fixation, research on nitrogenase has the ability to create a firm foundation for future advances in agriculture and food security in support of the NSF’s mission to “…advance the national health, prosperity, and welfare…”

Associate Dean Dr. Teresa Thiel and her lab in the Department of Biology at the University of Missouri – St. Louis study a type of cyanobacteria called Anabaena variabilis. Uniquely, this cyanobacterium has three different nitrogenase enzymes, each capable of performing nitrogen fixation in different environmental conditions. The Thiel team previously studied each of the three nitrogenases and characterized a group of fifteen genes (called the nif1 gene cluster) whose expression through transcription (DNA to RNA) and translation (RNA to protein) is necessary to make the primary nitrogenase in Anabaena variabilis. They also identified potential sites of regulation; cells often regulate discrete steps in the protein production process as a way to conserve cellular resources by limiting the amount of protein produced when it is not needed. For years, scientists knew the important role nitrogenase played in nitrogen fixation, but had yet to uncover how cyanobacterial regulation of production of this important enzyme.

In a recent publication, Dr. Thiel and her team describe their research on one regulatory site called an RNA stem-loop. The investigators predicted this secondary structure would occur before an important gene in the nif1 cluster (called nifH1). The nifH1 gene encodes a protein largely responsible for nitrogenase enzyme assembly and function. Using a process called polymerase chain reaction (PCR) to mutate the RNA stem-loop, they studied how changes in the stem-loop altered nifH1 transcript stability and processing. The Thiel team found that mutations impacting the structure or sequence of the RNA stem-loop also severely inhibited the levels of nifH1 transcript, and most importantly, limited cyanobacteria’s ability to perform nitrogen fixation.

These findings have potential for modulating the efficiency of nitrogen fixation in cyanobacteria, leading to more fertilizer production, and a potential source of renewable energy by harnessing the hydrogen created during nitrogen fixation. This work also may impact an exciting area of bioengineering research. As described in a MCB awarded US and UK research BBSRC-collaboration Ideas Lab proposal, bioengineers are attempting to create a “nitroplast” cellular structure, patterned after the nitrogenase in cyanobacteria, to allow plants to make their own fertilizer.

When asked about the broader impacts of her research, Dr. Thiel responded:

The engagement of scientists with the larger scientific and non-scientific community is critical to promoting a public understanding of science and in attracting students to careers in science. To do so, the broader impacts of my research include integrating research within graduate, undergraduate, and high school education. Students from Jennings Senior High School, a predominantly African-American high school located in North St. Louis County, have participated in 6 weeks of summer research as part of the Jennings at UMSL Program, which is designed to help students succeed in college. Additionally, a student from the UMSL SUCCEED program, which supports vocational experiences for students with intellectual or developmental disabilities works as a laboratory aide in my lab. Furthermore, I participate in educational outreach activities in the St. Louis community, working with local high school teachers to incorporate hands-on microbiology activities in their classrooms.

This work is partially funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1052241.

MCB WELCOMES DR. CASONYA JOHNSON, PROGRAM DIRECTOR FOR THE GENETIC MECHANISMS CLUSTER

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What were you doing before you came to the NSF?

I am an associate professor in the Department of Biology at Georgia State University. I teach courses in genetics to students at all levels and conduct research with my students to investigate the underlying mechanisms by which transcriptional regulators direct post-embryonic development—in other words, we want to understand how the molecules that regulate the process of making RNA from DNA affect the development of an organism after the embryo stage.

What attracted you to work for NSF?

I was attracted by the opportunity to be at the forefront of cutting edge research, to expand my own knowledge of my research field, and to understand how funding trends are directed.

What was your first impression of NSF? Has this impression changed since you began serving as a rotator?

My first impression was that the impact of NSF (on science as a whole) extends far beyond the individual research laboratory. I have only been here a month, but my impression stands.

What are the personal goals you most want to accomplish while at NSF?

I want to learn as much as I can, about everything I can; to find ways to broaden my research focus; to find ways to communicate to the research community the ways in which NSF supports research; and to find ways to better engage the general public so that everyone can understand the need for and benefits of basic scientific research.

What has surprised you most about working at NSF?

I think I am most surprised about how much support – from IT to administrative to security – is offered here. That type of support is sometimes missing in academia, so I am used to spending time trying to figure things out for myself, when here all I need to do is ask for help.

What are some of the challenges of serving as a rotator?

The learning curve is very steep. The biggest challenge is fighting the feeling that I’m not moving fast enough to get things done. The other challenge is making sure that my students and my personal research do not suffer while I am here.

What would you tell someone who is thinking about serving as a program director at NSF?

Do it! Your colleagues at NSF will help you succeed and at a minimum, you will leave with a much better understanding of how NSF works.

When your friends/colleagues find out that you work at NSF, what do they say or ask?

All have responded “What an amazing opportunity!” Then, they ask if I like it and who is taking care of my lab.

SHARING MCB SCIENCE: DISCOVERY OF A NON-BASE FLIPPING MECHANISM IN DNA REPAIR

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Eichman Lab members involved in the study (from left to right): Dr. Elwood Mullins, Dr. Brandt Eichman, Rongxin Shi, and Dr. Zachary Parsons. Photo Credit: Susan Urmy/Vanderbilt

The DNA of humans, like that of all other organisms, can be damaged, acquiring what are referred to as “lesions.” A common form of DNA damage is DNA alkylation, where a small group of carbons and hydrogens (alkyl group) are chemically bound to the base of DNA nucleotides (the As, Ts, Cs, and Gs that make up DNA). When a DNA base is alkylated, the normal function of the cell’s DNA is disrupted and the genetic information being stored is mutated, which has the potential to develop into some types of cancer and threaten the survival of the organism.

To protect the organism from the effects of DNA lesions, cells have processes to repair DNA. One such process is called base excision repair, which was one subject of last year’s Nobel Prize in Chemistry. As shown in the figure below, base excision repair begins with DNA glycosylase (ie. a protein with enzymatic function that initiates a process), which is able to bind to double-stranded DNA and look for DNA lesions using a base-flipping mechanism. In base-flipping, a DNA nucleotide that is suspected of containing an alkyl group is flipped away from its base pair partner and into the active site of the DNA glycosylase. If the DNA glycosylase sees a lesion, it severs the chemical bond that links the DNA base to the DNA backbone and initiates subsequent repair steps, ultimately restoring the DNA to an undamaged state.

Until recently, it was thought that all DNA glycosylases used base-flipping to repair damaged DNA. A paradigm shift occurred in the DNA repair field when a non-base-flipping DNA glycosylase enzyme, called AlkD, was discovered by Professor Dr. Brandt Eichman in the Department of Biological Sciences and Center for Structural Biology at Vanderbilt University and his research group, in collaboration with Professor Dr. Sheila David and her research group at University of California Davis and Professor Dr. Yasuhiro Igarashi at the Toyama Prefectural University in Japan. Repair that does not involve base-flipping has also been shown by the Eichman team to uniquely allow the repair of bulky DNA lesions.

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Space-filling models (left) and illustrations (right) showing base-flipping excision repair (top) and non-base-flipping excision repair (bottom). Top: A damaged DNA base (blue) from a double stranded DNA helix (orange and yellow) is inserted, or “flipped,” into the active site of the DNA glycosylase enzyme (white or grey). Bottom: A bulky chemical group (purple) attached to a DNA base (blue) results in a lesion within a double stranded DNA helix (orange and yellow) that is repaired without base-flipping by a DNA glycosylase enzyme (AlkD) (white or grey).

As described in a recent publication in Nature, the Eichman research team used a technique called X-ray crystallography to capture a series of time-lapsed 3D renderings of AlkD as it repaired a lesion. The Eichman team’s conclusion that AlkD removes DNA damage using a non-base-flipping mechanism was supported by their crystallographic analysis which showed the AlkD enzyme mainly contacted the DNA backbone, not the DNA lesion. Thus, non-base-flipping broadens the spectrum of DNA damage that DNA glycosylases are known to repair. Also, the 3D structure of AlkD is common to proteins that do not have enzymatic functions, which makes it difficult for researchers to identify non-base-flipping DNA glycosylases just based on their structure. Therefore, there is a strong possibility there are other DNA repair proteins that scientists have yet to identify.

When asked about the broader impacts of his research, Dr. Eichman responded: “This research program has involved trainees from all levels—undergraduate, graduate, and postdoctoral—several of whom have continued on in a number of scientific careers, including medical school, science policy, and industry. Most importantly, it has enabled us to expose undergraduates to cutting edge structural biology and to the practical aspects of X-ray crystallography, both in the classroom and in the lab.”

This work is funded jointly by the Genetic Mechanisms program in the Division of Molecular and Cellular Biology (MCB) and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate of Mathematical and Physical Sciences, Award #MCB-1122098 and Award #MCB-1517695.

FAREWELL TO DR. MANJU HINGORANI

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First Row (Left to Right): Dr. Karen Cone, Dr. Theresa Good, Dr. Manju Hingorani, Dr. Charlie Cunningham; Second Row (Left to Right): Keshanti Tidwell, Dr. Stacy Kelley, Dr. Linda Hyman, Dr. Susanne von Bodman, and Dr. Wilson Francisco

The Division of Molecular and Cellular Biosciences (MCB) gave a warm send off to Dr. Manju Hingorani, former Program Director in the MCB Genetic Mechanisms program.

During her two year tenure at the NSF, Dr. Hingorani worked with investigator-driven proposals submitted to both the Genetic Mechanisms and the Cellular Dynamics and Function programs. As a rotating Program Director, Dr. Hingorani managed proposal reviews and awards and responded to inquiries from principal investigators conducting fundamental research related to the central dogma of biology. Dr. Hingorani noted she particularly enjoyed managing CAREER proposal reviews because it gave her glimpses of potential future leaders in science and education. Dr. Hingorani also aided in the review of NSF Graduate Research Fellowship Program proposals, appreciating the chance to serve in a program that has benefitted students from her home institution.

As Dr. Hingorani returns to her position as Professor of Molecular Biology and Biochemistry at Wesleyan University, she looks forward to reconnecting with her students “in 3D,” in her laboratory, and in classes. Unfortunately for us, she will take most of her Swiss chocolate stash back with her!

MCB would like to thank Dr. Manju Hingorani for her service, and we wish her all the best in the future. If you are interested in serving like Dr. Hingorani as a rotating MCB Program Director, please contact us at 703-292-8440 and read the rotator Dear Colleague Letter.

Mr. Casey Bethel: Recipient of Georgia’s 2017 Teacher of the Year Award Following a NSF Research Experience for Teachers (RET)

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Dr. Raquel Lieberman (Left) and Mr. Casey Bethel, Georgia’s 2017 Teacher of the Year (Right)

Mr. Casey Bethel was recently honored as Georgia’s 2017 Teacher of the Year. He teaches advanced placement (AP) Biology, AP Physics, Biology, and Physical Sciences at New Manchester High School in Douglasville, Georgia. Recipients of this prestigious award are outstanding local and state public school teachers in Georgia who serve as shining examples of excellence in education, and Mr. Bethel is the first STEM teacher in over a decade to receive this award. He notes, “This award is a huge honor, and in many ways it serves as validation of the hard work and sacrifices I have put into growing in this career. I hope that it further inspires my students to work hard and pursue their dreams.”

Mr. Bethel credits his accomplishment and growth as an educator to the many summers he spent working in Dr. Raquel Lieberman’s lab supported in part by a Division of Molecular and Cellular Biosciences (MCB)-funded Research Experience for Teachers (RET) supplement. As described in the Dear Colleague Letter (NSF 12-075), RET supplements enable K-12 science educators to participate in NSF-funded scientific research projects with the goal of enhancing their professional development through the experience of conducting research at the emerging frontiers of science in order to bring new knowledge to the classroom. Dr. Lieberman actively recruited Mr. Bethel and requested a RET supplement when designing the broader impacts of her MCB-funded 2009 CAREER award. You can find out more about the Faculty Early Career Development Program (CAREER) award here.

The Lieberman lab uses techniques, such as protein crystallography and computer modeling, to determine structure–function relationships of proteins associated with Alzheimer’s disease and glaucoma. Mr. Bethel notes, “Dr. Lieberman welcomed me and made me a contributing member of her team. Every year since, my wealth of knowledge has grown and my teaching practices have improved. My students are better prepared for college science courses now, and more than 50 of them are excelling in STEM majors and careers.” Additional outcomes of the RET experience for Mr. Bethel and Dr. Lieberman include co-authorship of a scientific research paper undergoing peer review, and the publication of a teaching unit describing multimedia-guided inquiry for high school science classrooms in the Journal of Chemical Education.

Join us in congratulating Mr. Casey Bethel as Georgia’s 2017 Teacher of the Year and acknowledging the commitment of Dr. Raquel Lieberman to expanding the broader impacts of her research as MCB celebrates this outstanding recognition.

This work is partially funded by the Division of Molecular and Cellular Biosciences, Award #MCB-0845445.

SHARING MCB SCIENCE: Cellular Polarization in Yeast

Many cellular processes are regulated by the ability of a cell to transmit and receive signaling molecules such as hormones, cytokines, and neurotransmitters. The binding of a signaling molecule to the extracellular portion of a receptor protein embedded in a cell’s membrane starts a signaling cascade inside the cell, which activates intracellular proteins and changes cellular responses. G proteins constitute a large family of intracellular proteins that act as molecular switches inside the cell upon coupling with G protein receptors in the membrane.

The mating process in Saccharomyces cerevisiae (budding yeast) requires the activation of a heterotrimeric G protein switch in response to the yeast cell’s G protein receptors binding pheromone signaling molecules released by a potential mate. This activation results in the growth of a mating projection–or shmoo–extending in the direction of the pheromone source. Cellular growth in response to a chemical stimulus is called chemotropism. The location and accumulation of G protein receptors across the cell membrane plays a critical role in the cell’s ability to detect the pheromone source and directional growth towards a mating partner. However, some of the molecular mechanisms underlying shmoo growth and G protein receptor accumulation have not been well understood until recently. A discovery by Dr. David Stone, a Professor in the Department of Biological Sciences at the University of Illinois at Chicago, and his research team has led to advancements in this area.

As mentioned previously, G protein receptors that bind pheromones are not uniformly distributed in the cell’s membrane. Instead, they cluster wherever the pheromone concentration is the highest and a shmoo grows in the direction of the cell’s suitor. The interaction between the pheromone and G protein receptor causes intracellular signaling, resulting in the delivery of secretory vesicles and cytoskeletal rearrangement. The delivery of the secretory vesicles is actin cable-directed. These processes ultimately lead to yeast cell fusion and reproduction. The mechanism that Dr. Stone and his team wanted to understand was how G protein receptors polarize towards the source of the pheromone gradient and determine the position of the shmoo growth site prior to the delivery of secretory vesicles and cytoskeletal rearrangement.

In an article published in Science Signaling, the Stone lab showed that the interaction of an enzyme called yeast casein kinase 1 (Yck1) with a G protein (Gβ) at the cell membrane plays an important role in the establishment of polarity by inhibiting phosphorylation (addition of a phosphoryl group) and internalization of the G protein receptor. The authors further showed differential phosphorylation of the receptor is essential to pheromone gradient sensing and generates receptor polarity independently of actin-cable nucleation and vesicle delivery. Dr. Stone and his team also proposed that differences in G protein receptor occupancy modulate the chemotropic growth site in budding yeast through signal amplification produced by two positive feedback loops that the authors describe in the publication.

When asked about the broader impacts of his research, Dr. Stone responded:

“My lab has contributed to an emerging paradigm in G-protein coupled receptor (GPCR) signaling which is of broad interest to the cell signaling community: GPCRs and their associated heterotrimeric G proteins are spatiotemporally regulated, and in turn, regulate spatiotemporal processes. We were among the first groups to show that heterotrimeric G proteins serve to recruit effector molecules to specific regions of the plasma membrane at specific times.

My NSF-sponsored research program has involved scientists in training at all levels — from middle school and high school students to undergraduate students, graduate students and postdoctoral fellows. Most notably, I worked closely with a group of 6th graders from a local middle school to formulate a proposed experiment to be carried out on the International Space Station. NASA picked our experiment from a national pool of applicants, and the experiment was performed by astronauts on the International Space Station. The students set up the experiment and analyzed the results in my lab. The launch of their experiment was covered by local media. Over the years, my research groups have included a significant number of women and underrepresented minorities (>50%). The training of my graduate students has been highly enriched by their visits to the labs of our numerous collaborators, who have provided them with experience in a range of approaches—e.g., proteomics, advanced imaging, microfluidics, and mathematical modeling.”

This work is partially funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1415589.

SHARING MCB SCIENCE: EVIDENCE FOR AUTOPHAGY-DEPENDENT PATHWAYS OF RIBOSOMAL RNA TURNOVER IN ARABIDOPSIS

Ribosomes play an essential role in protein manufacturing in the cell, and are made up of ribosomal RNA (rRNA) and proteins. While scientists understand a great deal about how ribosomes are created, surprisingly little is known about how they are broken down at the end of their useful life. Understanding ribosome turnover is important, because each cell invests a lot of energy and resources to maintain a sufficient number of ribosomes to keep up with its protein production demands.

As described in a recent publication in the journal Autophagy, a collaboration between Iowa State University’s Loomis Professor of Plant Physiology Dr. Diane Bassham, Associate Professor of Biochemistry Dr. Gustavo MacIntosh, and their research groups resulted in the discovery that eukaryotic cells may be using a process called autophagy to break down ribosomes. In autophagy, a compartment (called an autophagosome) is created by autophagy-related proteins (ATGs) around the cargo slated for destruction, and the autophagosome with its cargo is trafficked from the cytoplasm of the cell to an organelle called a vacuole (in plant cells) or a lysosome (in animal cells). Fusion with the vacuole or lysosome results in the cargo being deposited inside the organelle with the enzymes required for its destruction. One such enzyme, called RNS2, is from the RNase T2 family of ribonucleases (enzymes that break down RNA into smaller components).

Hypothesizing that the process of autophagy may play a role in breakdown of ribosomes, the research team developed a method to measure ribosomal RNA accumulation in the vacuole of mutant plant (Arabidopsis thaliana) cells lacking the RNS2 ribonuclease. The mutant is called rns2-2. The researchers used confocal microscopy to look for evidence of autophagy activation and the accumulation of autophagosomes. Fluorescent labeling of an ATG protein on the surface of autophagosomes provided evidence of an increased number of autophagosomes (indicated by small, fluorescent blue dots in the image) in the rns2-2 mutant when compared to normal, wild type (WT) Arabidopsis thaliana plant cells. Bassham DataThis result allowed the research team to further hypothesize that autophagy activation in the rns2-2 mutant was compensation for the plant cells’ inability to degrade rRNA with the vacuolar ribonuclease RNS2.

The research team also found evidence of the involvement of more than one autophagy pathway in the breakdown of ribosomes. As described in their publication, mutations in an autophagy gene (ATG5) blocked the activity of the autophagy pathway and prevented accumulation of rRNA in the vacuole. But, mutations in a different autophagy gene (ATG9), did not prevent accumulation of the rRNA in the vacuole, suggesting the pathway used by ATG5 and ATG9 to deliver rRNA to the vacuole may be different. As Dr. Bassham notes, “Our results shed light on the mechanisms by which ribosomal components are recycled and in turn, on the way in which ribosome number and quality are controlled.”  Dr. Bassham credits NSF MCB support to “allow Dr. Gustavo MacIntosh and I to reinforce and expand our collaboration by establishing a group consisting of a post-doctoral researcher, several graduate students, and undergraduate students to work together in the analysis of the relationship between autophagy and RNA degradation, allowing progress that would have not been possible had our research groups continued to work independently.”

When asked about the broader impacts of her research, Dr. Bassham responded:

“A major impact of our project has been training students in research. In addition to training several graduate students and a post-doc who worked full time on the research project, ten undergraduate students participated in laboratory research in our summer internship program, including several from the primarily undergraduate institution Grand View University in Des Moines, IA, who would otherwise not have the opportunity to gain research experience. A second outcome is the continued development, headed by Iowa State University Professor Eve Wurtele, of an educational video game called Meta!Blast that is used to teach cell biology to undergraduate and high school students. In the game, the player navigates a three-dimensional cellular environment within a plant and completes tasks based on cell functions and biochemical reactions. Interactivity aids retention and understanding of concepts and the use of multimedia allows us to reach diverse populations of students. The process of autophagy was incorporated into the game as a result of this project.”

This work is funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1051818.

Types of Submissions

The Proposal & Award Policies & Procedures Guide (PAPPG) comprises documents relating to the Foundation’s proposal and award process.

In this ongoing segment of our blog, we will provide a series of infographic snapshots to highlight some of the important information found in the PAPPG. This in no way replaces the need to read the PAPPG before submitting a proposal.  The goal of this ongoing blog theme is to make the information in the PAPPG more accessible to our readers by providing clear, concise, colorful, and informative graphics.

For complete and official information about type of submissions, please refer to the PAPPG (effective January 25, 2016).

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* Preliminary proposals are part of the proposal process in the Division of Environmental Biology and the Division of Integrative Organismal Systems in the Directorate for Biological Sciences.

Welcome to MCB Kelly Ann Parshall!

Hear from Program Specialist Kelly Ann Parshall

What is your educational background?

I majored in English writing with a concentration in African Studies. My objective was to work in development in sub-Saharan Africa. However, when I received my Peace Corps invitation, it was to the South Pacific. Despite the surprise, I had a great experience working in health and environmental initiatives in the small island nation of Vanuatu. This fall I will start attending American University part-time to pursue a masters in Global Environmental Policy.

What is your position? When did you start working in MCB?

As a Program Specialist, I support the Genetic Mechanisms cluster as well as the Systems and Synthetic Biology cluster. I started working in MCB in April 2016.

What attracted you to work for NSF?

When my Peace Corps service concluded, I knew I wanted to work for the government or at a nonprofit. I particularly loved my third year assignment working for the German government on climate change, conservation and natural resource management initiatives. In the biology community in Vanuatu, I saw extraordinary technology-facilitated advances like drones zapping invasive crown of thorns starfish to save reefs. While the projects MCB funds are a bit different, I am happy to support such a wonderful mission.

What have you learned so far from your position?

As someone who has worked in aid, I have spent a significant amount of time applying to grants, assembling funding leads and liaising with donors. It’s nice to be on the other side! NSF operates on a significantly larger scale than any organization I’ve ever been a part of before. It’s amazing to see the thoroughness and transparency with which grantees are selected. I’m looking forward to supporting the entire proposal process as the year progresses.

Categories of Funding Opportunities

The Proposal & Award Policies & Procedures Guide (PAPPG) comprises documents relating to the Foundation’s proposal and award process.

In this new segment of our blog, we will provide a series of infographic snapshots to highlight some of the important information found in the PAPPG. This in no way replaces the need to read the PAPPG before submitting a proposal.  The goal of this ongoing blog theme is to make the information in the PAPPG more accessible to our readers by providing clear, concise, colorful, and informative graphics.

PAPPG

Click here for the direct link to the “Categories of Funding Opportunities” section.