Sharing Science

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.

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.

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.

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. This 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.

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