Sharing MCB Science

SPOTLIGHT ON MCB FUNDED SCIENCE

A spotlight illuminates the words 'Spotlight on MCB-funded Science.'

Photo Credit: Matusciac Alexandru/Shutterstock.com

Sharing MCB Science is one of our six blog themes where you can learn about exciting MCB-funded research submitted by our investigators (via this webform). We greatly appreciate the overwhelmingly positive response of the MCB scientific community and have received many more submissions than can be featured in long form on the blog. Enjoy this shorter spotlight of submissions we have received!

a closeup of a cell, it is shapes like a lollypop, a long thin stalk with a rounded top which is darker in color

Joseph K. E. Ortega – Photograph of a stage IV sporangiophore of Phycomyces blakesleeanus with the micro-capillary tip of a pressure probe.

Algal, fungal, and plant cells interact with their environment by regulating their size and shape through expansive growth, an increase in cell volume due predominately to an increase in water uptake. This process presents a special challenge for algae, fungi, and plants because cells in these organisms have an exoskeleton-like cell wall that provides support, protection, and shape.  When water enters these cells, turgor pressure builds up, which stretches (deforms) the cell wall; at the same time, new material is added to the cell wall to fill in the expanded regions and thereby control the size and shape of the enlarging cell. The interconnected processes of water uptake, wall deformation, and control of cell size and shape are crucial for algal, fungal, and plant survival.

Previous research has provided a good description of the molecular and mechanical changes accompanying expansive growth.  Taking advantage of this foundation, Dr. Joseph K. E. Ortega, Professor of Mechanical Engineering at the University of Colorado, Denver, is bringing a new dimension to quantify cellular changes during expansive growth.  He has developed a mathematical model of the interconnected processes, called the Augmented Growth Equations (AGE). As described in his new publication, the model organizes multiple equations to represent the relationships between variables and uses dimensional analysis to produce dimensionless coefficients. The dimensionless coefficients enable researchers to more easily quantify the biophysical processes and better predict how changes in water absorption and cell wall deformation regulate expansive growth. While the model does not address the shape of the cells, the mathematical framework provides insight as to how water uptake and wall deformation are regulated in algal, fungal, and plant cells to control expansive growth during normal conditions and in response to changes in the environment.

This work is partially funded by the Molecular Biophysics Cluster of the Division of Molecular and Cellular Biosciences, Awards #MCB-0948921.

on the left is a still image from an MRI of a chest with the heart and lungs visible. On the right is a graph with the quick heartbead and slower respiration plotted as curves.

Dr. Steven Van Doren – Example of how the TREND software can distinguish the patterns of a heart beating and lungs breathing from an MRI movie

Visual outputs, such as photographs or movies, contain important data from scientific experiments. For example, identifying biologically relevant signals over time (trends) can be challenging, as they may be subtle or mixed into background movements or noise. To identify trends, researchers scour the data looking for specific features, such as peaks, and either mark them by hand, which is time consuming and subjective, or set specific background thresholds in instrumentation, which can result in mistaking signal for noise. In a new publication, Dr. Steven Van Doren, Professor of Biochemistry at the University of Missouri, and his post-doctoral researcher, Dr. Jia Xu, describe a new software program called TREND (Tracking and Resolving Equilibrium and Nonequilibrium population shifts in Data). The software allows researchers to objectively extract information from two-dimensional images or videos, and in another recent publication, they describe extending the analysis to several different types of data.

TREND uses a statistical approach, called principal component analysis (PCA), on a series of individual measurements to compress and organize multivariate data so that researchers can select for and detect changes in a variety of factors over time. The factors can be anything: characteristics of a stream, grades in a class, or gene variants. When applied to movies, such as this sample video of a sunset, a trend in the data, such as the movement of the sun across the sky, can be plotted after removing background noise, such as motion from clouds. Another example is separating the pattern of a heart beating from lungs breathing using TREND (a photo of this video is shown above). TREND is available for licensing and download at http://biochem.missouri.edu/trend and https://nmrbox.org/.

 

This work is partially funded by the Molecular Biophysics Cluster of the Division of Molecular and Cellular Biosciences, Awards #MCB-1409898.

Spotlight on MCB-funded Science

 

A spotlight illuminates the words 'Spotlight on MCB-funded Science.'

Photo Credit: Matusciac Alexandru/Shutterstock.com

Sharing MCB Science is one of our six blog themes where you can learn about exciting MCB-funded research submitted by our investigators (via this webform). We greatly appreciate the overwhelmingly positive response of the MCB scientific community and have received many more submissions than can be featured in long form on the blog. Enjoy this shorter spotlight of submissions we have received!

Ever wonder how a cell makes a tough decision? When food is scarce, Bacillus subtilis (a common soil bacteria) faces a difficult choice of when to shut down cellular processes and become dormant via sporulation (spore formation). Timing is key: wait too long and die from starvation; sporulate too early and die from crowding by rapidly dividing neighboring bacteria. What serves as the trigger – a specific biochemical signal or a more general physiological response – to enable starvation sensing and sporulation was unknown. As part of a collaborative project, Dr. Oleg Igoshin, an Associate Professor in the Department of Bioengineering at Rice University, Dr. Masaya Fujita, an Associate Professor in the Department of Biology and Biochemistry at the University of Houston, and their research teams applied computational and mathematical tools to this biological question. As described in this publication, they discovered the rate at which the cell grows may serve as a signal of starvation, triggering spore formation. This work could lessen food spoilage and control food-borne pathogens by offering new ways to inhibit sporulation in close relatives of B. subtilis that live on food.

This work is partially funded by the Systems and Synthetic Biology Cluster of the Division of Molecular and Cellular Biosciences, Awards #MCB – 1244135 and #MCB – 1244423.

Diatoms (a unicellular photosynthetic microalgae) are an important part of food webs, especially in areas of the ocean with an abundance of fish frequented by the fishing industry. Because conditions and availability of environmental resources change, diatoms regulate physiological functions (such as the carbon-concentrating mechanisms (CCMs) and photorespiration previously described) at the level of gene expression. Instead of focusing on one environmental condition or type of diatom, Dr. Justin Ashworth (Post-doctoral Fellow),  Dr. Monica Orellana (Principal Scientist) and Dr. Nitin Baliga (Senior Vice President and Director) of the Institute for Systems Biology integrated all publicly available microarray data (displaying gene expression levels) from multiple conditions for the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum to look for trends. As described in this publication and in the resulting integrative analysis available online at the Diatom Portal, the research team uncovered common patterns of gene expression and function. They also identified potential cis-regulatory DNA sequence motifs and distinct regions induced in response to changes in ocean pH levels and the availability of nitrate, silicic acid, and carbon. A greater understanding of this fundamental level of regulation enables scientists to better support diatoms in their role as biogeochemical nutrient recyclers.

This work is partially funded by the Cellular Dynamics and Function Cluster of the Division of Molecular and Cellular Biosciences, Award #MCB – 1316206.

As we previously described on the MCB Blog, the laboratory of Dr. Alexander Mankin and Dr. Nora Vázquez-Laslop at the Center for Biomolecular Sciences, University of Illinois – Chicago, studies fundamental mechanisms in protein synthesis. Ribosomes inside the cell read three mRNA nucleotides at a time (a reading frame) during protein synthesis (translation). Sometimes, the ribosome slips one or two nucleotides on the mRNA to a different reading frame (frameshift). Recent work on the E. coli bacterial copper transporter gene (copA) by Drs. Mankin, Vázquez-Laslop, and their research team uncovered a slippery sequence in the mRNA that led to “programmed frameshifts.” Depending on whether or not the ribosome slipped, two different proteins were made – a previously unidentified copper chaperon protein or a copper transporter protein. Together, the copper chaperon and transporter proteins help protect the bacterial cell from internalizing too much copper. This work provides new insight into how bacteria change gene expression in different environmental conditions and offers training for student researchers such as lead author Sezen Meydan, who was highlighted in the ‘Meet the Author’ section of Molecular Cell.

This work is partially funded by the Genetic Mechanisms Cluster of the Division of Molecular and Cellular Biosciences, Awards #MCB – 1244455 and #MCB – 1615851.

SHARING MCB SCIENCE: UNRAVELING THE DYNAMICS OF TRANSMEMBRANE PROTEINS

Alt Caption (for ADA compliance): Undergraduate honors student Armin Mortazavi, wearing a blue dress shirt, and faculty mentor Dr. Roger Koeppe, wearing a black suitcoat, dress shirt, and glasses, are seated in a laboratory in front of a computer screen looking at deuterium nuclear magnetic resonance data (not shown).

An alpha helix is a stretch of amino acids coiled in three-dimensional space, similar to a spring, which can serve a variety of functions in transmembrane proteins (proteins that span the membrane of a cell). For example, a protein may be anchored into the membrane by one or more alpha helices. Or, since alpha helices physically link the interior and exterior of the cell, they can stimulate the initiation of a signaling cascade inside the cell in response to an external binding event. In addition, multiple alpha helices bundled together can form a channel, enabling ions to move across the hydrophobic interior of the cell membrane where they are otherwise excluded. These are just a small sample of the large number of biological processes and pathways thought to involve alpha helices in transmembrane proteins; therefore, researchers like Dr. Roger Koeppe II and his students are studying alpha helices to fill in the gaps in our fundamental understanding of the dynamics of transmembrane proteins.

One such gap involves identifying the factors that stabilize tilted transmembrane alpha helices. Alpha helices display a variety of orientations within a membrane. Some may reside in the membrane vertical to its axis; others tilt across the membrane at different angles. In his lab in the Department of Chemistry and Biochemistry at the University of Arkansas, Fayetteville, Dr. Koeppe and his students established an experimental model and used a biophysical technique to determine the positions of amino acid residues inside tilted alpha helices in a membrane environment.

In a recent publication, the team described their efforts to design, synthesize, and purify a series of nearly-identical peptides (23 amino acid strands that only varied slightly in amino acid composition). At the ends of each peptide, the team placed deuterium (radioactive isotope) labels on two alanine amino acid residues (at positions 3 and 21). When the team exposed these peptides to lipids (fatty acids that compose the interior of a cell membrane), they formed tilted alpha helices. Using a technique called solid-state deuterium nuclear magnetic resonance (NMR), a series of spectra were gathered for each helix.

As the cover for the March 2016 issue of ChemBioChem depicts, the team detected a deviation between actual and expected orientations of the deuterium-labeled alanine residues at positions 3 and 21 – they actually appeared far from their expected orientations as part of the alpha helix (blue curve). In this way, Dr. Koeppe and his research team discovered that the first and last few amino acids at each end of the tilted alpha helix were unraveled from and no longer part of the alpha helix.

A driving force behind the formation of an alpha helix is the increased stability that results from numerous interactions between the amino acids in the helix, so unraveling these interactions at the ends of the helix may seem to disrupt stability. However, Dr. Koeppe and his team noted that unwinding the helix at both ends creates a larger surface area, enabling new interactions to occur between the amino acids in the helix and the surrounding membrane lipids – providing additional conformational stability in support of the tilt and limiting the helix’s motion. These research findings contribute to our understanding of fundamental biological processes including lipid-protein interactions, membrane protein stability, and membrane biophysics.

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

“Notably, much of this research involved an undergraduate honors student, Armin Mortazavi. The opportunities for undergraduate students to conduct cutting-edge research alongside graduate students provide students with valuable hands on experience, exposure to new techniques, and mentoring at an early stage in their scientific careers. We have also increased the exposure of communities in Arkansas to science – establishing science cafés and statewide infrastructure projects such as a laboratory where you can explore proteins using virtual reality technology. These efforts broaden the participation of non-traditional or underrepresented groups in science.”

This work is partially funded by the Molecular Biophysics Cluster of the Division of Molecular and Cellular Biosciences, Award #MCB – 1327611.

SHARING MCB SCIENCE: REGULATING NITROGENASE FORMATION IN CYANOBACTERIA

thiel-1b

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

Eichmangroup1

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.

research-summary.png

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. 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.

SHARING MCB SCIENCE: QQS Orphan Gene: a new avenue for sustainable protein sources?

Dr. Eve Syrkin Wurtele (left) and Dr. Ling Li (right). Photo by Christopher Gannon.

Dr. Eve Syrkin Wurtele (left) and Dr. Ling Li (right). Photo by Christopher Gannon.

Genes that are unique to one species and are not found in the genome of any other organism are called orphan genes. Because they are rare, orphan genes are perceived to lack evolutionary significance and, as a result, their origins and functions are not well-understood.

In 2004, Iowa State University Adjunct Assistant Professor Dr. Ling Li discovered the orphan gene QQS in Arabidopsis, a small flowering plant that is frequently used in scientific research. There was not an obvious indication from the QQS gene sequence as to its function in plants. Studies conducted in collaboration with Iowa State University Professor of Genetics, Development, and Cell Biology Dr. Eve Wurtele identified the function of the Arabidopsis QQS orphan gene – but in soybeans! The research team inserted the QQS gene into soybeans and found QQS increased leaf and seed protein production. This was an unexpected, exciting discovery and Li and Wurtele’s collaboration on QQS resulted in multiple publications and pending patents on expressing the QQS gene in plants.

Li and Wurtele were also curious about the behavior and functionality of the QQS orphan gene in other plants humans eat. If QQS were placed in other plants, would it increase protein production as it did in soybeans?

As described in an article published in the Proceedings of the National Academy of Sciences (PNAS), Li and Wurtele showed that the introduction of QQS into staple crops such as rice and corn also resulted in increased protein content without affecting crop yields. On a larger scale, this work could help meet the nutritional needs of people living in areas of the world where dietary protein is often insufficient. Protein-heavy plants present a more sustainable source of protein than animals.

With help from Professor of Genetics, Development, and Cell Biology Dr. Yanhai Yin, the investigators also discovered that QQS binds to a protein common to plants and animals, known as NF-YC4; an increase in NF-YC4 production also increases leaf and seed protein.  The production of this protein can be altered in crop species using an approach that doesn’t require safety testing. As a result, researchers think NF-YC4 may be the key to creating high-protein crops faster and cheaper, saving years of research and hundreds of millions of dollars. Dr. Li noted, “By producing more of the NF-YC4 gene in staple crops, researchers can increase the protein value of plants without using transgenes (exogenous genes), which could save time and money in the regulatory process.”

Work such as this, can open the door to many more discoveries, leading scientists to better appreciate the potential and value of studying orphan genes. “This is one orphan gene that we’ve shown has big potential,” Wurtele said. “And we believe there will be many more discoveries related to other orphan genes in the future.”

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

“Our goal is to use our finding to increase the protein content of staple crops in regions where humans have high rates of protein deficiency. Five graduate students and 17 undergraduate students have participated in computational and experimental research as part of our research. Four postdocs have gone directly on to promising academic or industrial careers.

We are also dedicated to disseminating results that that have been achieved with the help of NSF-MCB support. In the past four years, we have presented over 35 invited seminars at universities, institutes, and conferences in eight countries sharing our research results. We have held fourteen hands-on workshops on the MetNet bioinformatics platform tools that helped us identify the role of QQS, NF-YC-4, and other genes.  We have also created an interactive computer game called Meta!Blast, which is designed to teach cross-cutting science concepts, and is being used in undergraduate classrooms and as an exhibit in science museums.”

This work is funded by the Division of Molecular and Cellular Biosciences, Award #MCB-0951170 and by the Division of Integrative Organismal Systems, Award #IOS-1257631 in the Directorate for Biological Sciences.

Sharing MCB Science: Single-molecule motions and interactions in live cells

The nucleotides (As, Ts, Cs, and Gs) of deoxyribonucleic acid (DNA) are copied in a process called DNA replication, which is performed by a complex of molecular machinery called the replisome. The coding strand of double stranded DNA acts as a blueprint for the replisome, storing information that is critical for explaining how to create sub-cellular molecules like proteins. This information-storage feature of the DNA coding strand is precious, because one small change in the sequence of nucleotides making up the DNA coding strand could result in a mutation that is harmful to the cell or organism.

To guard against mutations, all cells contain various DNA repair systems. DNA repair works to preserve the information in the DNA coding strand by maintaining the order of nucleotides. For decades, a protein known as MutS has been shown to play a key role in DNA repair. MutS binds to mispaired bases or “errors” that occur in the DNA sequence and initiates their correction. Like searching for a needle in a haystack, MutS must search for and identify a single error in the DNA sequence that occurs just once in tens of millions of correct pairings.

For many years, the process by which MutS searches for an error in the DNA sequence has been unknown to scientists, because they have lacked the resolution necessary to detect the searching behavior of MutS. A research collaboration between University of Michigan Associate Professor Dr. Lyle Simmons, Assistant Professor Dr. Julie Biteen, and their co-workers in the Departments of Molecular, Cellular, and Developmental Biology and Chemistry, resulted in a recent publication in the Proceedings of the National Academy of Sciences (PNAS). It details the discovery of how a single MutS molecule conducts its search for DNA in need of repair inside a living bacterial cell.

MutS movement to the replisomeUsing real time super-resolution microscopy and single-molecule tracking in living bacterial cells, the Simmons and Biteen research teams discovered MutS moves around the cell very rapidly looking for DNA in need of repair. Yet, as seen in this image on the left depicting the movement of MutS in a bacterial cell (shown in black), MutS also moves to the replisome (circled in white) and pauses to search for errors before moving away.  The behavior of MutS is indicated in the image by tracking its movements (colored purple to red).  Strikingly, the Simmons and Biteen research teams show that MutS is also positioned at the replisome in cells during normal growth, prior to the formation of an error in DNA.  Such a localized search behavior at the replisome by MutS allows for the rapid and efficient recognition of errors in the DNA sequence, and allows MutS to initiate DNA repair before those errors become permanent mutations.  Dr. Simmons notes, “This work provides a new fundamental insight into how MutS searches for rare base-pairing errors in the complex environment of a living cell.”

Dr. Simmons and his lab members bring their excitement about science discovery from their laboratory to elementary, high school, and university classes through hands on experiments in K-5 classes, analysis of genomic data with high school students, and design of new curriculum to ensure the success of diverse students pursuing science PhDs.

This work is funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1050948.

Sharing MCB Science: Mechanosensitive Channel MSL8 Regulates Osmotic Forces During Pollen Hydration and Germination

Flowering plants are all around us and use pollen to reproduce. Pollen, from the male part of a plant (stamen) is transported in a variety of ways to the sticky surface (stigma) of the female part of a plant (pistil).  In many species of flowering plants, pollen granules arrive at the stigma dehydrated and dormant. When pollen sticks to the stigma, it sucks up water to rehydrate and produces a long tube for reproduction. The pollen tube pushes through the stigma, reaching inside the pistil to fertilize an ovule and form a seed. Of course, the process is a little more complicated than this. As the pollen rehydrates, water rushes in and swells the granule, causing an imbalance in the ratio of ions and water found inside and outside the granule (osmotic stress). To successfully produce a seed, pollen must have a mechanism to detect and reduce this stress.

Dr. Elizabeth Haswell, Associate Professor in the Department of Biology at Washington University in St. Louis, and members of the Haswell lab, including graduate student Eric Hamilton and undergraduate Andrew Katims, turned to bacteria for insight about this mechanism and recently discovered how pollen handles the stress. E. coli bacteria use a mechanosensitive channel of small conductance (MscS) to create a connection between the interior of the cell and the outside world through the cell membrane by sensing and responding to physical or mechanical forces. During times of cell swelling brought on by external environmental stressors like rain, the MscS channel senses osmotic stress, opens, and allows ions to exit. As ions flow out through the channel, water also flows out of the cell through other mechanisms. Bacteria, fungi, archaea, and plants are all known to have MscS-like (MSL) proteins, but in most cases their physiological roles are unknown.

Dr. Haswell noted, “We recently discovered a related, but distinct, role for MSL proteins in pollen.” Instead of using mechanosensitive channels to detect stress brought on by changes in the environment, the Haswell team discovered that plants use these channels to sense and reduce osmotic stress during reproduction.

As described in an article published in Science, the Haswell lab used various techniques, including in vivo confocal imaging of fluorescent MscS-like 8 (MSL8) protein fusions, to show that MSL8 is located in the membrane of pollen. Next, studies were conducted to determine if MSL8 could produce a mechanosensitive ion channel. The researchers also showed that MSL8 protein produced in Xenopus laevis (African clawed frog) oocytes generated currents like those in a small-conductance mechanosensitive ion channel (MscS).

To determine if the MSL8 protein functions as a mechanosensitive ion channel during rehydration of pollen granules, the Haswell lab isolated two mutants of the MSL8 protein (msl8-1 and msl8-4). The msl8-1 mutant produced reduced levels of MSL8 transcripts and the msl8-4 mutant generated no detectable levels of MSL8 transcripts. The researchers rehydrated pollens containing MSL8 and pollens containing the mutants in distilled water for two hours and looked for an effect on the viability of pollen. Pollen containing natural levels of MSL8 transcripts was 83-95% viable, but for pollen with a reduced level of transcripts (msl8-1) viability diminished to 46% and for pollen lacking detectable transcripts (msl8-4) viability was only 21-38%. Thus, the Haswell lab concluded MSL8 protein is a mechanosensitive channel important for the survivability of pollen during rehydration.

MSL8 plays another important role in germination. In vitro germination assays showed pollen containing the msl8-4 mutant form of MSL8 burst 26% of the time, compared to only a 3% bursting rate in natural pollen with normal MSL8 protein. Bursting in msl8 mutant pollen may be due to the inability to modulate osmotic stress during pollen tube production. Also, overexpression of fluorescent MSL8-YFP protein in pollen inhibited the rate of germination to only 4-39% of natural pollen. As Dr. Haswell noted, “MSL8 negatively regulates pollen germination, but is required for cellular integrity during germination and tube growth.”

This study shows the important role of mechanosensitive ion channels in plants and the delicate balance achieved by MSL8 channels during pollen rehydration and germination. In addition, Dr. Haswell and her students have provided evidence of a homolog of the prokaryotic E. coli MscS channel that suits the needs of a significantly more complex organism. Dr. Haswell noted “Pollen development is a stage of plant development that is highly sensitive to environmental stress, and a better understanding of how pollen grains handle water stress may help mitigate the effects of climate change.”

In an effort to share their work with broader audiences, the Haswell lab has created a YouTube whiteboard animation depicting their discovery of the MSL8 mechanosensitive channel and its regulation of osmotic forces during pollen rehydration and tube creation.

This work is partially funded by the Division of Molecular and Cellular Biosciences, Award #MCB-1253103.