Genomic Gymnastics of a Single-Celled Ciliate and How It Relates to Humans

Laura Landweber
Credit: Denise Applewhite.
Laura Landweber
Grew up in: Princeton, New Jersey
Job site: Columbia University, New York City
Favorite food: Dark chocolate and dark leafy greens
Favorite music: 1940’s style big band jazz
Favorite hobby: Swing dancing
If I weren’t a scientist I would be a: Chocolatier (see “Experiments in Chocolate” sidebar at bottom of story)

One day last fall, molecular biologist Laura Landweber Link to external web site surveyed the Princeton University lab where she’d worked for 22 years. She and her team members had spent many hours that day laboriously affixing yellow Post-it notes to the laboratory equipment—microscopes, centrifuges, computers—they would bring with them to Columbia University, where Landweber had just been appointed full professor. Each Post-it specified the machinery’s location in the new lab. Items that would be left behind—glassware, chemical solutions, furniture, office supplies—were left unlabeled.

As Landweber viewed the lab, decorated with a field of sunny squares, her thoughts turned to another sorting process—the one used by her primary research subject, a microscopic organism, to sift through excess DNA following mating. Rather than using Post-it notes, the creature, a type of single-celled organism called a ciliate, uses small pieces of RNA to tag which bits of genetic material to keep and which to toss.

Landweber is particularly fond of Oxytricha trifallax, a ciliate with relatives that live in soil, ponds and oceans all over the world. The kidney-shaped cell is covered with hair-like projections called cilia that help it move around and devour bacteria and algae. Oxytricha is not only bizarre in appearance, it’s also genetically creative.

Unlike humans, whose cells are programmed to die rather than pass on genomic errors, Oxytricha cells appear to delight in genomic chaos. During sexual reproduction, the ciliate shatters the DNA in one of its two nuclei into hundreds of thousands of pieces, descrambles the DNA letters, throws most away, then recombines the rest to create a new genome.

Landweber has set out to understand how—and possibly why—Oxytricha performs these unusual genomic acrobatics. Ultimately, she hopes that learning how Oxytricha rearranges its genome can illuminate some of the events that go awry during cancer, a disease in which the genome often suffers significant reorganization and damage.

Oxytricha’s Unique Features

Oxytricha carries two separate nuclei—a macronucleus and a micronucleus. The macronucleus, by far the larger of the two, functions like a typical genome, the source of gene transcription for proteins. The tiny micronucleus only sees action occasionally, when Oxytricha reproduces sexually.

Oxytricha trifallax cells in the process of mating
Two Oxytricha trifallax cells in the process of mating. Credit, Robert Hammersmith.

What really makes Oxytricha stand out is what it does with its DNA during the rare occasions that it has sex. When food is readily available, Oxytricha procreates without a partner, like a plant grown from a cutting. But when food is scarce, or the cell is stressed, it seeks a mate. When two Oxytricha cells mate, the micronuclear genomes in each cell swap DNA, then replicate. One copy of the new hybrid micronucleus remains intact, while the other breaks its DNA into hundreds of thousands of pieces, some of which are tagged, recombined, then copied another thousand-fold to form a new macronucleus. Continue reading

The Changing Needs of a Cell: No Membrane? No Problem!

Russian nesting dolls. Credit: iStock.

How “membrane-less” organelles help with key cellular functions

Scientists have long known that animal and plant cells have specialized subdivisions called organelles. These organelles are surrounded by a semi-permeable barrier, called a membrane, that both organizes the organelles and insulates them from the rest of the cell’s mix of proteins, salt, and water. This set-up helps to make cells efficient and productive, aiding in energy production and other specialized functions. But, because of their semi-permeable membranes, organelles can’t regroup and reform in response to stress or other outside changes. Cells need a rapid response team working alongside the membrane-bound organelles to meet these fluctuating needs. Until recently, who those rapid responders were and how they worked has been a mystery.

Recent research has led biologists to learn that the inside of a cell or an organelle is not just a lot of different molecules dissolved in water. Instead, we now know that cells contain many pockets of liquid droplets (one type of liquid surrounded by a liquid of different density) with specialized composition and function that are not surrounded by membranes. Because these “membrane-less organelles” are not confined, they can rapidly come together in response to chemical signals, such as those that indicate stress, and equally rapidly fall apart when they are no longer needed, or when the cell is about to divide. This enables membrane-less organelles to be “rapid responders.” They can have complex, multilayered structures that help them to perform many critical cell functions with multiple steps, just like membrane-bound organelles. Scientists even suspect that the way these organelles form as droplets may shed light on how life on Earth first took shape (see sidebar “Could This Be How Life First Took Shape?” at bottom of page).

The Many Membrane-less Organelles

Scientists have identified more than a dozen membrane-less organelles at work in mammalian cells. Several kinds found inside the nucleus—including nuclear speckles, paraspeckles, and Cajal bodies—help with cell growth, stress response, the metabolizing (breaking down) of RNA, and the control of gene expression—the process by which information in a gene is used in the synthesis of a protein. Out in the cytoplasm, P-bodies, germ granules, and stress granules are membrane-less organelles that are involved in metabolizing or protecting messenger RNA (mRNA), controlling which mRNAs are made into proteins, and in maintaining balance, or homeostasis, of the cell’s overall health.

The nucleolus, located inside the nucleus, is probably the largest of the membrane-less organelles. It acts as a factory to assemble ribosomes, the giant molecular machines that “translate” messenger RNAs to make all cellular proteins. Continue reading

The Endoplasmic Reticulum: Networking Inside the Cell

Like a successful business networker, a cell’s endoplasmic reticulum (ER) is the structure that reaches out—quite literally—to form connections with many different parts of a cell. In several important ways, the ER enables those other parts, or organelles, to do their jobs. Exciting new images of this key member of the cellular workforce may clarify how it performs its roles. Such knowledge will also help studies of motor neuron and other disorders, such as amyotrophic lateral sclerosis (ALS), that are associated with abnormalities in ER functioning.

Structure Follows Function

Illustration of some of the jobs that the ER performs in the cell.

An illustration of some of the jobs that the endoplasmic reticulum (ER) performs in the cell. Some ER membranes (purple) host ribosomes on their surface. Other ER membranes (blue) extending into the cytoplasm are the site of lipid synthesis and protein folding. The ER passes on newly created lipids and proteins to the Golgi apparatus (green), which packages them into vesicles for distribution throughout the cell. Credit: Judith Stoffer.

Initiated in 1965, the Postdoctoral Research Associate Program (PRAT) is a competitive postdoctoral fellowship program to pursue research in one of the laboratories of the National Institutes of Health. PRAT is a 3-year program providing outstanding laboratory experiences, access to NIH’s extensive resources, mentorship, career development activities and networking. The program places special emphasis on training fellows in all areas supported by NIGMS, including cell biology, biophysics, genetics, developmental biology, pharmacology, physiology, biological chemistry, computational biology, immunology, neuroscience, technology development and bioinformatics

The ER is a continuous membrane that extends like a net from the envelope of the nucleus outward to the cell membrane. Tiny RNA- and protein-laden particles called ribosomes sit on its surface in some places, translating genetic code from the nucleus into amino acid chains. The chains then get folded inside the ER into their three-dimensional protein structures and delivered to the ER membrane or to other organelles to start their work. The ER is also the site where lipids—essential elements of the membranes within and surrounding a cell—are made. The ER interacts with the cytoskeleton—a network of protein fibers that gives the cell its shape—when a cell divides, moves or changes shape. Further, the ER stores calcium ions in cells, which are vital for signaling and other work.

To do so many jobs, the ER needs a flexible structure that can adapt quickly in response to changing situations. It also needs a lot of surface area where lipids and proteins can be made and stored. Scientists have thought that ER structure combined nets of tubules, or small tubes, with areas of membrane sheets. However, recent NIGMS PRAT (Postdoctoral Research Associate; see side bar) fellow Aubrey Weigel, working with her mentor and former PRAT fellow Jennifer Lippincott-Schwartz of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (currently at the Howard Hughes Medical Institute in Virginia) and colleagues, including Nobel laureate Eric Betzig, wondered whether limitations in existing imaging technologies were hiding a better answer to how the ER meets its surface-area structural needs in the periphery, the portion of the cell not immediately surrounding the nucleus. Continue reading

CRISPR Serves Up More than DNA

Marine bacterium Marinomonas mediterranea
The marine bacterium Marinomonas mediterranea uses a CRISPR system to spot invading RNAs and store a memory of the invasion event in its genome. Research team member Antonio Sanchez-Amat was the first to isolate and characterize this bacterial species. Credit: Antonio Sanchez-Amat, University of Murcia.

A new study has added another twist to the CRISPR story. As we’ve highlighted in several recent posts, CRISPR is an immune system in bacteria that recognizes and destroys viral DNA and other invading DNA elements, such as transposons. Scientists have adapted CRISPR into an indispensable gene-editing tool now widely used in both basic and applied research.

Many previously described CRISPR systems detect and cut viral DNA, insert the DNA pieces into the bacterial genome and then use them as molecular “mug shots” to flag and destroy the virus if it attacks again. But various viruses use RNA, not DNA, as genetic material. Although research has shown that some CRISPR systems also can target RNA, how these systems can archive harmful RNA encounters in the bacterial genome was unknown. Continue reading

How Cells Manage Chance

We asked the heads of our scientific divisions to tell us about some of the big questions in fundamental biomedical science that researchers are investigating with NIGMS support. This article is the second in an occasional series that explores these questions and explains how pursuing the answers could advance understanding of important biological processes.

Sample slide, variability of mRNA in yeast cells
The number of copies of mRNA molecules (bright green) observed here in yeast cells (dark blue) fluctuates randomly. Credit: David Ball, Virginia Tech.

For some health conditions, the cause is clear: A single altered gene is responsible. But for many others, the path to disease is more complex. Scientists are working to understand how factors like genetics, lifestyle and environmental exposures all contribute to disease. Another important, but less well-known, area of investigation is the role of chance at the molecular level.

One team working in this field is led by John Tyson Exit icon at Virginia Tech. The group focuses on how chance events affect the cell division cycle, in which a cell duplicates its contents and splits into two. This cycle is the basis for normal growth, reproduction and the replenishment of skin, blood and other cells throughout the body. Errors in the cycle are associated with a number of conditions, including birth defects and cancer. Continue reading

Field Focus: Progress in RNA Interference Research

Scientists first noticed what would later prove to be RNA interference when puzzling over an unexpected loss of color in petunia petals. Subsequent studies in roundworms revealed that double-stranded RNA can inactivate specific genes. Credit: Alisa Z. Machalek.

In less than two decades, RNA interference (RNAi)—a natural process cells use to inactivate, or silence, specific genes—has progressed from a fundamental finding to a powerful research tool and a potential therapeutic approach. To check in on this fast-moving field, I spoke to geneticists Craig Mello Exit icon of the University of Massachusetts Medical School and Michael Bender of NIGMS. Mello shared the 2006 Nobel Prize in physiology or medicine with Andrew Fire Exit icon of Stanford University School of Medicine for the discovery of RNAi. Bender manages NIGMS grants in areas that include RNAi research.

How have researchers built on the initial discovery of RNAi?

A scientific floodgate opened after the 1998 discovery that it was possible to switch off specific genes by feeding microscopic worms called C. elegans double-stranded RNA that had the same sequence of genetic building blocks as a target gene. (Double-stranded RNA is a type of RNA molecule often found in, or produced by, viruses.) Scientists investigating gene function quickly began to test RNAi as a gene-silencing technique in other organisms and found that they could use it to manipulate gene activity in many different model systems. Additional studies led the way toward getting the technique to work in cells from mammals, which scientists first demonstrated in 2001. Soon, researchers were exploring the potential of RNAi to treat human disease. Continue reading

Meet Jennifer Doudna

Jennifer Doudna
Credit: Jennifer Doudna
Jennifer Doudna
Fields: Biochemistry and structural biology
Studies: New genome editing tool called CRISPR
Works at: University of California, Berkeley
Raised in: Hilo, Hawaii
Studied at: Pomona College, Harvard University
Recent honors: Winner of the Lurie Prize in the Biomedical Sciences Exit icon, an annual award that recognizes outstanding achievement by promising scientists age 52 or younger
If she couldn’t be a scientist, she’d like to be: A papaya farmer or an architect

Jennifer Doudna likes to get her hands dirty. Literally. When she’s not in her laboratory, she can often be found amid glossy green leaves and brightly colored fruit in her Berkeley garden. She recently harvested her first three strawberry guavas.

Coaxing tropical fruit plants from her childhood home in Hawaii to grow in Northern California is more than just a hobby—it’s an intellectual challenge.

“I like solving puzzles, I like the process of figuring things out, and I enjoy working with my hands,” says Doudna. “Those things were what really drew me to science in the beginning.”

Since she was a graduate student, Doudna’s professional puzzle has been RNA, a type of genetic material inside our cells. Recently, there has been an explosion of discoveries about the many roles RNA molecules play in the body. Doudna’s work probes into how RNA molecules work, what 3-D shapes they form and how their structures drive their functions.

“I’ve been fascinated by understanding RNA at a mechanistic level,” Doudna says.

While teasing out answers to these fundamental questions, Doudna’s lab has played a leading role in a discovery that is upending the field of genetic engineering, with exciting implications for human health.

Her Findings

The discovery started with bacteriophages—viruses that infect bacteria, just like the common cold infects humans. About 10 years ago, researchers using high-powered computing to sift through bacterial genomes began to find mysterious repetitive gene sequences that matched those from viruses known to infect the bacteria. The researchers named these sequences “clustered regularly interspaced short palindromic repeats,” or CRISPRs for short.

Over the next few years, scientists came to understand that these CRISPR sequences are part of something not previously thought to exist—an adaptive bacterial immune system, which remembers viruses fought off before and raises a response to fight them when exposed again. CRISPRs were this immune system’s reference library, holding records of viral exposure.

Somehow, bacteria with a CRISPR-based immune system (there are three types now known to scientists) use these records to command certain proteins to recognize and chop up DNA from returning viruses.

Wanting to know more about this process, Doudna’s team picked one protein in a CRISPR-based defense system to study. This protein, called Cas9, had been identified by other researchers as being essential for protection against viral invasion.

To their delight, Doudna’s group had hit the jackpot. Cas9 turned out to be the system’s scalpel. Once CRISPR identifies a DNA sequence from the invading virus, Cas9 slices the sequence out of the viral genome, destroying the virus’s ability to copy itself.

Doudna’s lab and their European collaborators also identified the other key components of the CRISPR-Cas9 system—two RNA molecules that guide Cas9 to the piece of viral DNA identified by CRISPR.

Even more importantly, the researchers showed that the two guide RNAs could be manipulated in the lab to create a tool that both recognizes any specified DNA sequence and carries Cas9 there to make its cut.

“That was really where we made the connection between the basic, curiosity-driven research that we were doing and recognizing that we had in our hands something that could be a very powerful technology for genome editing,” remembers Doudna.

She was right. After publication of their 2012 paper, the field of CRISPR-guided genetic manipulation exploded. Labs around the world now use the tool Doudna’s team developed to cut target gene sequences in organisms ranging from plants to humans. The technique is already replacing more time-consuming, less-reliable methods of creating ‘knock-out’ model organisms (those missing a specific gene) for laboratory research. CRISPR-based editing even allows more than one gene to be knocked out at the same time, something that was not possible with previous genome-editing techniques.

The ability of CRISPR systems to recognize DNA sequences with extraordinary precision also holds potential for human therapeutics. For example, a paper from another laboratory published early this year showed that, in a mouse model, CRISPR-based editing could cut out and replace a defective gene responsible for a type of muscular dystrophy. Researchers are testing similar CRISPR-based techniques in models of human diseases ranging from cystic fibrosis to blood disorders.

Doudna is a co-founder of two biotechnology companies hoping to harness the potential of CRISPR-based genome editing. Although the technology holds great promise, she acknowledges that much work needs to be done before CRISPR can be considered safe for human trials. Major challenges include assuring that no off-target cuts are made in the genome and finding a safe way to deliver the editing system to living tissues.

She is also excited to continue working with her research team, advancing the basic understanding of the CRISPR-based system.

“I’m very interested in seeing what we can contribute to the whole question about how you deliver a technology like this, how you can use it therapeutically in an organism. That’s an area where we hope that our biochemical understanding of this system will be able to contribute,” she concludes.

An RNA Molecule That Cues the Internal Clock

Dysfunction in our internal clocks may lead to insufficient sleep, which has been linked to an increased risk for chronic diseases. Credit: Stock image.

Our internal clocks tell us when to sleep and when to eat. Because they are sensitive to changes in daytime and nighttime cues, they can get thrown off by activities like traveling across time zones or working the late shift. Dysfunction in our internal clocks may lead to insufficient sleep, which has been linked to an increased risk for chronic diseases like high blood pressure, diabetes, depression and cancer.

Researchers led by Yi Liu Exit icon of the University of Texas Southwestern Medical Center have uncovered a previously unknown mechanism by which internal clocks run and are tuned to light cues. Using the model organism Neurospora crassa (a.k.a., bread mold), the scientists identified a type of RNA molecule called long non-coding RNA (lncRNA) that helps wind the internal clock by regulating how genes are expressed. When it’s produced, the lncRNA identified by Liu and his colleagues blocks a gene that makes a specific clock protein.

This inhibition works the other way, too: The production of the clock protein blocks the production of the lncRNA. This rhythmic gene expression helps the body stay tuned to whether it’s day or night.

The researchers suggest that a similar mechanism likely exists in the internal clocks of other organisms, including mammals. They also think that lncRNA-protein pairs may contribute to the regulation of other biologic processes.

Learn more:
University of Texas Southwestern Medical Center News Release Exit icon
Circadian Rhythms Fact Sheet
Resetting Our Clocks: New Details About How the Body Tells Time Article from Inside Life Science
Remarkable RNAs Article from Inside Life Science

Meet Scott Poethig

Scott Poethig
Fields: Plant biology, cell and developmental biology, genetics
Works at: University of Pennsylvania
Studied at: College of Wooster, Yale University
Favorite musicians: Nick Drake and Bruce Springsteen
High school job: Radio D.J.
Favorite book: “The Little Prince,” by Antoine de Saint-Exupéry

When Scott Poethig signed up for a developmental biology course in his senior year of college, he expected to learn how organisms transition from single cells to juveniles to adults. He did not expect to learn just how much scientists still didn’t know about this process.

“It was the first course I had taken as an undergraduate where I felt that I could ask a question that there wasn’t an answer to already,” he recalls. “I thought, ‘Wow! This is amazing.’”

Poethig already had an interest in plant biology and an independent research project studying corn viruses. He immediately saw the potential in combining his knowledge of plants with his questions about how organisms grow. “There seemed to be a lot of low hanging fruit in plant development,” he says.

Today, Poethig is the head of a plant development lab at the University of Pennsylvania. His work probes the complex molecular mechanisms that drive the transition from a young seedling to an adult plant that hasn’t yet produced seeds.

“The analogous period in human development is the interval between birth and puberty,” he explains. “People think of puberty as the major developmental transition in postnatal human development, but a lot of change happens before that point.”

His Findings

Poethig discovered that for the mustard plant Arabidopsis, a model organism frequently studied by geneticists, change begins early. Before these plants begin to flower—a sign of reproductive maturity—they undergo a process of vegetative maturation. In Arabidopsis, Poethig found that juvenile plants can be distinguished from adult plants by where hairs are produced on a leaf. Juvenile plants only produce hairs on the upper surface of the leaf, whereas adult plants produce leaves with hairs on both the upper and lower surfaces.

By studying mutant Arabidopsis plants where the adult pattern of hair development is either delayed or advanced, Poethig identified microRNAs as key players in this developmental transition.

MicroRNA molecules commonly block the expression of specific genes. Poethig found that in Arabidopsis, a type of microRNA prevents development. Young plants have high levels of this microRNA and cannot fully mature. When those levels drop, plants progress to adulthood.

MicroRNAs similarly control development in the nematode C. elegans. Scientists study the genetics of this tiny worm to better understand related developmental processes in more complex organisms. Because plants also use microRNAs to regulate development, Poethig’s discoveries may contribute to our understanding of how these molecules govern development in animals, including humans.

Poethig now wants to learn what determines the timing of developmental changes. He asks: “Why do microRNA levels drop? What’s the signal that causes that? What is the plant measuring?” His current hypothesis: sugar.

In a recent study, he found that giving plants additional sugar reduced microRNA levels and sped up development. Meanwhile, mutant plants that couldn’t produce enough sugar on their own through photosynthesis had increased microRNA levels and delayed development compared to normal plants.

This research may one day advance our understanding of how nutrition and genetics interact to affect human development. “In essentially all organisms, aging and the timing of developmental processes are strongly affected by nutrition,” Poethig explains. “In humans, childhood obesity is sometimes associated with early puberty, and it is important to understand the molecular basis for this effect.”

Poethig believes that studying microRNAs in plants may also lead to discoveries in human genetics outside of developmental biology. “MicroRNAs control a wide range of gene activity in plants and animals,” Poethig explains. “In humans, these molecules control the activity of as many as 30 percent of our genes. So understanding how microRNAs work in plants could help us understand their function in humans.”

Besides studying the Arabidopsis plants in his lab, Poethig also studies the plants in his kitchen, and uses his fascination with the history, culture and politics of food to excite others about science. Watch video.

Field Focus: Precision Gene Editing with CRISPR

Bacterial cells infected by viruses.
Bacterial cells can be infected by viruses (shown in red and purple) and have evolved ways to defend themselves. Credit: Stock image.

Like humans, bacteria can be infected by viruses and have evolved ways to defend themselves. Researchers are now adapting this bacterial “immune system” to precisely and efficiently edit genes in cells from humans and a wide range of other organisms. Scientists are excited about the tremendous potential of this powerful tool for advancing biomedical research and treating diseases.

The bacterial defense system is called CRISPR, for clustered regularly interspaced short palindromic repeats. A breakthrough in understanding CRISPR came from examining bacteria used by the dairy industry for the production of yogurt and cheese. In a study published in 2007, researchers showed that these bacteria insert viral DNA sequences into their own genomes and use that information to disarm the virus when it attacks again. Subsequent research has shown that the CRISPR system consists of small RNA molecules that target specific viral DNA sequences and proteins that cut the DNA, thus destroying the virus.

Researchers have already adapted CRISPR into a gene-editing tool that’s quicker, cheaper and more precise than existing methods. Researchers can use CRISPR to add, delete, rev up or tone down certain genes as well as create animal models for studying human diseases. The ability to precisely target genes in human cells is expected to speed progress in the development of gene-based therapies.

Although much is known about CRISPR, we still have a lot to learn. For example, how do bacterial cells obtain and insert the viral DNA into their genome? What triggers production of the CRISPR RNA molecules? How are invading viral DNAs targeted for destruction? This last question is answered in part by a pair of findings described in an earlier post, A Crisper View of the CRISPR Gene-Editing Mechanism. We also want to figure out how we can make the CRISPR gene-editing tool even more versatile and precise.

The CRISPR story offers a good example of how studying basic biological processes leads to new—and sometimes unexpected—insights and applications.

Emily Carlson also contributed to this blog post.

Related advances:
CRISPR/Cas9 Protein Complex Can Be Programmed to Recognize and Cleave RNA Exit icon
CRISPR System Adapted to Reversibly Regulate Gene Expression Exit icon