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The Perfect Cytoskeletal Storm

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Ever thought about giving cell biology a whirl? If so, I suggest you sit down and take a look at this full-blown cytoskeletal “storm,” which provides a spectacular dynamic view of the choreography of life.

Before a cell divides, it undergoes a process called mitosis that copies its chromosomes and produces two identical nuclei. As part of this process, microtubules, which are structural proteins that help make up the cell’s cytoskeleton, reorganize the newly copied chromosomes into a dense, football-shaped spindle. The position of this mitotic spindle tells the cell where to divide, allowing each daughter cell to contain its own identical set of DNA.

To gain a more detailed view of microtubules in action, researchers designed an experimental system that utilizes an extract of cells from the African clawed frog (Xenopus laevis). As the video begins, a star-like array of microtubules (red) radiate outward in an apparent effort to prepare for cell division. In this configuration, the microtubules continually adjust their lengths with the help of the protein EB-1 (green) at their tips. As the microtubules grow and bump into the walls of a lab-generated, jelly-textured enclosure (dark outline), they buckle—and the whole array then whirls around the center.

Abdullah Bashar Sami, a Ph.D. student in the NIH-supported lab of Jesse “Jay” Gatlin, University of Wyoming, Laramie, shot this movie as a part his basic research to explore the still poorly understood physical forces generated by microtubules. The movie won first place in the 2019 Green Fluorescent Protein Image and Video Contest sponsored by the American Society for Cell Biology. The contest honors the 25th anniversary of the discovery of green fluorescent protein (GFP), which transformed cell biology and earned the 2008 Nobel Prize in Chemistry for three scientists who had been supported by NIH.

Like many movies, the setting was key to this video’s success. The video was shot inside a microfluidic chamber, designed in the Gatlin lab, to study the physics of microtubule assembly just before cells divide. The tiny chamber holds a liquid droplet filled with the cell extract.

When the liquid is exposed to an ultra-thin beam of light, it forms a jelly-textured wall, which traps the molecular contents inside [1]. Then, using time-lapse microscopy, the researchers watch the mechanical behavior of GFP-labeled microtubules [2] to see how they work to position the mitotic spindle. To do this, microtubules act like shapeshifters—scaling to adjust to differences in cell size and geometry.

The Gatlin lab is continuing to use their X. laevis system to ask fundamental questions about microtubule assembly. For many decades, both GFP and this amphibian model have provided cell biologists with important insights into the choreography of life, and, as this work shows, we can expect much more to come!

References:

[1] Microtubule growth rates are sensitive to global and local changes in microtubule plus-end density. Geisterfer ZM, Zhu D, Mitchison T, Oakey J, Gatlin JC. November 20, 2019.

[2] Tau-based fluorescent protein fusions to visualize microtubules. Mooney P, Sulerud T, Pelletier JF, Dilsaver MR, et al. Cytoskeleton (Hoboken). 2017 Jun;74(6):221-232.

Links:

Mitosis (National Human Genome Research Institute/NIH)

Gatlin Lab (University of Wyoming, Laramie)

Green Fluorescent Protein Image and Video Contest (American Society for Cell Biology, Bethesda, MD)

2008 Nobel Prize in Chemistry (Nobel Foundation, Stockholm, Sweden)

NIH Support: National Institute of General Medical Sciences


Largest-Ever Genetic Study of Autism Yields New Insights

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Baby and DNA Strands

Anyone who’s spent time with people affected by autism spectrum disorder (ASD) can tell you that it’s a very complex puzzle. The wide variability seen among individuals with this group of developmental brain disorders, which can disrupt communication, behavior control, and social skills, has also posed a huge challenge for researchers trying to identify underlying genetic and environmental factors. So, it’s no surprise that there’s been considerable interest in the recent findings of the largest-ever genetic study of ASD.

In a landmark study that analyzed the DNA of more than 35,000 people from around the world, the NIH-funded international Autism Sequencing Consortium (ASC) identified variants in 102 genes associated with increased risk of developing ASD, up from 65 identified previously. Of the 102 genes, 60 had not been previously linked to ASD and 53 appeared to be primarily connected to ASD as opposed to other types of intellectual disability or developmental delay. It is expected that this newfound genetic knowledge will serve to improve understanding of the complex biological mechanisms involved in ASD, ultimately paving the way for new approaches to diagnosis and treatment.

The study reported in the journal Cell was led by Joseph Buxbaum, Icahn School of Medicine at Mount Sinai, New York; Stephan Sanders, University of California, San Francisco; Kathryn Roeder, Carnegie Mellon University, Pittsburgh, PA; and Mark Daly, Massachusetts General Hospital, Boston, MA and the Broad Institute of MIT and Harvard, Cambridge, MA. These researchers and their teams faced what might seem like a rather daunting task.

While common genetic variants collectively are known to contribute substantially to ASD, rare variants have been recognized individually as more major contributors to a person’s risk of developing ASD. The challenge was how to find such rare variants—whether inherited or newly arising.

To do so, the researchers needed to analyze a enormous amount of DNA data. Fortunately, they and their ASC colleagues already had assembled a vast trove of data. Over the last decade, the ASC had collected DNA samples with full consent from thousands of people with and without ASD, including unaffected siblings and parents. All were aggregated with other studies, and, at the time of this investigation, they had gathered 35,584 unique samples. Those included more than 21,000 family-based samples and almost 12,000 samples from people diagnosed with ASD.

In search of rare genetic alterations, they sequenced whole exomes, the approximately 1.5 percent of the genome that codes for proteins. Their search produced a list of 102 ASD-associated genes, including 30 that had never been implicated in any developmental brain disorder previously.

But that was just the beginning. Next, the ASC team dug deeper into this list. The researchers knew from previous work that up to half of people with ASD also have an intellectual disability or developmental delay. Many of the associated genes overlap, meaning they play roles in both outcomes. So, in one set of analyses, the team compared the list to the results of another genetic study of people diagnosed with developmental delays, including problems with learning or gross motor skills such as delayed walking.

The detailed comparison allowed them to discern genes that are more associated with features of ASD, as opposed to those that are more specific to these developmental delays. It turns out that 49 of the 102 autism-associated genes were altered more often in people with developmental delay than in those diagnosed with ASD. The other 53 were altered more often in ASD, suggesting that they may be more closely linked to this condition’s unique features.

Further study also showed that people who carried alterations in genes found predominantly in ASD also had better intellectual function. They also were more likely to have learned to walk without a developmental delay.

The 102 new genes fell primarily into one of two categories. Many play a role in the brain’s neural connections. The rest are involved primarily in switching other genes on and off in brain development. Interestingly, they are expressed both in excitatory neurons, which are active in sending signals in the brain, and in inhibitory neurons that squelch such activity. Many of these genes are also commonly expressed in the brain’s cerebral cortex, the outermost part of the brain that is responsible for many complex behaviors.

Overall, these findings underscore that ASD truly does exist on a spectrum. Indeed, there are many molecular paths to this disorder. The ASC researchers continue to collect samples, so we can expect this list of 102 genes will continue to expand in the future.

With these gene discoveries in hand, the researchers will now also turn their attention to unravelling additional details about how these genes function in the brain. The hope is that this growing list of genes will converge on a smaller number of important molecular pathways, pointing the way to new and more precise ways of treating ASD in all its complexity.

Reference:

[1] Large-scale exome sequencing study implicates both developmental and functional changes in the neurobiology of autism. Satterstrom FK, Kosmicki JA, Wang J, Breen MS, De Rubeis S, An JY, Peng M, Collins R, Grove J, Klei L, Stevens C, Reichert J, Mulhern MS, Artomov M, Gerges S, Sheppard B, Xu X, Bhaduri A, Norman U, Brand H, Schwartz G, Nguyen R, Guerrero EE, Dias C; Autism Sequencing Consortium; iPSYCH-Broad Consortium, Betancur C, Cook EH, Gallagher L, Gill M, Sutcliffe JS, Thurm A, Zwick ME, Børglum AD, State MW, Cicek AE, Talkowski ME, Cutler DJ, Devlin B, Sanders SJ, Roeder K, Daly MJ, Buxbaum JD.Cell. 2020 Jan 23. {Epub ahead of print]

Links:

Autism Spectrum Disorder (NIH/National Institute of Mental Health)

Joseph Buxbaum (Icahn School of Medicine at Mount Sinai, New York)

Sanders Lab (University of California, San Francisco)

Kathryn Roeder (Carnegie Mellon University, Pittsburgh, PA)

Mark Daly (Broad Institute of MIT and Harvard, Cambridge, MA)

Autism Sequencing Consortium (Emory University, Atlanta)

NIH Support: National Institute Mental Health; National Human Genome Research Institute


Finding New Genetic Mutations Amid Healthy Cells

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Po-Ruh Loh
Po-Ru Loh Courtesy of Loh Lab

You might recall learning in biology class that the cells constantly replicating and dividing in our bodies all carry the same DNA, inherited in equal parts from each parent. But it’s become increasingly clear in recent years that even seemingly healthy tissues contain neighborhoods of cells bearing their own acquired genetic mutations. The question is: What do all those altered cells mean for our health?

With support from a 2018 NIH Director’s New Innovator Award, Po-Ru Loh, Harvard Medical School, Boston, is on a quest to find out, though without the need for sequencing lots of DNA in his own lab. Loh will instead develop ultrasensitive computational tools to pick up on those often-subtle alterations within the vast troves of genomic data already stored in databases around the world.

How is that possible? The math behind it might be complex, but the underlying idea is surprisingly simple. His algorithms look for spots in the genome where a slight imbalance exists in the quantity of DNA inherited from mom versus dad.

Actually, Loh can’t tell from the data which parent provided any snippet of chromosomal DNA. But looking at DNA sequenced from a mixture of many cells, he can infer which stretches of DNA were most likely inherited together from a single parent.

Any slight skew in those quantities point the way to genomic territory where a tiny portion of chromosomal DNA either went missing or became duplicated in some cells. This common occurrence, especially in older adults, leads to a condition called genetic mosaicism, meaning that, contrary to most biology textbooks, all cells aren’t exactly the same.

By detecting those subtle imbalances in the data, Loh can pinpoint small DNA alterations, even when they occur in 1 in 1,000 cells collected from a person’s bloodstream, saliva, or tissues. That’s the kind of sensitivity that most scientists would not have thought possible.

Loh has already begun putting his new computational approach to work, as reported in Nature last year [1]. In DNA data from blood samples of more than 150,000 participants in the United Kingdom Biobank, his method uncovered well over 8,000 mosaic chromosomal alterations.

The study showed that some of those alterations were associated with an increased risk of developing blood cancers. However, it’s important to note that most people with evidence of mosaicism won’t go on to develop cancer. The researchers also made the unexpected discovery that some individuals carried genetic variants that made them more prone than others to pick up new mutations in their blood cells.

What’s especially exciting is Loh’s computational tools now make it possible to search for signs of mosaicism within all the genetic data that’s ever been generated. Even more importantly, these tools will allow Loh and other researchers to ask and answer important questions about the consequences of mosaicism for a wide range of diseases.

Reference:

[1] Insights into clonal haematopoiesis from 8,342 mosaic chromosomal alterations. Loh PR, Genovese G, Handsaker RE, Finucane HK, Reshef YA, Palamara PF, Birmann BM, Talkowski ME, Bakhoum SF, McCarroll SA, Price AL. Nature. 2018 Jul;559(7714):350-355.

Links:

Loh Lab (Harvard Medical School, Boston)

Loh Project Information (NIH RePORTER)

NIH Director’s New Innovator Award (Common Fund)

NIH Support: Common Fund; National Institute of Environmental Health Sciences


Study Finds Genetic Mutations in Healthy Human Tissues

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General mutations throughout the body

The standard view of biology is that every normal cell copies its DNA instruction book with complete accuracy every time it divides. And thus, with a few exceptions like the immune system, cells in normal, healthy tissue continue to contain exactly the same genome sequence as was present in the initial single-cell embryo that gave rise to that individual. But new evidence suggests it may be time to revise that view.

By analyzing genetic information collected throughout the bodies of nearly 500 different individuals, researchers discovered that almost all had some seemingly healthy tissue that contained pockets of cells bearing particular genetic mutations. Some even harbored mutations in genes linked to cancer. The findings suggest that nearly all of us are walking around with genetic mutations within various parts of our bodies that, under certain circumstances, may have the potential to give rise to cancer or other health conditions.

Efforts such as NIH’s The Cancer Genome Atlas (TCGA) have extensively characterized the many molecular and genomic alterations underlying various types of cancer. But it has remained difficult to pinpoint the precise sequence of events that lead to cancer, and there are hints that so-called normal tissues, including blood and skin, might contain a surprising number of mutations —perhaps starting down a path that would eventually lead to trouble.

In the study published in Science, a team from the Broad Institute at MIT and Harvard, led by Gad Getz and postdoctoral fellow Keren Yizhak, along with colleagues from Massachusetts General Hospital, decided to take a closer look. They turned their attention to the NIH’s Genotype-Tissue Expression (GTEx) project.

The GTEx is a comprehensive public resource that shows how genes are expressed and controlled differently in various tissues throughout the body. To capture those important differences, GTEx researchers analyzed messenger RNA sequences within thousands of healthy tissue samples collected from people who died of causes other than cancer.

Getz, Yizhak, and colleagues wanted to use that extensive RNA data in another way: to detect mutations that had arisen in the DNA genomes of cells within those tissues. To do it, they devised a method for comparing those tissue-derived RNA samples to the matched normal DNA. They call the new method RNA-MuTect.

All told, the researchers analyzed RNA sequences from 29 tissues, including heart, stomach, pancreas, and fat, and matched DNA from 488 individuals in the GTEx database. Those analyses showed that the vast majority of people—a whopping 95 percent—had one or more tissues with pockets of cells carrying new genetic mutations.

While many of those genetic mutations are most likely harmless, some have known links to cancer. The data show that genetic mutations arise most often in the skin, esophagus, and lung tissues. This suggests that exposure to environmental elements—such as air pollution in the lung, carcinogenic dietary substances in the esophagus, or the ultraviolet radiation in sunlight that hits the skin—may play important roles in causing genetic mutations in different parts of the body.

The findings clearly show that, even within normal tissues, the DNA in the cells of our bodies isn’t perfectly identical. Rather, mutations constantly arise, and that makes our cells more of a mosaic of different mutational events. Sometimes those altered cells may have a subtle growth advantage, and thus continue dividing to form larger groups of cells with slightly changed genomic profiles. In other cases, those altered cells may remain in small numbers or perhaps even disappear.

It’s not yet clear to what extent such pockets of altered cells may put people at greater risk for developing cancer down the road. But the presence of these genetic mutations does have potentially important implications for early cancer detection. For instance, it may be difficult to distinguish mutations that are truly red flags for cancer from those that are harmless and part of a new idea of what’s “normal.”

To further explore such questions, it will be useful to study the evolution of normal mutations in healthy human tissues over time. It’s worth noting that so far, the researchers have only detected these mutations in large populations of cells. As the technology advances, it will be interesting to explore such questions at the higher resolution of single cells.

Getz’s team will continue to pursue such questions, in part via participation in the recently launched NIH Pre-Cancer Atlas. It is designed to explore and characterize pre-malignant human tumors comprehensively. While considerable progress has been made in studying cancer and other chronic diseases, it’s clear we still have much to learn about the origins and development of illness to build better tools for early detection and control.

Reference:

[1] RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues. Yizhak K, Aguet F, Kim J, Hess JM, Kübler K, Grimsby J, Frazer R, Zhang H, Haradhvala NJ, Rosebrock D, Livitz D, Li X, Arich-Landkof E, Shoresh N, Stewart C, Segrè AV, Branton PA, Polak P, Ardlie KG, Getz G. Science. 2019 Jun 7;364(6444).

Links:

Genotype-Tissue Expression Program

The Cancer Genome Atlas (National Cancer Institute/NIH)

Pre-Cancer Atlas (National Cancer Institute/NIH)

Getz Lab (Broad Institute, Cambridge, MA)

NIH Support: Common Fund; National Heart, Lung, and Blood Institute; National Human Genome Research Institute; National Institute of Mental Health; National Cancer Institute; National Library of Medicine; National Institute on Drug Abuse; National Institute of Neurological Diseases and Stroke


Taking In Some Science-Inspired Art

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Dr. Francis Collins poses with the artist Nana Bagdavadzeand her work depicting DNA
Enjoyed visiting Artists & Makers Studio 2 in Rockville, MD, where Nana Bagdavadze has an exhibit of DNA art that opened May 3, 2019. The artist’s work was inspired by her experience as a bone marrow donor for her sister, who’s doing great! Credit: NIH

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