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Life Is an N of 1

Michael Rosbash in his labView Large Image | View Hi-Res Image | Download PowerPoint SlideIt is a strange feeling not to anticipate begging, pleading, sweating, or getting angry at a rejection letter/email when crafting a manuscript for Cell. This manuscript will presumably escape that torture as it is a brief, personal story invited by the Cell editor Marta Koch. I was sorely tempted to tell the tale of my long and colorful history with Cell. However, it cannot be brief, so here is a different piece of personal history.Our Nobel Prize can be seen from several perspectives. It is for making important contributions to the mechanistic underpinnings of circadian rhythms. It celebrates basic science, emphasizes the importance of model organisms to research, and elevates Drosophila once again to a starring role. It is the fifth Prize for this remarkable, little beast and its first as a solo performer since 1995. (The metaphor makes me think of Meryl Streep and the Oscars.) Lastly, the Prize can be seen as underscoring the relationship between genes and behavior.The inheritance of behavioral traits—the “nature” in the nature-nurture debate—has a murky history. Eugenics gave it a bad rap, and it was overtaken in the US by Skinnerism and its emphasis on the environment—or “nurture”—in determining behavior. Twin studies as well as the dubious nature of many of those psychological studies contributed to swinging the pendulum back in the other direction toward inheritance. It has been followed by the current zeitgeist of epigenetics, which takes us back to the nurture camp. Although the word epigenetics has been hijacked by the histone-chromatin field, the term as originally coined by C.H. Waddington can be conveniently viewed in this context as environmental influence or developmental noise, i.e., anything that influences phenotype other than a fixed genetic path. In addition, determinism is generally unattractive, which adds to the appeal of non-genetic influences.It is against this backdrop that I entered the story. I was studying gene expression and RNA at Brandeis when I met Jeff Hall, also a professor at Brandeis, and began some years later to collaborate on circadian rhythms and the Drosophila period gene. Among the many reasons I began this work was the fact that I had always been interested in the relationship of genes and behavior. In particular, why did I always seem to court trouble? Was it nature or nurture, my genes or some environmental event?“I had always been interested in the relationship of genes and behavior. In particular, why did I always seem to court trouble?”Ever since I can remember, mostly since middle school, I had trouble at school. I do not mean academic trouble; I was always a good student. (Good does not mean outstanding, as I was largely indifferent to learning until mid-way through my second year at Caltech when I was bitten by the academic bug. My delayed path was probably not unrelated to my behavioral issues, but this is another story for another time.) However, I had quasi-serious behavioral issues in the Newton Public Schools before Caltech. I had a big mouth, was rowdy, and most importantly, really disliked authority—being told what to do. I also pushed buttons, and limits. Remarkably in hindsight, this caused me little trouble at Caltech as an undergraduate and at MIT as a PhD student. Both institutions were filled with weird characters and were remarkably tolerant meritocracies; if you could perform, everyone ignored all but the worst behavioral issues. And some iconoclastic behavior, a lack of respect for the status quo, is part of doing original research. So I thrived at both institutions.However, my character traits contributed to some trouble during my post-doc in Edinburgh and especially as an assistant professor at Brandeis. Although a much longer and more complicated story, I really lacked respect for my elders, which was frequently visible if not audible. Although some of this was due to my having been a politically engaged student in the late 60s (“Never trust anyone over 40”) as well as some very different and complicating local factors, my issues from adolescence still existed and were not irrelevant.These conflicts with authority led to my being denied tenure in the Rosenstiel Center, where my laboratory had resided since I had been hired in 1974. The negative recommendation was despite my strong publication and grant record. There were at the time two parallel tenure decisions required for Rosenstiel Center assistant professors: one in the Center and the other in the academic department of the candidate—in my case, Biology. In contrast to the Center decision, the Biology Department voted to recommend me for tenure albeit narrowly. This was a novel conflict, which landed on the desk of the Brandeis President for adjudication. He granted me University tenure but required that my lab vacate the Rosenstiel Center and that I move to Biology. The move was delayed because the University had some trouble finding and then outfitting adequate space, but I finally moved in August of 1982, to the Bassine Biology building. Strictly by chance, my new space turned out to be on the same floor as the lab of my pal Jeff Hall. We even shared a seminar-lunchroom, which was situated between the two labs. About 2 months later, in October of 1982, we began our collaborative work.“There are of course no control experiments in life; everything is an N of 1 . . . .”As I indicated above, one motivation to begin this gene-behavior work was my own character: how did it come about? For reasons that are too complicated to explain in a short essay. I was always partial to genetic or “hard-wiring” as responsible for human behavior. Our own gene-behavior research on fruit flies reinforced this prejudice, despite the sea of differences between this work and the kind of human question I had been pondering. However, there was one substantial argument in favor of an environmental explanation for my character: my father died of a sudden heart attack in 1954, when I was 10 years old and in 6th grade. This tragedy not only left me without a father and male role model but also shook our nuclear family to its core. I was set adrift and had a hard time finding some emotional center of gravity. Moreover, the world or at least my mother handled this tragedy much differently in the 1950s than one would today; for example, my father was never mentioned in our house again after his death. As the timing coincided roughly with middle school, indeed with my first memories of school troubles, this seemed a good candidate for a major contribution to my character. It was certainly a viable explanation, which resonated with a couple of my more-sophisticated friends over the years and even with me from time to time.Of course I never figured out how to investigate a human character question of this complexity, and therefore I left it behind as our circadian work progressed over the decades. Aging—some would say maturing—also left behind many of those difficult character traits that caused me so much trouble for so long. However, here is a counterargument to the aging or maturing description: my family to this day has been unable to tolerate Larry David because of his similarity to the contemporary Michael Rosbash. The resemblance is not only physical. My daughter said to someone not long ago: “Watch Curb Your Enthusiasm? Get real; we live with this kind of behavior all the time.” So maybe the changes over the 35 years since tenure and my laboratory move are more superficial than profound.Most of the original circadian crew, circa 1985. (L to R) Will Zehring, Tony (A.A.) James, Jeff Hall, Pranitha Reddy, Michael Rosbash, Dave Wheeler.View Large Image | View Hi-Res Image | Download PowerPoint SlideNonetheless, the question of my character and its origins reemerged in 2009. I had a 65th birthday symposium and party for the Biology Department and for my many trainees who returned to campus for the symposium. As my final talk required some reflection, I decided to explore this issue. I also thought it might amuse my trainees and colleagues, all of whom had experienced some version of my unvarnished character.“‘Watch Curb Your Enthusiasm? Get real; we live with this kind of behavior all the time.’”I was aided in this quest—and not without some irony you will appreciate—by my mother’s death the year before in 2008, when she was 94 years old. (My Lord, would she be kvelling had she lived to see this day!) She had kept all sorts of memorabilia from my childhood, including most notably my report cards and notes from my earliest years. Note that with the possible exception of my 6th grade report card, they all occurred well before my father’s death and therefore provide in my opinion unambiguous data in favor of a hard-wiring explanation.School report cardsView Large Image | View Hi-Res Image | Download PowerPoint SlideThere are of course no control experiments in life; everything is an N of 1, so maybe some earlier environmental event strongly contributed to my character. I also note that my preference for genetics or hard-wiring does not absolve me of responsibility, although I am still not a fan of free will as a biological-philosophical construct. And I tend to doubt almost everything, so all of these issues as well as most others must be associated with probability coefficients; there is no certainty. Or as I like to say in the lab: “I’ll give you 5-to-1 odds that it’s hard-wiring.” And to hedge even more strongly, “I’ll give you 5:1 that it’s mostly hard-wiring.” Don’t forget: doubt as well as curiosity is a motive for doing scientific research.

Reflections on Covers

Look at this month’s cover, the leftmost image in Figure 1Figure 1. Can you figure out what it means? Five months ago, the Cell Systems editorial team revised our guidelines for authors who are interested in submitting cover images. Now, with a full hand of covers to reflect on, I’d like to highlight these new guidelines and their broader significance.Figure 1Left to Right: Volume 5 Issue 5, November 22, 2017; Volume 5 Issue 4, October 25, 2017; Volume 5 Issue 3, September 27, 2017; Volume 5 Issue 1, July 26, 2017; Volume 2 Issue 1, January 27, 2016 (copyright William Rainey).View Large Image | View Hi-Res Image | Download PowerPoint SlideThe inset white diamond on the November cover is a radar chart. The purple and grey quadrilaterals drawn on the chart each plot four parameters of an oscillating waveform: its period (top vertex), amplitude (bottom vertex), rise time (left vertex), and decay time (right vertex). The purple shape is squashed on the bottom because that waveform oscillates with a much shorter amplitude. The gray shape doesn’t extend as high as the purple because it has a shorter period.Now, let’s layer on more information. The gray quadrilateral represents the behavior of a synthetic gene circuit containing a single branch of negative feedback. The purple quadrilateral represents the circuit with two parallel feedback branches. So the differences in shapes tell us that the oscillatory behavior of the circuit changes—predictably, as it turns out—when the circuit’s components are modified. This captures the essence of the work by Ping Wei, Wendell Lim, and Chao Tang on page 445 of this issue.Our new cover guidelines request images based on data and photos instead of original artwork. The previous guidelines specified, “Although submissions featuring original artwork will be considered, editors will look more favorably toward submissions that feature actual scientific data.” The new guidelines read, “The ideal cover image will either feature actual scientific data from the paper or high-quality photographs that convey the paper’s message.”So why the shift? Simply put, we believe in powerful stories told through data, clearly presented and set center stage. The use of visual design principles and an artistic touch can enhance the visual appeal of a piece of data. But beneath this surface appeal, we are looking for images that shine a spotlight on a crucial idea or piece of data central to a paper.Three recent covers provide examples of different approaches to featuring data. In some cases, a single figure panel showing a central result of the paper is visually striking and can be used on its own, as in the case of our October 2017 cover (second image from left; dark brown lines show the accumulation of metabolites secreted by yeast and then consumed by lactic acid bacteria). In other cases, an image can be rearranged, rotated, or repositioned to create an attractive composition (middle image; an initial oval-shaped group of cells was shifted down and left, and some cells were “wrapped around” to the top right corner). Alternatively, the strong use of color and background can accentuate an interesting shape (the silhouette of a tomato plant on the July 2017 cover; second image from right). For the inspiration behind this cover, see Henri Matisse's “Algue blanche sur fond orange et rouge.”Photos can also often be used to effectively create an enticing visual puzzle, and it is relatively easy to achieve polished results using them. For instance, for the cover of our January 2016 issue, which highlights engineering in biology, we chose a cover of a beaver—nature’s engineer—drinking at water’s edge.In contrast to photos, it can be more challenging to generate a scientifically meaningful and aesthetically appealing cover in which original artwork is used. We have used original artwork in the past, but going forward, we now request that authors use either original data or photos, which provide, in our experience, the most straightforward route to produce a compelling cover.To facilitate the creation of a cover image, we provide a template that shows the placement of the journal title and that indicates what parts of the image will be lost when the cover is trimmed to generate an online image (hint: make sure the important parts of the image are contained within the box labeled “trim”).I hope that exposing our approach to thinking about cover images will help those of you creating your own image for our covers. Going forward, we aim to further expose how we think about other aspects of running the journal, ranging from nuts-and-bolts decisions (such as how we choose the cover) to deeper issues (such as editorial decision-making criteria). Please write with any comments or suggestions for other aspects of our thinking that you would like to hear about (systems@cell.com).

The Coming Peptide Tidal Wave

Design, build, test, repeat—the essence of design thinking and a guiding principle of synthetic biology. Given the complexities of biological systems, there are bottlenecks that slow this virtuous design cycle for many of its potential applications. Yet, in the realm of small protein design and testing, the floodgates have just been thrust open.The recent study by David Baker’s group creates a platform for the design, testing, and refinement of small therapeutic proteins (Chevalier et al., 2017xChevalier, A., Silva, D.A., Rocklin, G.J., Hicks, D.R., Vergara, R., Murapa, P., Bernard, S.M., Zhang, L., Lam, K.H., Yao, G. et al. Nature. 2017; 550: 74–79PubMedSee all ReferencesChevalier et al., 2017) and uses this to create de novo designed protein inhibitors of influenza and botulinum toxin.A designed small protein (Bot.0671.2) bound the receptor binding domain (HcB) of botulinum neurotoxin. White is the design prediction; rainbow is the X-ray structure bound to the target (gray surface). Image courtesy of G. Rocklin.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe key element to their work is that it is “massively parallel” in most of its facets. In the initial stage, they design and screen hundreds of thousands of peptides computationally for likelihood of high-affinity binding to a target using Rosetta, a program developed in the Baker lab that predicts protein structure (https://www.rosettacommons.org/). Although protein structures have long been used to computationally aid in the design of small molecules, chemical space is fantastically vast, and our ability to build and test in that space is constrained by the limits of chemical synthesis or the availability of compounds in existing libraries. These limits are much less pronounced in protein space and the effort by Chevalier et al. illustrates that making and testing designed proteins should be far easier to scale.The authors point to the convergence of two technical advances that enable this massively parallel pipeline. First is the ability to accurately model the structure of small proteins in the size range of 40 amino acids, and second, the capacity to synthesize oligonucleotides large enough to encode them. After computationally identifying approximately 10,000 peptides with high-predicted binding and stability for the two targets, they are able to directly make the oligonucleotides encoding them. These are then assembled in yeast libraries from which cells expressing potential binders are sorted and the proteins identified using deep sequencing. This pipeline ultimately results in the identification of thousands of high-affinity binders. Further analysis of which predicted binders do bind and which don’t is used to refine the computational model, thus completing the design cycle.These landmark efforts build off the group’s long-term goal of figuring out how sequence impacts folding and stability and their use of iterative cycles to improve computational-based design (e.g., Rocklin et al., 2017xRocklin, G.J., Chidyausiku, T.M., Goreshnik, I., Ford, A., Houliston, S., Lemak, A., Carter, L., Ravichandran, R., Mulligan, V.K., Chevalier, A. et al. Science. 2017; 357: 168–175Crossref | PubMed | Scopus (6)See all ReferencesRocklin et al., 2017).To cap off their latest study, the authors choose individual peptides for additional proof-of-principle testing, including confirmatory crystal structures and evidence of in vitro and in vivo activity. Specifically, the influenza inhibitor is shown to have impressive efficacy in mouse models of infection, and the toxin inhibitor protects cultured rat cortical neurons.It is also very important to note that repeated administration of the influenza-targeting small proteins does not elicit a strong immune response. This portends well for de novo designed small proteins as potential therapeutics, although further work is needed to fully explore their immunogenicity and determine whether additional steps in the design stages will be needed to filter out peptides with high immunogenic potential. It is also an open question whether computational analysis can be used to filter out small proteins that have greater potential for off-target effects.Time to take out the crystal ball. What could this mean for a future bench researcher, who for example, might want to make a new inhibitor for a target protein? The starting point for such a project might one day be as simple as pulling up the structure, running a computer program, and ordering a few thousand oligonucleotides—perhaps an afternoon’s work. What could a shift to massively parallel peptide design mean for the larger scientific community? The number of clinical trials based on small peptide therapeutics has been increasing in recent years. This should only accelerate given the speed and ease of protein synthesis methods coupled with these new computational tools. Perhaps in a decade, the majority of new filings for clinical trials will be for small designer proteins and not small molecules or antibodies.

Tiny Answers to Big Questions

The most abundant biological entities on Earth still have a few tricks up their sleeve, and a recent surge of research is reinvigorating the field. With the potential to dramatically expand known protein sequence space, give insights into unprecedented biological processes, and provide means to wipe out antibiotic-resistant superbugs, bacteriophages may be tiny, but they are mighty.Image from iStock/Chima_Borealis.View Large Image | View Hi-Res Image | Download PowerPoint SlideBacteriophages, the ubiquitous and extremely diverse viruses that infect bacteria, are estimated to outnumber their bacterial hosts by a factor of ten. Anywhere bacteria thrive—from deep-ocean hydrothermal vents, to Arctic permafrost, to our own intestines—phages are abundant. With 1023 phage infections happening every second on Earth, it is clear that the extreme evolutionary pressures imposed by phages have shaped bacterial evolution. By transferring genetic material between bacteria, phages profoundly influence the emergence and spread of antibiotic resistance and virulence traits.Decades ago, phages helped scientists make many of the seminal discoveries in molecular biology and genetics. From the revelation that DNA is the hereditary material of cellular life, to the existence of a triplet code in translation, to foundational principles in DNA replication and recombination, phages were an integral part of the molecular biologist’s toolkit. Although they are often underappreciated, we can largely thank phages for the field of molecular biology itself.The phage legacy carries on even today, a century after their discovery. Notably, the study of interactions between bacteria used in the dairy industry and the phages that infect them led to the first evidence of the true function of CRISPR-Cas systems (Barrangou et al., 2007xBarrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. Science. 2007; 315: 1709–1712Crossref | PubMed | Scopus (1663)See all ReferencesBarrangou et al., 2007). Through ten years of intense research, these fascinating bacterial defense systems have been developed for genome engineering applications in countless eukaryotic organisms—even human embryos—and their potential impact on biomedical research, agriculture, and therapeutics is astounding.But phages aren’t just tools we can use to study fundamental biological processes. Apart from being the most abundant biological entities on the planet, phages are also the most diverse. Although many of us may conjure images of phage lambda or T7 from undergraduate textbooks, these well-studied examples are nowhere close to being representative. With more genome sequences becoming available at an unprecedented pace, scientists have a wealth of genetic “dark matter” to explore. Phages remain one of the largest untapped resources of completely novel genetic material, which is sure to contain many surprises.By exploring this incredible diversity, we are still learning unexpected things about what makes phages such effective bacterial killing machines. Early in 2017, a study in Nature reported the first evidence that phages communicate with each other. By producing and sensing small peptides as chemical messengers, phages make decisions about whether to proceed with the lytic or lysogenic infection cycle based on the abundance of phages present, in a mechanism analogous to bacterial quorum sensing. Although many different phages have this system, the sequence-specific production and recognition of the communication peptide ensures that each type of phage speaks and understands its own “language.” By controlling the dimerization of a phage-encoded transcriptional regulator, the peptide concentration can elicit different transcriptional responses, tuning the infection dynamics depending on the phage population density (Erez et al., 2017xErez, Z., Steinberger-Levy, I., Shamir, M., Doron, S., Stokar-Avihail, A., Peleg, Y., Melamed, S., Leavitt, A., Savidor, A., Albeck, S. et al. Nature. 2017; 541: 488–493Crossref | PubMed | Scopus (26)See all ReferencesErez et al., 2017).A study published in Science demonstrated that some phages assemble a specialized compartment within their host’s cytoplasm to facilitate spatial and temporal control over the phage replication cycle. This nucleus-like structure, composed of a phage-encoded protein shell, sequesters the phage genome and machinery involved in DNA replication and transcription. Remarkably, the authors observed phage capsids (heads) docking on the surface of the compartment to be packaged with phage DNA, then retreating back to the cytoplasm to assemble with tails into mature phage particles (Chaikeeratisak et al., 2017xChaikeeratisak, V., Nguyen, K., Khanna, K., Brilot, A.F., Erb, M.L., Coker, J.K.C., Vavilina, A., Newton, G.L., Buschauer, R., Pogliano, K. et al. Science. 2017; 355: 194–197Crossref | PubMed | Scopus (10)See all ReferencesChaikeeratisak et al., 2017). It remains to be determined how common this process is, how different components are trafficked in and out of the compartment, and whether it may play a role in protecting phages from bacterial defenses like CRISPR during the infection process.Apart from investigating these intriguing, natural biological processes, research into phage infection mechanisms has laid the groundwork for novel therapeutics. For many years, phage-based therapies for bacterial infections only gained traction in former Soviet nations, where a lack of western antibiotics necessitated the use of alternative treatments. No phage-based therapies have yet been approved in the United States for infectious disease treatment, but with the rise of antibiotic-resistant infections, many scientists are turning their attention back to nature to harness the incredible power of phages to kill bacteria. Just this spring, news articles emerged about a patient in San Diego, California, with a severe systemic bacterial infection that was resistant to all antibiotics. Doctors had run out of options to fight the infection, and as a last resort, the patient was treated intravenously with an experimental cocktail of phages. Remarkably, the patient awoke from his septic-shock-induced coma within three days of treatment and was later released from hospital with no apparent adverse effects of the phage injections (University of California, San Diego, 2017xSee all ReferencesUniversity of California, San Diego, 2017). Despite being only a single patient, this success will undoubtedly fuel future efforts into phage therapy.Phages really have it all: an impressive track record in molecular biology, a wealth of completely novel and unexpected biology, and a new renaissance of therapeutic potential. It has never been clearer that groundbreaking phage research is sure to carry on for the next hundred years and beyond.

Sorting Out Complex Thoughts and Messy Emotions

Cognition. Intelligence. Emotion. Sexuality. These are not merely complicated traits, invoking awed respect. These are aspects of animal life and human nature that are daunting in their biological complexity and in their existential importance. Curious biologists have been tackling animal behavior for centuries, but some topics and behaviors still remain opaque to biological understanding. While the demystification of human nature might give some unease, more of us are simply skeptical of any attempt to unravel the genetic underpinnings of such things.Image from iStock/Photo2008.View Large Image | View Hi-Res Image | Download PowerPoint SlideFor years now, genome-wide association studies (GWAS) have been mining ever-growing genetic datasets for clues to the genetic bases of complex traits and diseases that include behaviors and disabilities in cognition, intelligence, sexuality, social interaction, and emotion. Along with a few breakthroughs, there have been significant disappointments and legitimate questions about the limits of potential success (Visscher et al., 2012xVisscher, P.M., Brown, M.A., McCarthy, M.I., and Yang, J. Am. J. Hum. Genet. 2012; 90: 7–24Abstract | Full Text | Full Text PDF | PubMed | Scopus (904)See all ReferencesVisscher et al., 2012).Some geneticists don’t seem to have gotten the memo.Discussing the biology of human intelligence is a good way to start a scholarly brawl, and yet this complex trait is strongly heritable. Previous GWAS have found hints and candidates for causative genes, but the results are thought to be statistically underpowered. However, in May an international collaborative group published a large meta-analysis of data combined from these previous GWAS (and with new data), and reported 30 new and very promising candidate genes influencing human intelligence (Sniekers et al., 2017xSniekers, S., Stringer, S., Watanabe, K., Jansen, P.R., Coleman, J.R.I., Krapohl, E., Taskesen, E., Hammerschlag, A.R., Okbay, A., Zabaneh, D. et al. Nat. Genet. 2017; Crossref | Scopus (4)See all ReferencesSniekers et al., 2017). By increasing the cohort (nearly 80,000 people) and using some new tools (such as MAGMA), the work substantially expanded the list of genetic players. One new candidate is FOXO3, a transcription factor involved in insulin/IGF signaling.What of love? Shakespeare claimed that the course of true love never did run smooth, but geneticists recently claimed that assortative mating occurs in humans, meaning that humans tend to select mates that resemble themselves to some extent (Robinson et al., 2017xRobinson, M., Kleinman, A., Graff, M., Vinkhuyzen, A., Couper, D., Miller, M., Peyrot, W., Abdellaoui, A., Zietsch, B., Nolte, I. et al. Nat. Hum. Behav. 2017; 1Crossref | Scopus (5)See all ReferencesRobinson et al., 2017). This operates phenotypically, separate from confounding influences (such as socialization), and has implications for human population genetics and evolution. Another recent report found 12 genes associated with human reproductive behavior, specifically age at first birth and number of children (Barban et al., 2016xBarban, N., Jansen, R., de Vlaming, R., Vaez, A., Mandemakers, J.J., Tropf, F.C., Shen, X., Wilson, J.F., Chasman, D.I., Nolte, I.M...., BIOS Consortium, and LifeLines Cohort Study. Nat. Genet. 2016; 48: 1462–1472Crossref | PubMed | Scopus (11)See all ReferencesBarban et al., 2016). Romantic.Not even parenting practices are sacred. Hopi Hoekstra and her group study the evolution and genetics of behavior in closely related species of mice—the mice exhibit significant behavioral differences but can interbreed. This facilitates quantitative genetics and whole-genome analysis of behavioral traits. In a paper in April, the group reports on the genetics of parenting (Bendesky et al., 2017xBendesky, A., Kwon, Y.M., Lassance, J.M., Lewarch, C.L., Yao, S., Peterson, B.K., He, M.X., Dulac, C., and Hoekstra, H.E. Nature. 2017; 544: 434–439Crossref | PubMed | Scopus (12)See all ReferencesBendesky et al., 2017). Their tour de force showed heritability of a suite of parental behaviors (such as nest-making and baby-licking) and then dissected the genetic infrastructure. Even though the behaviors seem very similar in males and females, the underlying genetics can differ significantly. One behavior, nest-building, stands out, both because it seems genetically independent of other parenting tasks and because it has evolved through changes in the expression of vasopressin.Quantitative genetics is bringing powerful tools to old questions, including some deemed sacred or hopelessly complex. More drama is certain to come. Be sure to get a good seat.

The Right Tool for the Job

When you need a Phillips head screw driver, you need a Phillips head screwdriver. You might try to press a flat head into service, but it will be largely ineffective. Tools in biology are no different. The right tool for the job can mean the difference between nailing a biological question or leaving a project in pieces. Chemical tools, in particular, provide ways to acutely probe biological processes. It’s difficult to imagine where the cytoskeleton field would be without nocodazole or latrunculin or how well we would understand mRNA transport without leptomycin B. Sadly, however, there’s no equivalent of a hardware store for biology, and the tool shelf (such as it is) is sparsely stocked. There are chemical libraries that can be sourced from national centers, commercial sources, or collaborators in industry and academia (see, for example, Jones and Bunnage, 2017xJones, L.H. and Bunnage, M.E. Nat. Rev. Drug Discov. 2017; 16: 285–296Crossref | PubMed | Scopus (12)See all ReferencesJones and Bunnage, 2017). Screening them does yield hits, but the hits may or may not have the necessary specificity, selectivity, or properties to enable use in cells, live animals, or humans. The dilemma is how to pluck effective tools from within the ranks of available molecules or to engineer them.Image from iStockphoto/Floortje.View Large Image | View Hi-Res Image | Download PowerPoint SlideFor many small molecules with phenotypic effects or impacts on specific pathways, the precise molecular targets aren’t known, or too many molecular targets have been implicated. New work from Jesse Gray and Michael Springer tackles the latter issue (Boswell et al., 2017xBoswell, S.A., Snavely, A., Landry, H.M., Churchman, L.S., Gray, J.M., and Springer, M. Nat. Chem. Biol. 2017; 13: 501–507Crossref | PubMed | Scopus (2)See all ReferencesBoswell et al., 2017). What can you do when a small molecule shows potent effects in multiple screens aimed at finding inhibitors of dramatically different molecular processes? Is the molecule really promiscuous, or are the individual screens too coarse? Can further studies refine the answer? Gray and Springer’s study offers an approach to deconvolute the mechanisms of action for molecules targeting key steps in gene expression—a challenge because transcription, splicing, processing, and transport are interlinked. Using metrics based on analyses of single-strand sequencing data that can distinguish effects on distinct steps, a molecule, isoginkgetin (previously associated with inhibition of both splicing and RNA decay), was surprisingly shown to block transcription elongation. Other elongation inhibitors block CDK9 and control of pause release, but this one is different, and appears to act on a regulatory step later in the transcription process. Although isoginkgetin’s molecular target isn’t identified, these results point to a new and distinct control point for transcriptional elongation. That’s one shiny new addition to the toolkit for probing mechanisms of gene expression.Even when a protein target for a small molecule is well defined, it can be a challenge to ensure it interacts effectively and selectively in vivo—a measure of its ultimate utility. One approach is to capitalize on cellular identity, for example, by targeting a molecule to a cell surface protein that’s restricted to a subset of cells. A twist on that is to ectopically tether a molecule to specific cells, where it can interact with endogenous proteins. A recent paper from Michael Tadross and his colleagues focuses on bringing small-molecule inhibitors to the surface of neurons to test how inhibitors affect signaling in specific neuronal populations (Shields et al., 2017xShields, B.C., Kahuno, E., Kim, C., Apostolides, P.F., Brown, J., Lindo, S., Mensh, B.D., Dudman, J.T., Lavis, L.D., and Tadross, M.R. Science. 2017; 356: eaaj2161Crossref | PubMed | Scopus (6)See all ReferencesShields et al., 2017). The approach relies on expression of an optimized version of the HaloTag protein (Los et al., 2008xLos, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M. et al. ACS Chem. Biol. 2008; 3: 373–382Crossref | PubMed | Scopus (602)See all ReferencesLos et al., 2008), a bacterial enzyme engineered to traffic to the cell surface where it interacts selectively with an exogenously supplied ligand. By fusing specific inhibitors to the HaloTag ligand, they become highly enriched on the cells of interest, while proximal cells that lack the expressed HaloTag experience a dramatically lower concentration of the inhibitor and do not respond to it. One application examines how dopamine receptor-expressing cells contribute to the motor symptoms in Parkinson’s disease. Impairing AMPAR glutamate receptor signaling in D2 cells of a PD mouse model selectively tempers motor control dysfunction in these mice, establishing dominance of these cells over D1 cells in the circuits controlling motor function. Rapid response is one benefit of this kind of pharmacology. In addition, the targeted proteins are endogenous (of necessity proximal to the ectopically expressed HaloTag) and can be cell-type specific in combination with other resources (e.g., specific Cre lines or precise geographical administration).Every DIY project demands a combination of the right tools and craftiness or inventive problem solving. Questions in biology are just like this. These recent papers showcase how using small-molecule tools can build mechanistic “know-how” around a biological process and, through crafty application, can control where and when molecules encounter their targets to yield answers at the organism scale.

Will Diet and Exercise Save Us All?

How many times have you been to the doctor and was told that a balanced lifestyle is key for a healthy, disease-free, long life? How many times did genetics take the blame? But do we really know whether genetic risk associated with complex diseases can be offset by behavioral changes? A recent study at the New England Journal of Medicine now brings us some answers.Image from iStock.com/udraView Large Image | View Hi-Res Image | Download PowerPoint SlideThe idea that most human diseases are the result of a complex interplay between genes and environment is not new. Complex genetic disorders, those in which the effects of multiple genes are at play and clear-cut patterns of inheritance are difficult to pinpoint, are particularly influenced by lifestyle and environmental factors. Take heart disease as an example. While one’s own risk of cardiovascular disease increases by 3- to 5-fold if a person’s parent is diseased, factors such as diet, physical activity, tobacco use, environmental stress, and host factors such as age and sex also play a significant role in the risk of developing disease. The problem is that quantifying the contribution of each of these factors is tricky. Khera et al. (2016)xKhera, A.V., Emdin, C.A., Drake, I., Natarajan, P., Bick, A.G., Cook, N.R., Chasman, D.I., Baber, U., Mehran, R., Rader, D.J. et al. N. Engl. J. Med. 2016; CrossrefSee all References)Khera et al. (2016) set up to try to answer the billion dollar question: can a healthy lifestyle compensate for a strong genetic risk of developing disease?Genetic risk in complex diseases results from the combination of incremental effects of a large number of DNA sequence polymorphisms. In the case of coronary artery disease and myocardial infarct—a common and severe complication of cardiac disease—over 50 independent loci have been associated with risk of disease in genome-wide analyses. The aggregate of these risk genes provides has proved useful in defining genetic susceptibility to cardiovascular events. Khera et al. make use of this strategy to stratify the ∼60,000 subjects in their prospective study into three genetic risk categories: low, intermediate, and high. Then they ask whether baseline heathy lifestyle choices impact the risk of developing coronary events and atherosclerosis. The definition of healthy lifestyle was drawn from the strategic goals of the American Heart Association (AHA) to reduce the risk of cardiovascular in the general population and include no current smoking, no obesity, physical activity at least once weekly, and a healthy diet pattern. Importantly, the subjects in the study were previously enrolled in independent studies and represent three completely independent cohorts, what helps to power their analyses.As one would expect, subjects with high polygenic risk score were at much higher risk of developing coronary events than those at low genetic risk. What may come as a surprise is that further dividing subjects of different genetic risk groups according to their lifestyle choices shows that those who make healthy lifestyle choices present ∼45%–47% lower relative risk of developing coronary events. Even subjects at high genetic risk have their risk reduced when they make healthy lifestyle choices. The association also seems to hold true in men and women, and it does not seem to be under strong influence of ancestry. Moreover, not only the risk of adverse coronary events is reduced by adherence to healthy lifestyle but also coronary artery calcification, which is a prelude to more severe disease.Does that mean that going to the gym and eating fresh fruit, vegetables, grains, nuts and fish (while avoiding saturated fat, red meat and sugar-sweetened beverages) can counteract completely the effect of genetic inheritance? Certainly not. The new study clearly shows that high genetic risk is a strong and independent risk factor that increases the risk of coronary events and atherosclerosis. What perhaps is unexpected is that genetics and healthy lifestyle choices seem to be independently contributing to the susceptibility of heart disease. While it is true that people with high genetic risk benefit the most from healthy choices, the positive effect is also seen in those with intermediate and low genetic risk. In other words, healthy lifestyle benefits everyone, and high genetic risk is not necessarily deterministic of disease outcome.What do these findings mean from a basic biology perspective? Will we be able to get passed associations and begin to understand relationships of causality between healthy habits, genetics and disease? Will we be able to move beyond generic prescriptions of diet and exercise and tailor one’s choices to his or her genetics? Recent data have already showed that the response to food, for instance, is highly individualized when it comes to metabolism and glucose response (Zeevi et al., 2015xPersonalized Nutrition by Prediction of Glycemic Responses. Zeevi, D., Korem, T., Zmora, N., Israeli, D., Rothschild, D., Weinberger, A., Ben-Yacov, O., Lador, D., Avnit-Sagi, T., Lotan-Pompan, M. et al. Cell. 2015; 163: 1079–1094Abstract | Full Text | Full Text PDF | PubMed | Scopus (69)See all ReferencesZeevi et al., 2015), speaking to the need a personalized approaches for nutrition and medicine. Further, can the risk to other complex diseases be modified by behavioral changes? While we eagerly wait for answers to some of these questions, perhaps it would be wise to stick with carrots.

The I in Scientist

Beyond the shared, and perhaps self-evident, characters of a scientist, such as an interminable child-like curiosity, a passion for understanding, and a quasi-obsessive single mindedness, each one of us has their own set of very personal drivers. Some less obvious, perhaps even weirder, than others.Bassem Hassan. Image courtesy of Einstein foundation.View Large Image | View Hi-Res Image | Download PowerPoint SlideAfter an almost idyllic childhood in what was back then West Germany, I grew up in the late seventies and eighties —between the ages of 6 and 22—in war-torn, and repeatedly occupied, Beirut, Lebanon. A city I adore and loathe in equal measure. It is impossible to describe to those who have not experienced it what it is like to spend your entire youth in a civil war. Aside from all the death and destruction, there is an absurdity to the notion of a society tearing itself apart, and inviting others to share in its destruction, that has left me with a disdain—to say the least—for all the ideas that separate people on the basis of manufactured identities or beliefs. At the same time, it engendered in me a desire for transcendence. A search for something bigger and more universal: more sharable, so to speak.“We grew up relatively privileged surrounded by all manner of books in a household that was a sort of a cultural oasis in the midst of war.”The culture of scientific research was essentially non-existent during the war. But I grew up in a family that (over)emphasized academic excellence and, more generally, high achievement, as almost the only meaningful reasons for being! My parents both came from poor backgrounds and climbed the social ladder through education and hard work. Growing up, my sisters and I would often be told that the only thing we will inherit is our education. Overall, we grew up relatively privileged surrounded by all manner of books in a household that was a sort of a cultural oasis in the midst of war. I quickly became an obsessive reader, mostly of poetry, novels, and the biographies of great historical figures (from which I developed my immense adoration for Beethoven). For as long as I can remember I wanted to be a poet and started writing poetry at the age of fourteen and still do to this day. There was a brief phase in fourth and fifth grades—in retrospect perhaps definitive—in which I found science the coolest subject in school, mainly thanks to our science teacher. Apart from that however, it was all about literature, especially Arabic poetry. There is a condensed beauty in poetry, and in the imagery and metaphor of great poems, that no other medium of expression parallels. There was no question in my mind that literature was my calling … until the last year of high school.Hassan with old lab colleagues at Hugo Bellen’s 60th birthday symposium.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn 1986 another wonderful teacher, a gray-haired Lebanese of Greek background, taught us genetics, and I was smitten! The logic of Mendelian inheritance, the encoding of traits in DNA, the concept of a gene. All that revealed to me an esthetic elegance that life itself harbors. But this revelation came at a price: a sort of existential internal struggle. Poet or scientist? Scientist or poet? Looking back at it, I suppose the way I resolved the quandary is itself telling. I rationalized that as a scientist I could still write poetry, but that as a poet, I could not “do science.” Never mind that back then I had no clue what “doing science” actually entailed, and the only mechanism I could think about is to follow in my father’s footsteps and become a medical doctor. I started Pre-Med with a Biology major at the American University of Beirut but as soon as I was confronted with courses of animal anatomy and histology I realized that there was no way I would spend the next few years of my life memorizing names of muscles and bones. To me personally medicine was noble, but not beautiful, and I did not want to heal, I wanted to understand. I wanted to decipher the language in which nature writes her poems. I knew, however, of no way to change what seemed like a pre-set course. Once again an inspiring teacher—our microbiology instructor—changed my life. The microbiology course contained a healthy dose of molecular genetics of microorganisms and the basics of gene regulation. More importantly, our teacher had the answer to how to “do science” without getting an M.D. first! “Why don’t you do a Ph.D. in genetics in the US”? he suggested. My fate was sealed and the rest, as they say, is history!“The first time I saw these neurons and their axons and dendrites I was literally breathless. I just couldn’t stop looking down the microscope.”Fast forward more than 25 years and two amazingly generous scientific mentors and the drivers of my decisions, the hatred of war and the pursuit of beauty remain with me as major sources of inspiration. In the intellectual and experimental practice of science—if certainly not always in its institutions—I find a refuge from all the nonsense of identity politics. In science I have a pursuit that, like music, I find to be universal and timeless. Likewise, I find it extremely difficult to take on a project if I am not esthetically moved by it. I was driven to start working on two genes in my career as a Ph.D. student and then as a postdoc, simply because I found their expression patterns strikingly “beautiful.” I recall both moments as if they happened yesterday. The first occurred when I was looking for a new lab to do my Ph.D. I had started my Ph.D. working in a cancer research lab not because I was moved by it, but because I thought it would be “useful.” I suppose I was still under the influence of my pre-med days. Needless to say, without real motivation, things didn’t go very well, but thanks to a very supportive graduate program director, I had the opportunity to switch labs without losing financial support. I decided to do neuroscience because I thought neurons were amazingly cool cells and I wanted to work on Drosophila because of my lingering love for genetics. I visited the lab of Harald Vaessin, who had just started as a faculty member at Ohio State, and he showed me an in situ hybridization on a Drosophila embryo for the mRNA of a gene called prospero. The pattern of blue dots on white background in which you could see each cell that expressed the gene was simply stunning. How could I not work on that?! During my postdoc, I was again looking for a new project after publishing my first paper, when I walked into Hugo Bellen’s office to discuss some possibilities. On his desk was an image of a mouse embryo with blue dots (is there a theme developing here?) all over its skin. “What’s that? It’s very beautiful,” I asked holding the printout. “Take a guess,” he said smiling. “I’ll give you a hint: it’s a LacZ knock-in mouse from Huda’s [Zoghbi] lab.” “If there were such a thing,” I said, “I’d guess this is the mouse version of atonal,” I said sarcastically. “That’s exactly what it is!” Hugo exclaimed. That was the beginning of a long-lasting relationship between me and Atonal (a key neural transcription factor) that continues to this day and that has infected a few of my students and postdocs, I am afraid!Toward the end of my postdoc, again because of in situ hybridization for Atonal on Drosophila larval brains (yes, yes, blue dots …), I generated an enhancer-reporter line and ended up discovering an unknown neuronal circuit in the fruit fly brain. The first time I saw these neurons and their axons and dendrites I was literally breathless. I just couldn’t stop looking down the microscope. Their cell bodies formed two clusters located dorsally in the fly brain with their axons and dendrites arborizing exquisitely into the fly’s visual system. Based on their cell body position I named them the “dorsal cluster neurons” (DCNs), a name which I must admit I regret today because it is neither imaginative nor informative. Be that as it may, right there and then, I decided I would not stop until I figured out—some day—the rules of their development and function simply because I was—and still am—in awe of the “beauty” of their connectivity pattern. My lab still works on these neurons, and using them, my students and postdocs have contributed to our understanding of the probabilistic development of brain connectivity, leading to the formation of individualized neuronal circuits. A new generation of young scientists in my lab is taking these discoveries to new frontiers asking whether individualized neuronal circuit anatomy contributes to individualized innate behavioral tendencies.Dorsal cluster neurons (in green) in the fly brain.View Large Image | View Hi-Res Image | Download PowerPoint Slide“There is poetry in biology in both its form and content …”There is poetry in biology in both its form and content, and the pursuit of that beauty is both rigorously rational and emotionally uplifting. It is difficult for me to imagine a calling more satisfactory than this, and then sharing that passion for discovery with members of my team, in the hope that I give forward to them what my teachers and mentors have given me.In writing all this, I am keenly aware that this is a very personal story that may very well not resonate with or apply to many, perhaps most people. But that is the entire point of searching for, and being nourished by, our very personal drives. On that note, it is maybe a lucky coincidence—or perhaps not at all—that the twists and turns of my life and career have lead me today to study the neurodevelopmental basis of individuality … in flies! Perhaps somewhere in there lies another captivating line of nature’s magnificent poem that I will be lucky enough to read.

2015 Victor A. McKusick Leadership Award

View Large Image | View Hi-Res Image | Download PowerPoint SlideLet me begin by expressing my thanks to The American Society of Human Genetics (ASHG) for the recognition it is giving with the McKusick Award. Thanks also to David Valle for his very kind words of introduction and also to Neil Risch for the way he managed to astonish me with the news.Previous recipients of the McKusick Award have set the bar very high, and I am surprised to be joining them. This is a good moment for me to thank those in charge of the nominations and selection of candidates. It is also a good moment to suggest that the ASHG is perhaps wrong in honoring only me for the award this year because what is being recognized is in fact the opportunities that by chance came my way. Indeed, the colleagues who translated opportunities into data, discoveries, and services are ultimately honored here.Thanks also to the man from Maine, Victor McKusick, whose style, energy, wisdom, and advice have influenced so many of us. Victor had a memorable way of saying things and of getting things done, and he would certainly have something to say about my speaking to you from Australia under bizarre technical conditions. I can hear Victor saying, “If I had to go to theyre, I would not start from heyah.”Recognition is precious, and it is received accordingly. The ASHG is recognizing me, but in fact, with this award, it is recognizing many, many persons and a team of good people who I will not name individually here so as to avoid errors of omission. My colleagues are identifiable in many other ways, for example, as co-authors of publications and as participants in programs of service and in initiatives in education. Indeed, we cooperated, making the whole more than the sum of its parts and letting cooperation be seen as a successful feature of human evolution. This is my opportunity to express my personal admiration and affection for all those colleagues. In the words of Thomas Merton, they have been “shining like the sun” and are recognized here accordingly.And then there is the family! It has been and remains an inspiration. The words are brief; the feeling is huge.Chance and opportunity, where and when, have played important roles in my life. For example, in 1962, my wife and I were traveling in our Volkswagen Beetle when an impatient driver tried to pass us on the two-lane highway. Unfortunately, there was oncoming traffic in the other lane. The impatient person smashed into us while trying to avoid a head-on collision. Our car veered, and in a fortunate break in the traffic, we rolled (yes, rolled!) through a gap in the oncoming lane of cars. Believing we were about to die, I called out to my wife, “It has been great!” We did survive, and the continuing gift of life has been and continues to be acknowledged in every breath.Where chance and opportunity played their important roles in my life and when they did are relevant facts. For example, I have worked all my academic life at the Montreal Children’s Hospital under the McGill University umbrella in Quebec, Canada. These proved to be enlightened environments. When they played their roles was also important. In the 1950s, Quebec was experiencing the Quiet Revolution, which transformed Quebec society; and while that was happening, the role of the DNA molecule in biology was being recognized. The incidental development of chromatography as a new technology, and its contribution to the discovery of inborn errors of metabolism, for example, greatly influenced me personally. I was participating as a fledgling clinician-scientist in what is now called translational science. I found it could serve scientific inquiry, medical services, and the health of society as a whole. The character of society is revealed in how we treat our weaker members, such as children, the aged, the disabled, and the chronically ill. Perhaps at some other time, I could describe how my own experiences illustrate a relatively humane society in progress. You would discover why it has been possible for me to work with all of those colleagues in this place called the Montreal Children’ s Hospital at McGill University in Montreal, Quebec, Canada.I have always enjoyed literature and poetry. I love this poem by the Russian author Osip Mandelstam; it was composed in 1909. It is a metaphor for the genetics of life itself:What shall I do with the body I have been givenSo much at one with me, so much my own?For the calm happiness of breathing, being ableTo be alive, tell me where I should be grateful.I am gardener, flower too, and un-aloneIn this vast dungeon.My breath, my glow, you can already seeOn the windowpanes of eternity.A pattern is imprinted thereUnknown ’til now.Let this muddle die down, this sediment flow outThe lovely pattern cannot be crossed out.And then there is this wonderfully honest brief comment by W.S. Merwin, the American poet, who at age 84 wrote, “I have with me all that I do not know / I have lost none of it.”Thank you again for the recognition and for this opportunity to share a thought or two.

2015 Victor A. McKusick Leadership Award Introduction: Charles Scriver

View Large Image | View Hi-Res Image | Download PowerPoint SlideGood morning.The Victor A. McKusick Leadership Award is presented on behalf of our society to an individual whose professional achievements have fostered and enriched the development of various human genetics disciplines. Recipients exemplify enduring leadership and the vision required for ensuring that human genetics will flourish and successfully assimilate into the broader context of science, medicine, and health.This year’s recipient of the McKusick Leadership Award is Dr. Charles Scriver.Charles is a Canadian through and through! He was born in Montreal, Quebec, Canada. Both of his parents were distinguished academic physicians on the faculty of McGill University. Charles followed their path: he received his B.A. and his M.D. from McGill and did his internship and residency in pediatrics at that same institution.His training at McGill had two important interruptions: he did one year of residency (1957–1958) at Harvard and one year of fellowship (1958–1959) at University College London (UCL). These brief times away from McGill were important for Charles because they launched his career as a biochemical geneticist. At Harvard, he met Mary Efron, with whom he described new inborn errors of amino acid transport and metabolism. At UCL, he worked with C.E. Dent on cystinuria and learned the fundamentals of amino acid transport.Returning to McGill, Charles completed his chief residency in pediatrics and joined the pediatrics faculty. He rapidly rose in the ranks to become the director of the deBelle Laboratory for Biochemical Genetics and the Alva Professor of Human Genetics. His group was incredibly productive and became one of the most prominent in the world. He has published more than 400 papers and many book chapters. He has received numerous honorary degrees and awards, including the ASHG William Allan Award in 1978 and the Excellence in Human Genetics Education Award in 2001. Early in his career, he worked closely with Leon Rosenberg at Yale, another recipient of the McKusick Award. Together, they wrote a seminal book, Amino Acid Metabolism, which educated a generation of biochemical geneticists on this topic.From the “spring board” of biochemical genetics, Charles developed a voracious interest in all aspects of genetics and, in particular, how it informs medicine. His many contributions include delineation of new disorders, refinement and application of newborn and population screening, development and testing of novel treatments of genetic disease, informing public policy, genetic education, and the like.Along the way, he served on the ASHG Board of Directors and as our president in 1987. He also served as president of the Canadian Society of Clinical Investigation, the Society for Pediatric Research, and the Society for the Study of Inherited Metabolic Diseases.He married his wife, Zipper, in 1956, and together they have four children. After spending time with them both, I know that her support has been essential for his success.My own direct interactions with Charles began in the 1970s, when I was looking for a fellowship in biochemical genetics. I visited several programs and went through the usual drill: talks and interviews, typically ending with a statement of interest and promise to be in touch in a few days. By contrast, when I first walked in the door of Charles’s office, he said, “We would love to have you join our group, and let me tell you what we are doing.” His openness, honesty, and gentle directness were not only refreshing but, I learned, also constant features of Charles’ personality and his leadership style.Although I did not accept his offer, to my great good fortune I ended up working with Charles in many ways on many different projects over the years, in particular serving along with Art Beaudet and Bill Sly as editors of The Metabolic and Molecular Bases of Inherited Disease for the last 30 years. The experiences we shared on this joint venture were career highlights for all of us.The McKusick Award recognizes leadership, and we all know that different leaders have different styles. Charles’s style is the quintessential model of leading by example: accessing a problem, seeking advice, making a plan, and participating directly in seeing it through. This model has served him, and consequently us, very well over the years.Finally, Charles is a true scholar and is as comfortable discussing philosophy, history, or literature as he is describing the intricacies of phenylalanine hydroxylase. As a consequence, interactions with him are always wonderful learning experiences.In these many ways, Charles personifies the features of the McKusick Award, and I am thrilled and proud to be able to introduce him to you!

Monitoring of Single Vesicle Cytochrome-c Release Illuminates BAK as a Novel Target of Aβ Oligomers

Intrinsic apoptosis is initiated by the BAX- or BAK-dependent permeabilization of the mitochondrial outer membrane and subsequent release of cytochrome c. In Alzheimer’s disease (AD), significant death of hippocampal and cortical neuronal populations occurs through multiple processes including activation of the intrinsic apoptosis pathway (1xApoptosin is a novel pro-apoptotic protein and mediates cell death in neurodegeneration. Zhang, H., Zhang, Y.W...., and Xu, H. J. Neurosci. 2012; 32: 15565–15576Crossref | PubMed | Scopus (18)See all References1). In particular, the expression of BAK is upregulated in AD hippocampal and cortical tissues when compared to age-matched control tissues (2xAlteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer’s disease. Kitamura, Y., Shimohama, S...., and Taniguchi, T. Brain Res. 1998; 780: 260–269Crossref | PubMed | Scopus (187)See all References, 3xApoptotic gene expression in Alzheimer’s disease hippocampal tissue. Sajan, F.D., Martiniuk, F...., and Freedman, M.L. Am. J. Alzheimers Dis. Other Demen. 2007; 22: 319–328Crossref | PubMed | Scopus (29)See all References). Prior studies have demonstrated that amyloid beta peptide 1–42 (Aβ1–42) oligomers induce cytochrome-c release and neuronal apoptosis via a BAX-dependent mechanism (4xSelective cytotoxicity of intracellular amyloid β peptide1–42 through p53 and Bax in cultured primary human neurons. Zhang, Y., McLaughlin, R...., and LeBlanc, A. J. Cell Biol. 2002; 156: 519–529Crossref | PubMed | Scopus (315)See all References4). In this issue of the Biophysical Journal, Kim et al. (5xBeta-amyloid oligomers activate apoptotic BAK pore for cytochrome-c release. Kim, J., Yang, Y...., and Shin, Y.-K. Biophys. J. 2014; 107: 1601–1608Abstract | Full Text | Full Text PDF | PubMed | Scopus (6)See all References5) identify a significant role for BAK in cytochrome-c release induced by Aβ1–42 oligomers. The authors demonstrate that Aβ1–42 oligomer-induced HT22 hippocampal cell death is significantly reduced by siRNA knockdown of BAK. Moreover, mitochondria isolated from BAK knockdown HT22 cells display reduced cytochrome-c release in vitro in response to either Aβ1–42 oligomers or truncated BID (tBID).Most impressively, the authors have developed a cell-free, liposome-based, fluorescence cytochrome-c release assay by which single vesicle release events can be accurately recorded and monitored over time. They carefully validate this assay using BAKΔC-term and tBID recombinant proteins and convincingly show that Aβ1–42 oligomers are capable of inducing cytochrome-c release from BAKΔC-term-containing vesicles as well as enhancing release in the presence of tBID. Finally, they show that Aβ1–42 oligomers bind directly to BAKΔC-term-containing vesicles but do not bind as significantly to vesicles containing tBID or protein-free liposomes. In regard to this latter point, although the authors suggest that the relatively weak binding of Aβ1–42 oligomers to protein-free liposomes represents nonspecific binding, this may in fact not be the case.Many previous studies have demonstrated that Aβ1–42 (or Aβ1–40) oligomers interact directly with lipid membranes and that these interactions are significantly influenced by a number of factors including oligomer concentration, Aβ1–40/Aβ1–42 ratio, cholesterol content, and charge on both the lipid surface and the peptide (6xInteraction between amyloid-β (1–42) peptide and phospholipid bilayers: a molecular dynamics study. Davis, C.H. and Berkowitz, M.L. Biophys. J. 2009; 96: 785–797Abstract | Full Text | Full Text PDF | PubMed | Scopus (60)See all References, 7xCholesterol modulates the interaction of β-amyloid peptide with lipid bilayers. Qiu, L., Lewis, A...., and Cheng, K.H. Biophys. J. 2009; 96: 4299–4307Abstract | Full Text | Full Text PDF | PubMed | Scopus (29)See all References, 8xβ-Amyloid (1–40) peptide interactions with supported phospholipid membranes: a single-molecule study. Ding, H., Schauerte, J.A...., and Gafni, A. Biophys. J. 2012; 103: 1500–1509Abstract | Full Text | Full Text PDF | PubMed | Scopus (21)See all References, 9xNanoscale electrostatic domains in cholesterol-laden lipid membranes create a target for amyloid binding. Drolle, E., Gaikwad, R.M., and Leonenko, Z. Biophys. J. 2012; 103: L27–L29Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References, 10xSingle-molecule imaging reveals Aβ42:Aβ40 ratio-dependent oligomer growth on neuronal processes. Johnson, R.D., Schauerte, J.A...., and Gafni, A. Biophys. J. 2013; 104: 894–903Abstract | Full Text | Full Text PDF | PubMed | Scopus (9)See all References). It would be interesting to examine how these different factors influence the Aβ1–42 oligomer interaction with BAK because the oligomerization process can be significantly modulated by the specific interactions of the Aβ1–42 peptide with the lipid membrane.We believe the results presented by Kim et al. (5xBeta-amyloid oligomers activate apoptotic BAK pore for cytochrome-c release. Kim, J., Yang, Y...., and Shin, Y.-K. Biophys. J. 2014; 107: 1601–1608Abstract | Full Text | Full Text PDF | PubMed | Scopus (6)See all References5) are novel and clearly demonstrate a central role for BAK in cytochrome-c release triggered by Aβ1–42 oligomers (Fig. 1Fig. 1). This is in contrast to previous studies suggesting that Aβ1–42 (or Aβ1–40) oligomers induce direct permeabilization of mitochondrial or other lipid membranes in the absence of any membrane-associated proteins (11xTwo-step mechanism of membrane disruption by Aβ through membrane fragmentation and pore formation. Sciacca, M.F., Kotler, S.A...., and Ramamoorthy, A. Biophys. J. 2012; 103: 702–710Abstract | Full Text | Full Text PDF | PubMed | Scopus (73)See all References, 12xMitochondrial membrane permeabilization by amyloid aggregates and protection by polyphenols. Camilleri, A., Zarb, C...., and Vassallo, N. Biochim. Biophys. Acta. 2013; 1828: 2532–2543Crossref | PubMed | Scopus (23)See all References). However, these prior studies used much higher concentrations of Aβ oligomers, which might account for the differences observed. Furthermore, the development of a liposome-based, fluorescence cytochrome-c release assay by which single vesicle release events can be monitored over time represents a significant technological advance that will allow the probing of additional pathways which regulate this process. Future studies that examine, in more detail, the mechanism by which Aβ1–42 oligomers activate BAK will be informative. Finally, the findings of Kim et al. (5xBeta-amyloid oligomers activate apoptotic BAK pore for cytochrome-c release. Kim, J., Yang, Y...., and Shin, Y.-K. Biophys. J. 2014; 107: 1601–1608Abstract | Full Text | Full Text PDF | PubMed | Scopus (6)See all References5) suggest that BAK may be a new therapeutic target for AD, and should prompt investigation into the effects of crossing BAK knockout mice with transgenic mouse models of high Aβ1–42 content such as the APPswe/PSEN1dE9 (APP/PS1) mouse.Figure 1A central role for BAK in cytochrome-c release triggered by Aβ1–42 oligomers. BAK oligomerization and pore formation in the mitochondrial outer membrane are triggered by interactions with either tBID or Aβ1–42 oligomers. Cytochrome c released through the BAK pore becomes an integral part of the apoptosome complex, which also includes APAF-1 and pro-caspase-9 (not shown). The apoptosome activates the intrinsic initiator caspase-9, which, in turn, cleaves and activates the executioner caspase-3, leading to apoptotic cell death. To see this figure in color, go online.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Behind the Scenes of CLC Gating: Deriving the Voltage Dependence of Membrane Proteins by Admittance Measurements

CLC channels and transporters are expressed in virtually every living cell and fulfill a number of housekeeping functions such as stabilizing the resting potential of skeletal muscles, controlling renal salt excretion, and regulating [Cl−] and pH in diverse cell organelles (1xCLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Jentsch, T.J. Crit. Rev. Biochem. Mol. Biol. 2008; 43: 3–36Crossref | PubMed | Scopus (239)See all References1). The physiological importance of the CLC family is emphasized by the existence of various human diseases associated with mutations in genes encoding CLC channels or transporters. The CLC family encompasses anion channels and secondary-active Cl−/H+ exchangers (2xSecondary active transport mediated by a prokaryotic homologue of ClC Cl− channels. Accardi, A. and Miller, C. Nature. 2004; 427: 803–807Crossref | PubMed | Scopus (400)See all References2), and thus contains proteins that are mediators of thermodynamically different transport processes. Despite these principal differences in function, all known CLC isoforms seem to be regulated by complex voltage-dependent gating. The importance of this regulation is illustrated by a large number of disease-associated mutations that specifically modify the voltage-dependence of various members of the CLC family.Gating of CLC channels and transporters are both related to conformational changes that underlie the coupled exchange cycle in the isoforms belonging to the transporter branch. High-resolution, three-dimensional structures of pro- and eukaryotic CLC transporters in different conformations provide insights into the structural rearrangement underlying coupled transport as well as the channel and transporter gating. This structural information revealed the existence of a highly conserved glutamate side chain at the extracellular side that can either project into the aqueous external solution or occupy two of three anion-binding sites in the CLC anion selectivity filter (3xGating the selectivity filter in ClC chloride channels. Dutzler, R., Campbell, E.B., and MacKinnon, R. Science. 2003; 300: 108–112Crossref | PubMed | Scopus (510)See all References, 4xStructure of a eukaryotic CLC transporter defines an intermediate state in the transport cycle. Feng, L., Campbell, E.B...., and MacKinnon, R. Science. 2010; 330: 635–641Crossref | PubMed | Scopus (115)See all References). This glutamate side chain—often referred to as “gating glutamate”—is apparently moving in two sequential steps from the outside into the anion conduction pathway of the protein (Fig. 1Fig. 1 A). Surprisingly, there are no functional data that demonstrate the existence of such sequential steps in mammalian CLC proteins. Gating of CLC transporters is perfectly well described with a simple two-state gating scheme. The nonlinear capacitances associated with activation gating are also monophasic and change upon voltage steps with monoexponential time dependences. This inconsistency is now addressed in an exciting new study by Grieschat and Alekov in this issue of the Biophysical Journal (5xMultiple discrete transitions underlie voltage-dependent activation in CLC Cl−/H+ antiporters. Grieschat, M. and Alekov, A.K. Biophys. J. 2014; 107: L13–L15Abstract | Full Text | Full Text PDF | PubMed | Scopus (4)See all References5), and it is used to provide novel insights into the mechanisms underlying CLC channel/transporter gating.Figure 1Movement of the gating glutamate and anion permeation in WT and mutant ClC-5. Gating charge movement (gc) consists of two components gc1 and gc2. In WT ClC-5 (A), the first component (gc1) arises from binding of the gating glutamate to the external Cl− binding site Sext and the simultaneous unbinding of one internal Cl− ion. The value gc2 is conferred by the movement of the gating glutamate to the central Cl− binding site Scen and unbinding of another internally bound Cl−. K201R and E211D disrupt the synchronization of gating glutamate movement and unbinding of the internal anion (B). To see this figure in color, go online.View Large Image | View Hi-Res Image | Download PowerPoint SlideCLC transporters do not only display pronounced time- and voltage-dependence (6xDeterminants of anion-proton coupling in mammalian endosomal CLC proteins. Zdebik, A.A., Zifarelli, G...., and Pusch, M. J. Biol. Chem. 2008; 283: 4219–4227Crossref | PubMed | Scopus (71)See all References, 7xChannel-like slippage modes in the human anion/proton exchanger ClC-4. Alekov, A.K. and Fahlke, C. J. Gen. Physiol. 2009; 133: 485–496Crossref | PubMed | Scopus (31)See all References) but also exhibit prominent gating charge movements (8xVoltage-dependent charge movement associated with activation of the CLC-5 2Cl−/1H+ exchanger. Smith, A.J. and Lippiat, J.D. FASEB J. 2010; 24: 3696–3705Crossref | PubMed | Scopus (20)See all References, 9xClC-3 is an intracellular chloride/proton exchanger with large voltage-dependent nonlinear capacitance. Guzman, R.E., Grieschat, M...., and Alekov, A.K. ACS Chem. Neurosci. 2013; 4: 994–1003Crossref | PubMed | Scopus (15)See all References) that are reminiscent of gating currents of voltage-gated cation channels. Grieschat and Alekov (5xMultiple discrete transitions underlie voltage-dependent activation in CLC Cl−/H+ antiporters. Grieschat, M. and Alekov, A.K. Biophys. J. 2014; 107: L13–L15Abstract | Full Text | Full Text PDF | PubMed | Scopus (4)See all References5) used these charge movements to study two mutants of the human ClC-5 exchanger by applying lock-in-based admittance measurements. Both mutations, K210R and E201D, perturb the motility of the external gate of the CLC transporter and lead to the appearance of two well-separated peaks in the voltage-dependent capacitances. The novel biphasic voltage dependence suggests that activation of mutant ClC-5 is not executed in one single step, but includes several distinct substeps. These steps depend on the concentrations of the permeant anion Cl−. Changes in internal but not external [Cl−] affected the apparent gating charge responsible for the activation of ClC-5. This asymmetry led the authors to conclude that internal anions directly contribute to the voltage sensitivity of the gating process. The different behavior of the two investigated mutations was attributed to a derailed activation sequence with lost synchronization between two intrinsically voltage-dependent steps—the sequential occupation of two stable conformations by the gating glutamate and the knock-off of Cl ions from the internal anion binding site (Fig. 1Fig. 1 B). Although the nature of processes that couple the conformational state of the gating glutamate and the anion occupancy of the internal binding site was not clarified, the reported findings convincingly demonstrate the discrete nature of gating processes in mammalian CLC transporters.The findings of Grieschat and Alekov (5xMultiple discrete transitions underlie voltage-dependent activation in CLC Cl−/H+ antiporters. Grieschat, M. and Alekov, A.K. Biophys. J. 2014; 107: L13–L15Abstract | Full Text | Full Text PDF | PubMed | Scopus (4)See all References5) provide novel important insights into the mechanisms of voltage-dependent gating in the CLC family. CLC proteins lack an obvious endogenous voltage sensor, and the origin of voltage-dependent channel gating has been debated for many years. Gating was suggested to be entirely conferred by the permeant anion; or by protons; or by the movement of charged amino-acid side chains. The reported data now suggest that all these processes jointly contribute to voltage-dependent CLC channel gating. In particular, proteins of the CLC family appear to sense voltage by both movements of the gating glutamate as well as by anion binding/unbinding reactions.The study not only provides exciting results about a highly important family of transport proteins, but also illustrates how lock-in based admittance measurements (10xPhase tracking: an improved phase detection technique for cell membrane capacitance measurements. Fidler, N. and Fernandez, J.M. Biophys. J. 1989; 56: 1153–1162Abstract | Full Text PDF | PubMedSee all References, 11xAdmittance-based measurement of membrane capacitance using the EPC-9 patch-clamp amplifier. Gillis, K.D. Pflugers Arch. 2000; 439: 655–664Crossref | PubMed | Scopus (75)See all References) can provide novel insights into complex conformational changes in membrane proteins. Admittance measurements can be used to separate nonlinear terms in the membrane capacitance that reflect movements of charges within the transmembrane electric field, and which depend on the responses of the investigated protein to voltage. In an oversimplified view, the nonlinear capacitance produced by voltage-dependent conformational changes is proportional to the first derivative of the electric charge that is moved during the underlying gating process. Mathematically, the ability of the derivative to detect not only local minima and maxima of a function, but also inflection points, makes such measurements more sensitive to experimentally separate individual steps within more complex gating schemes, even when the steps do not exhibit strongly differing voltage dependences.Similar strategies might prove advantageous to explore the gating sequence of any arbitrary protein when an increased resolution of its voltage dependence is desired. A huge number of novel transport proteins with complex properties have been identified in recent years that await biophysical investigation and interpretation. Some of us will use the methods elegantly applied in this study, and we are all looking forward to studies that apply high-resolution electrical recordings to understand the complex function of many other ion channels, coupled transporters, or pumps.

Marie T. Filbin: in memoriam.

Marie T. Filbin, Distinguished Professor of Biology at Hunter College in New York City, died on January 15, 2014 while visiting her family in Ireland. Following some early breakthrough work on the biochemistry of peripheral myelination, Marie’s most significant scientific contributions were in the understanding of the molecular mechanisms of the inhibition of axonal growth and the facilitation of axonal regeneration. Marie was a passionate and highly effective advocate for research in spinal cord injury and a tireless campaigner on behalf of patients with spinal cord disease, with some of whom she developed a deeply caring and life-long personal relationship. Marie was the very antithesis of a “stuffy” scientist—she lived her life with flair and style, infectious charm, great (and occasionally irreverent) good humor, passion for her work, devotion to her friends and family, and was a remarkable mentor to her students.Marie T. Filbin: 1955–2014View Large Image | View Hi-Res Image | Download PowerPoint SlideMarie Therese Filbin was born in 1955 in the town of Lurgan, Northern Ireland, where her family had been in business for generations. She attended the University of Bath, where she received a PhD in Biochemistry in 1981 for work on the characterization of nicotinic acetylcholine receptors in the locust. In 1982, she moved to the University of Maryland for postdoctoral training without (she admitted) much detailed career planning but driven by a strong sense of adventure and discovery. The move to Baltimore proved fortuitous, however, when in 1984 she joined Gihan Tennekoon in the Department of Neurology at Johns Hopkins. In a seminal paper published with Dr. Tennekoon, Marie showed that that the tightly wound myelin leaflets that surround peripheral nerves are bound together by P0, the most abundant myelin protein, which functions as a hemophilic adhesion molecule (Filbin et al., 1990xFilbin, M.T., Walsh, F.S., Trapp, B.D., Pizzey, J.A., and Tennekoon, G.I. Nature. 1990; 344: 871–872CrossRef | PubMedSee all ReferencesFilbin et al., 1990). At Hopkins, Marie was exposed to a group of researchers and clinicians with a strong interest in disorders of myelin in the peripheral and central nervous systems, and this experience helped to shape the direction of her future career. It was also a time during which she cemented some of the strongest and longest bonds of friendship and collaboration that would remain throughout her life.In 1990, Marie moved to New York City to take a faculty position in the Biology Department at Hunter College, the flagship institution of the City College of New York system. She continued to work on P0 in her own lab for the next several years, defining the conditions necessary for P0 to mediate myelin adhesion—including demonstrating that the protein needs to interact with the myelin cytoskeleton, directly or indirectly, for peripheral myelin adhesion to take place. As she began to focus on the integrated role of different myelin proteins during the process of remyelination, Marie became aware of the molecular dissonance between the mechanisms of axonal regrowth and remyelination. At this point, her focus changed to the role of inhibitory molecules within the white matter of the CNS that retard or prevent neural regeneration. She was the first to show that myelin-associated glycoprotein (MAG)—a transmembrane protein in both the central and peripheral nervous system—is an important inhibitor of neurite growth after injury (Mukhopadhyay et al., 1994xMukhopadhyay, G., Doherty, P., Walsh, F.S., Crocker, P.R., and Filbin, M.T. Neuron. 1994; 13: 757–767Abstract | Full Text PDF | PubMed | Scopus (769)See all ReferencesMukhopadhyay et al., 1994). After several meticulous and elegant papers aimed at elucidating underlying mechanisms, she eventually showed that the inhibitory effect of MAG is mediated through the NoGo receptor (Domeniconi et al., 2002xDomeniconi, M., Cao, Z., Spencer, T., Sivasankaran, R., Wang, K., Nikulina, E., Kimura, N., Cai, H., Deng, K., Gao, Y. et al. Neuron. 2002; 35: 283–290Abstract | Full Text | Full Text PDF | PubMed | Scopus (419)See all ReferencesDomeniconi et al., 2002). Realizing that myelin is present in varying degrees in any in vivo system in which regeneration occurs after injury, Marie’s growing multinational lab focused most of its efforts on investigating molecular manipulations that enhance axonal regrowth in the presence of myelin inhibition. Her primary finding that increasing levels of endogenous cAMP could neutralize the natural inhibitory effects of other molecules (Cai et al., 2001xCai, D., Qiu, J., Cao, Z., McAtee, M., Bregman, B.S., and Filbin, M.T. J. Neurosci. 2001; 21: 4731–4739PubMedSee all ReferencesCai et al., 2001) was highly controversial at the time but is now considered to be a major breakthrough. For this work, she was awarded the Ameritech Prize in 2001 and shortly thereafter received a prestigious Javits Investigator Award from the NIH. Within the past decade, much of Marie’s work concentrated on experimental manipulations that might be more immediately useful in treating spinal cord injury in human subjects and thus moved toward in vivo models both in her own lab and in collaboration with many other labs using complimentary injury models (Pearse et al., 2004xPearse, D.D., Pereira, F.C., Marcillo, A.E., Bates, M.L., Berrocal, Y.A., Filbin, M.T., and Bunge, M.B. Nat. Med. 2004; 10: 610–616CrossRef | PubMed | Scopus (452)See all ReferencesPearse et al., 2004).At Hunter College, a primarily undergraduate institution not known for its vibrant research program, Dr. Filbin found a wonderful environment in which she could develop her career with the full support and encouragement of the administration. Although given the opportunity to move several times, she chose not to because she recognized the special environment that Hunter provided both her and her trainees. Marie was proud to represent Hunter in the international research community and as its representative, her face, for a time, decorated the sides of New York City buses, a distinction that few of us can claim, to her great delight and the amusement of her friends. At Hunter, Marie was the long-term Director of the NIH-sponsored Specialized Neuroscience Research Program (SNRP), which supports research at institutions with a high enrollment of minority students and has been instrumental in raising the general profile of research at the institution and launching the scientific careers of many. Her own lab became a “mini United Nations,” carrying the faces and names from the melting pot of ethnicities drawn to Hunter; each of them became absorbed into her lab family, as a member of which they were expected to work as hard as she did and, in return, Marie cared for them and went to unusual lengths to promote their work and careers and provide them the support they needed to succeed in science.Marie as science ambassador and local transport celebrityView Large Image | View Hi-Res Image | Download PowerPoint SlideMarie expected the same high standards of her students and collaborators that she demanded of herself; she was direct, honest, and famously blunt in her scientific dealings. On review panels, she was a staunch champion for grants she believed in and regularly gave the benefit of the doubt to young scientists with ambition and vision. She was remarkably generous with her time and her heart and showed tremendous concern for the welfare of her students—one of whom she nursed (in loco parentis) through a terminal illness. In the case of another, she successfully campaigned to have an immediate deportation order rescinded, which required her to put together in short order a coalition of senior scientists and politicians willing to support her position, and she made several court appearances on the student’s behalf.Marie maintained close and frequent contact with her family in Ireland as with her friends in many parts of the world, and over the years, she accumulated a large collection of godchildren who adored her. About 15 years ago, on a whim, Marie bought a West Highland terrier and christened him McDuff; although he barked frequently at imaginary intruders spotted through her living room window, he brought her great joy and was her constant companion at home and at work. Marie loved the vitality of New York City—its food, art, theater, and, particularly, its people. She enjoyed entertaining and was a wonderful hostess and an enthusiastic and exacting cook. Her apartment had a revolving door that welcomed colleagues and friends from across the globe. She did not believe in the value of “beauty sleep,” perhaps because she did not need it. If the phase had not become such a cliche, you might say that she was the life and soul of the party. Nevertheless, Marie was certainly reluctant to “call it a night” if there was at least one other person present who believed the night was still young. Having kept cancer at bay for over a decade, during the last few years of her life as her disease progressed, she became more and more disabled, and yet she bore this burden with grace, courage, good humor, and optimism. Marie was a passionate and accomplished scientist who lived a full and spirited life in which all who knew her were blessed by her warm smile and kind heart. Because she loved poetry, in particular the work of Seamus Heaney, this is for Marie:Late August, given rain and sunFor a full week, the blackberries would ripen.At first, just one, a glossy purple clotAmong others, red, green, hard as a knot.You ate the first one and its flesh was sweetLike thickened wine: summer’s blood was in itLeaving stains upon the tongue and lust forPicking. Then red ones inked up and that hungerSent us out with milk-cans, pea tins, jam-potsWhere briars scratched and wet grass bleached our boots.(From Blackberry-Picking by Seamus Heaney)

The Importance of Intrinsic Order in a Disordered Protein Ligand

Over the last decade, it has become increasingly apparent that a significant number of proteins do not form stable tertiary structures, but instead dynamically sample a broad range of largely disordered states (1xIntrinsic protein disorder in complete genomes. Dunker, A.K., Obradovic, Z...., and Brown, C.J. Genome Inform. Ser. Workshop Genome Inform. 2000; 11: 161–171PubMedSee all References1). Generally referred to as intrinsically disordered proteins (IDPs), these species can be associated with a range of functions, and often serve as hub proteins that bind to multiple targets (2xFrom sequence and forces to structure, function, and evolution of intrinsically disordered proteins. Forman-Kay, J.D. and Mittag, T. Structure. 2013; 21: 1492–1499Abstract | Full Text | Full Text PDF | PubMed | Scopus (37)See all References2). Binding events involving IDPs are commonly accompanied by the formation of defined structure. In many cases, these appear to follow an induced-fit model of binding, where the loss of conformational entropy (in going from a largely disordered free state to a structured bound state) is balanced by the extensive number of interactions that can be accessed by the structurally malleable IDP. However, over the last decade it has become clear that the free state does not sample the full range of conformations that would be predicted from random chain diffusion (e.g., the kinase inhibitory domain from Sic (3xCompaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain. Brocca, S., Testa, L...., and Grandori, R. Biophys. J. 2011; 100: 2243–2252Abstract | Full Text | Full Text PDF | PubMed | Scopus (30)See all References3) and a polyglutamine binding protein (4xSolution model of the intrinsically disordered polyglutamine tract-binding protein-1. Rees, M., Gorba, C...., and Chen, Y.W. Biophys. J. 2012; 102: 1608–1616Abstract | Full Text | Full Text PDF | PubMed | Scopus (4)See all References4)). Instead, these systems exhibit bias toward more compact, transiently structured states, suggesting that the loss of conformational entropy in the interaction is not as drastic as originally thought.In some cases, the structural propensities of the unbound IDP play a more active role in determining the affinity of the interaction, with significant sampling of conformations resembling the bound state structure (5xp15 (PAF) is an intrinsically disordered protein with nonrandom structural preferences at sites of interaction with other proteins. De Biasio, A., Ibanez de Opakua, A...., and Blanco, F.J. Biophys. J. 2014; 106: 865–874Abstract | Full Text | Full Text PDF | PubMed | Scopus (5)See all References, 6xNMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein. Haba, N.Y., Gross, R...., and Chill, J.H. Biophys. J. 2013; 105: 481–493Abstract | Full Text | Full Text PDF | PubMed | Scopus (7)See all References). This has renewed interest in the possibility that conformational selection also plays a role in IDP interactions, where binding could be favored by the transient formation of a structured region in the free state that is preferentially bound by the target protein (7xCoupled folding and binding with α-helix-forming molecular recognition elements. Oldfield, C.J., Cheng, Y...., and Dunker, A.K. Biochemistry. 2005; 44: 12454–12470Crossref | PubMed | Scopus (321)See all References7). Evidence that conformational selection may be important for some IDP interactions has been accumulating, in particular for IDPs that form helical structures in the bound state.These studies have shown that perturbations altering the ability of the unbound state to sample a helical conformation correlate with binding affinity (8xMechanism of induced folding: both folding before binding and binding before folding can be realized in staphylococcal nuclease mutants. Onitsuka, M., Kamikubo, H...., and Kataoka, M. Proteins. 2008; 72: 837–847Crossref | PubMed | Scopus (34)See all References, 9xInternal dynamics control activation and activity of the autoinhibited Vav DH domain. Li, P., Martins, I.R.S...., and Rosen, M.K. Nat. Struct. Mol. Biol. 2008; 15: 613–618Crossref | PubMed | Scopus (67)See all References, 10xHelical propensity in an intrinsically disordered protein accelerates ligand binding. Iesmantavicius, V., Dogan, J...., and Kjaergaard, M. Angew. Chem. Int. Ed. Engl. 2014; 53: 1548–1551Crossref | PubMed | Scopus (32)See all References). In this issue of the Biophysical Journal, a similar relationship is demonstrated for an IDP that interacts with its target via an extended structure, providing evidence that conformational selection may also play a role in IDP interactions involving nonhelical binding motifs (11xConformational recognition of an intrinsically disordered protein. Krieger, J.R., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References11).The work in this issue, by Krieger et al. (11xConformational recognition of an intrinsically disordered protein. Krieger, J.R., Fusco, G...., and De Simone, A. Biophys. J. 2014; 106: 1771–1779Abstract | Full Text | Full Text PDF | PubMed | Scopus (15)See all References11), takes advantage of the information provided by NMR chemical shifts to characterize the unbound state of a peptide from the multisite scaffolding protein Gab2. In its SH3 domain-bound state, an N-terminal polyproline sequence in the Gab2 peptide forms an extended structure that interacts with a hydrophobic groove on the SH3 surface, while its C-terminal RxxK sequence binds via a 310 helix (12xDistinct binding modes of two epitopes in Gab2 that interact with the SH3C domain of Grb2. Harkiolaki, M., Tsirka, T...., and Feller, S.M. Structure. 2009; 17: 809–822Abstract | Full Text | Full Text PDF | PubMed | Scopus (39)See all References12). To characterize the structure of the free state, backbone chemical shifts from the Gab2 peptide were assigned and analyzed for secondary structure content. Two regions of residual structure were identified that contained significant populations of extended β- and polyproline type II structures (highlighted in Fig. 1Fig. 1). In addition, molecular-dynamics simulations were employed using replica-averaged chemical shifts as structural restraints (13xCharacterization of the conformational equilibrium between the two major substates of RNase A using NMR chemical shifts. Camilloni, C., Robustelli, P...., and Vendruscolo, M. J. Am. Chem. Soc. 2012; 134: 3968–3971Crossref | PubMed | Scopus (51)See all References13) in a procedure that has been shown to bias sampling in a way that accurately recapitulates the conformational tendencies of the system (14xAssessment of the use of NMR chemical shifts as replica-averaged structural restraints in molecular dynamics simulations to characterize the dynamics of proteins. Camilloni, C., Cavalli, A., and Vendruscolo, M. J. Phys. Chem. B. 2013; 117: 1838–1843Crossref | PubMed | Scopus (23)See all References14). The application of this chemical shift-restrained molecular-dynamics method, combined with a statistical mechanics analysis of the ensemble, revealed a free energy landscape for Gab2 that favors the formation of a significant population of extended structures resembling the bound state.Figure 1Structure of the Grb2 C-terminal SH3 domain (gray surface; green online) bound to a peptide spanning residues 508–521 from Gab2 (IQPPPVNRNLKPDR). The two regions of the Gab2 peptide that showed extended β- and polyproline II structure in the free state are highlighted (black; red online) for the N-terminal polyproline sequence (dark gray; orange online) for the region C-terminal to the RxxK motif. (PDB:2VWF.) To see this figure in color, go online.View Large Image | View Hi-Res Image | Download PowerPoint SlideFurther raising the significance of these findings were results from a series of mutant peptides that was also subjected to the same measurements and analyses. A reasonable correlation was observed between peptide binding affinity and the propensity of the free state to sample conformations resembling the bound state. The consistency of the trend for free-state conformational tendencies versus binding affinities over the range of mutants tested provides a strong case for the importance of the residual structure in this interaction. These results are in agreement with a model of binding that is facilitated by conformational selection of this bound state structure. Alternatively, the conformational propensities of the free state could influence binding affinity by increasing the rate of binding-induced folding, and/or decreasing the rate of unfolding-coupled dissociation—an interesting possibility that could potentially be resolved by kinetics experiments in the future.These results should be of general interest for groups that study fundamental principles of protein-protein interactions, and particularly those that involve intrinsically structured domains. Although there is a growing body of evidence that bound-state structures can be sampled in IDP ligands, relatively few studies have shown a relationship between binding affinity and the presence of residual bound-state structure, particularly in nonhelical binding motifs. The work presented in this issue helps address this deficiency, and contributes to the evolving view of IDP interactions that utilize a combination of conformational selection and induced fit to increase the affinity and specificity of these interactions (15xMultiscaled exploration of coupled folding and binding of an intrinsically disordered molecular recognition element in measles virus nucleoprotein. Wang, Y., Chu, X...., and Wang, J. Proc. Natl. Acad. Sci. USA. 2013; 110: E3743–E3752Crossref | PubMed | Scopus (36)See all References15).

2013 Curt Stern Award Introduction: John Moran

It is a great honor and a distinct pleasure to introduce the 2013 recipient of the Curt Stern Award, my good friend John Moran. This award honors the memory of Curt Stern as a pioneering human geneticist and is presented annually for outstanding scientific achievements in human genetics over the past 10 years.View Large Image | View Hi-Res Image | Download PowerPoint SlideStern was a leading Drosophila geneticist trained by Thomas Hunt Morgan, who was first to demonstrate crossing-over in homologous chromosomes in the fruit fly. He taught first at the University of Rochester and then at the University of California, Berkeley. After World War II, he turned to human genetics and in 1949 published a renowned textbook of human genetics. I first attended The American Society of Human Genetics (ASHG) annual meeting in 1971, and I remember when Curt Stern received the William Allan Award in 1974. It was a grand occasion honoring a pioneer in our field.Today we honor another outstanding scientist, John Moran, principally for his work in determining the importance of mobile DNA, or “jumping genes,” in the human genome. I have been very fortunate in the quality of graduate students and postdocs who have come to my lab. John Moran was right at the top of the list. Before I had even heard of John, Jef Boeke, an esteemed Johns Hopkins colleague, told me that he had met Moran and that Moran was going to apply to join my lab. I remember Jef’s comment that John was “really a terrific person and scientist.”Boy, was I fortunate that after Moran had done his graduate work on mobile DNA in yeast, he decided to work on human transposition as a postdoc and to join my lab. We had isolated two potentially active human L1 elements that had retrotransposed into F8 and DMD. John decided that he would set up a cell-culture assay for retrotransposition. Because he had meticulously assembled suitable reagents, plasmids, and cell lines and had a great scheme for detecting real retrotransposition events, his assay actually worked marvelously on the first try. Frankly, I couldn’t believe that all those HeLa cell colonies he obtained in that first experiment contained a retrotransposition event. Yet indeed they did! His experiments always seemed to work. He used his assay to show that both L1-encoded proteins were critical for retrotransposition and that during evolution, L1s could shuffle exons throughout the genome. John left my lab in 1998 for the University of Michigan after getting job offers from over ten different universities, and he continues to use his in vitro retrotransposition assay in much of his work at Michigan.That work has been amazing! He has discovered that virtually all of the findings made in cell culture recapitulate what happens in human beings. He has also found that in the battle for survival between retrotransposons and their hosts, retrotransposons, or “jumping genes,” can affect genome evolution in many ways.Along the way, John has published over 60 papers, including ten in the very top journals, Cell, Science, and Nature, and won many honors. In 1998, he won the ASHG Postdoctoral Research Award. He has been a Damon Runyan fellow and a Keck Foundation Distinguished Young Scholar. In the past 15 years at the University of Michigan, he has been awarded numerous institutional honors, including the Dean’s Award for Basic Science Research and an endowed university-wide chair, the Gil Omenn Collegiate Professorship of Human Genetics. For the past 5 years, he has been a Howard Hughes Medical Institute investigator, and in 2012 he was elected a fellow of the American Association for the Advancement of Science. John has been involved in many activities in support of the academic community, including fulfilling considerable teaching responsibilities at Michigan, serving as the associate director and soon-to-be director of the Predoctoral Genetics Training Grant at Michigan, and being very active in the affairs of ASHG. Because of his gregarious and exuberant style and clear presentations, Moran has been in high demand as a speaker at seminars and national and international symposia, leading him to average eight to ten talks per year.John has a number of personal characteristics that are important to his success as a scientist. First, he loves to talk about science, both yours and his. Second, in the lab he always carries out every possible control that anyone could think of to make sure that his claims are correct. He also holds his many trainees to this very high standard. Third, he is loaded with ideas, most of which are really good. I’ve never met any scientist with as many good experimental ideas that could be readily tested. On the other hand, John does have a few faults. First, he is a diehard fan of the New York Yankees. John was insufferable in continually asserting that Lou Gehrig was a better player than Cal Ripken. Second, Moran loves to talk. When you speak with John by phone, you need to block off at least 30 min for the conversation. If you haven’t got 30 min, you need to cut him off in mid-sentence. Seriously, I’ve always enjoyed conversations with Moran because his language is so colorful and his topics, science and sports, are so interesting. Another important characteristic of John is his devotion to his family. Moran has a wonderful wife, Robin, and three great children. In the summer of 2012, Jessica, age 10 at the time, accompanied John on a business trip to Japan. On that trip, Moran’s colleagues got to see what a wonderful relationship he had with his daughter!To conclude, it is truly fitting that John Moran receive the 2013 Curt Stern Award for his impeccable science and the impact he has had on human genetics and evolutionary biology through his work on human mobile DNA. Please join me in congratulating him for his stellar achievements!

Activation Pathways of Kinases Reveal Intermediate States as Novel Targets for Drug Design

Unregulated activation of kinases leads to aberrant signaling, uncontrolled growth, and differentiation of cancerous cells. Reaching a complete mechanistic understanding of the large scale conformational transformations underlying the activation of kinases could greatly help in the development of therapeutic drugs for the treatment of these pathologies. In princi- ple, the nature of conformational transition could be modeled by in silico via atomistic molecular dynamics simulations, although this is very challenging due to the long timescales (100s of μs) associated with activation. In this study, we employ a computational paradigm that couples transition pathway generation techniques and Markov State Model (MSM) based massively (total simulation time of ∼3 milliseconds) distributed simulations for mapping the conformational landscape of several key tyrosine kinases. The computations provide the thermodynamics and kinetics of kinase activation for the first time, and help identify key intermediates along the activation pathway. Furthermore, the presence of a novel allosteric sites in an intermediate states of kinases that could be potentially utilized for drug design is predicted.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Super-Resolution Imaging of Cav1.2 Channel Clusters

L-type Cav1.2 channels regulate multiple processes in neurons, muscle and endocrine cells, including action potential duration, excitation-contraction coupling, gene expression, neurotransmitter and insulin release. The amplitude and functional impact of local Ca2+ signals (“Cav1.2 sparklets”) depends on the spatial distribution of Cav1.2 channels in the surface membrane. Here, we used TIRF microscopy in combination with STORM and GSD super-resolution imaging to determine the organization of Cav1.2 channels in the surface membrane of arterial myocytes and tsA-201 cells. The spatial resolution of our super-resolution maps was ∼30 nm. The localization of Cav1.2 was determined based on immunofluorescence or EGFP fluorescence. As shown in the super-resolution map in Fig. 1, we found that in arterial myocytes, Cav1.2 channels were expressed in clusters broadly throughout the cell membrane. However, the size and geometry of these clusters varied, suggesting that number of Cav1.2 channels within clusters was dissimilar. The average size of the clusters was ∼3 μm2. Up to 12 channels could occupy clusters of this size assuming a monomer occupies ∼240 nm2. Similar findings were obtained from tsA-201 cells expressing Cav1.2-EGFP or immuno-labeled Cav1.2 channels. Data will be presented on the functional implications of these findings.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Continuous Flow AFM Imaging Reveals Fluidity and Time Dependent Interactions of Antimicrobial Dendrimer with Model Lipid Membranes

The alarming increase in multi-drug resistant bacteria worldwide is a major threat to modern life and there is thus an urgent need to address this issue by novel approaches in order to improve and tailor alternative drug formulations in the future. The nanoparticle studied here is a newly synthesized antimicrobial peptide-based dendrimer that acts by disrupting cell surfaces in a non-specific manner, thus rendering it less susceptible to resistance-forming mechanisms in bacteria.The effects of bilayer fluidity and presence of domains have been studied using neutron reflection and an atomic force microscope setup optimized for continuous flow imaging, developed specifically for investigating interactions with fast kinetics.Using these approaches we observed a requirement for membrane fluidity for dendrimers to induce high curvature and solubilize the membrane in a detergent-like manner. Domain coexistence led to a sequence of events initiated by the formation of a dense thread-like network and followed by membrane solubilisation via spherical aggregates from bilayer edges.In contrast, for gel-phase membranes, the antimicrobial dendrimers adsorbed and caused areas of locally depressed regions - a mechanism resembling membrane interdigitation or membrane thinning.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HIPS-CM's): An Expression Model System for Investigating Cardiac Channelopathies

Background and rationale:Until recently the study of dysfunctional ion channels in their native environment was only possible by using transgenic animal models, which do not recapitulate human physiology. Here, we have investigated the use of hiPS-CM's as a model system for investigating cardiac channelopathies, specifically long QT Syndrome-9 (LQT9) causing Cav3 mutation (F97C-Cav3).Methods:Commercially available hiPS-CM's were obtained from Cellular Dynamic International. hiPS-CM's were infected with WT-Cav3 or F97C-Cav3 and studied with standard patch-clamp techniques.Results:Using HEK293 cells and in rat myocytes we previously reported that LQT9 causing mutations did not affect peak-sodium current (INa-P), but increased late-sodium current (INa-L). In the HEK293 heterologous cell system we transfected the key components nNOS, α1-syntrophin and WT-Cav3/F97C-Cav3 to elucidate mechanism. To investigate the use of hiPS-CM's as a model, we infected hiPS-CM's with WT-Cav3/F97C-Cav3. We report that, INa-p was unchanged (A&B) but report an increase in INa-L (C&D).Conclusion:hiPS-CM's are a relevant physiologic expression model system to study cardiac channelopathies that recapitulates the native environment of a human ventricular myocyte.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Determining the 3D Topologies of Heteromeric Protein Assemblies by a Mass-Spectrometry Based Hybrid Approach

Describing, understanding, and modulating the function of the cell require elucidation of the networks and structure of its macromolecular assemblies. Here, we describe an integrative approach to determine the topologies of heteromeric assemblies using structural information derived from native mass spectrometry (MS), proteomics and chemical cross-linking MS. The method was developed and further assessed for robustness and accuracy using a benchmark of five hypothetical assemblies with simulated data and two assemblies with previously published MS data. With this benchmark in hand, we purify and characterize the yeast eukaryotic initiation factor 3 (eIF3) complex whose complete 3D topology has not been previously defined. Using a combination of MS-based data, we establish sub-stoichiometric binding of eIF5 and derive a high-likelihood structural model for the five subunit eIF3 complex. Our results further reveal two interaction modules within the eIF3 and are in accord with its role as a scaffold for other initiation factors. In conclusion, here we highlight the utility of a MS-based integrative approach for modelling complexes with unknown interactions and topology. The corresponding computational algorithm is implemented in the open source Integrative Modeling Platform (IMP).View Large Image | View Hi-Res Image | Download PowerPoint Slide